Functional proteins are known to undergo natural selection processes preserving their function and hence their structure. The protein energy landscape for a protein sequence is molded by evolution such that its native protein structure is conserved; however, point mutations can reshape the energy landscape of a protein populating certain (un)folding pathways. Evolutionary information, encapsulate within multiple sequence alignments (MSAs), can be used to identify conservation and mutation patterns, which are evidence of structural constraints plus mutational drift. The main hypothesis is that residues in physical contact coevolve (i.e. they show correlated mutational behavior) and that some of them may be both structurally or functionally important positions within protein folds and consequently could be targets for disease-associated point mutations. Coevolution-based strategies to disentangling indirect long-range dependencies between amino acids have received a new twist thanks to the increasing availability of protein sequences and to new methodological advances in the handling of phylogenetic information. In this work, we aim the comprehensive characterization of co-evolving residues leading to disease when disrupted by a point mutation. To this end we retrieve from protein MSAs homologous sequences containing experimentally found mutations and we compute a coevolution-based strategy, called normalized point-wise mutual information, to capture non-trivial covariations or geometric conservations responsible of (un)folding processes. The proposed protocol was tested with G-protein-coupled receptors proteins (GPCR) and it was able to identify co-variate residues e interactions that significantly (de)stabilize the native structure of these proteins and we were able to characterize and analyze these correlations with respect to
Isothermal titration calorimetry (ITC) is a powerful tool for acquiring both thermodynamic and kinetic data for biological systems. ITC offers several advantages over other experimental kinetics methods as it can be performed entirely in solution under physiological conditions, does not require spectroscopically-active (eg. fluorescent) molecules, it is compatible with spectroscopically opaque solutions, and can be applied to relatively dilute samples. Despite its long history and technical advantages, kinetic applications of ITC remain fairly rare. In order to expand the use of ITC kinetics we have developed several techniques in order to measure physical properties of enzymes and enzyme inhibitors. These techniques allow us to measure both michaelis-menten and non-michaelis menten properties of rapidly evolving enzyme reactions, measurement of the mode and strength of enzyme inhibitors in a single experiment and, lastly, permits the direct measurement of the association and dissociation rates of enzyme inhibitors. These experiments are simple and allow for rapid and complete characterization of enzymes and enzyme inhibitors and will broaden the overall applicability of ITC.
Organ functions decline during aging, and the most profound changes occur in the kidney. Kidneys are continuously exposed to oxidative stress. They are particularly vulnerable to physiological and environmental insults.
The proper response to stress is crucial for cell and organ survival. The formation of cytoplasmic stress granules (SGs) is a conserved reaction that helps eukaryotic cells to survive stress. Aging impairs the stress response, but little is known about the underlying mechanisms.
It is our goal to define how aging compromises the kidney’s ability to cope with stress. To this end, we developed two models of renal proximal tubule cell aging. They are based on the chemical or pharmacological induction of senescence. We demonstrated that both model systems display hallmarks of aging.
Using these models, we assessed SG formation and stress-induced signaling. We showed that aging impairs SG assembly. Moreover, our studies uncovered the underlying molecular mechanisms.
Taken together, our research provides a better understanding of the aging-dependent changes in kidney physiology. We identified new biomarkers that can score the stress response in kidney cells. Long-term, this information will help to develop new diagnostic and therapeutic tools to evaluate cellular aging.
Ribonucleases are enzymes involved in ribonucleic acid degradation forming a protein superfamily in which all members share similar characteristics. Bovine RNase A is the most studied enzyme of the family. Previous studies have shown that this enzyme is dynamic with movements particularly pertaining to histidine 48 and loop 1 residues. These movements are involved in substrate fixation and product release mechanisms. Such dynamic properties have been demonstrated for human members of the ribonuclease family but their involvement in biological functions remains unknown.
Among the eight human members of this superfamily, four possess a biological activity of therapeutic interest: RNase 2 (Eosinophil Derived Neurotoxin), RNase 3 (Eosinophil Cationic Protein), RNase 4 and RNase 5 (Angiogenin). Indeed, these proteins display antibacterial, antiviral, cytotoxic and angiogenic activities, respectively. In order to modulate these biological activities, allosteric regulation could offer the advantage of fine tuning enzyme activity without a direct interaction with substrate binding site.
Fragment based drug discovery (FBDD), a technique that can screen small chemical fragments, could be used to identify lead compounds that could be allosteric modulators. The effect of these chemical entities on protein dynamics and enzymatic activity will allow to determine parameters necessary to design efficient allosteric modulators of these ribonucleases.
In mammalian cells, the incorporation of the 21st amino acid selenocysteine into proteins is guided by the selenosome. The function of this protein complex requires several protein-protein and protein-RNA interactions, leading to the incorporation of selenocysteine at UGA codons. It is guided by stem-loop structures localized in the 3’ untranslated regions of the selenoprotein-encoding genes. Here, we conducted a global analysis of interactions between selenosome components using a bioluminescence resonance energy transfer assay in mammalian cells that showed that selenocysteine synthase (SecS), SECp43, and selenophosphate synthetases SPS1 and SPS2 form oligomers in eukaryotic cells. We also showed that SPS2 interacts with SecS and SPS1; these interactions were confirmed by co-immunoprecipitation. To further analyze the interactions of SECp43, the protein was expressed in Escherichia coli and small angle X-ray scattering analysis revealed that it is a globular protein comprising two RNA-binding domains. Using phage display, we identified potential interaction sites and highlighted two residues (K166 and R169) required for its dimerization. The SECp43 structural model presented here constitutes the basis of future exploration of the protein-protein interactions among early components of the selenocysteine biosynthesis and incorporation pathway
Infectious diseases are a major problem worldwide. In order to promote infection, many bacterial pathogens employ multicomponent protein complexes to deliver macromolecules directly into their eukaryotic host cell, like type IV secretion systems (T4SS). T4SS are important for two reasons: genetic exchange and release of effectors into the target cell. These functions allow adaptation of pathogens to environmental changes or disruption of host defense mechanisms.
Using phage display, BTH and FRET-based assays, our laboratory has already identified two distinct peptides of VirB6 that interact with VirB8 or with VirB10. These results suggest that VirB6 anchors VirB8 and VirB10 to the T4SS at the inner membrane. This led to the hypothesis that VirB6 acts in concert with VirB8 and VirB10 to deliver substrates to the periplasmic core of the T4SS.
In my research, I analyze the role of the interaction of VirB6 with VirB8 and VirB10 in the T4SS using a three-pronged approach to gain biochemical and structural information on these interactions and their functions. First, an Alanine-scanning mutagenesis of the 24 amino acids region of the interacting domains of VirB6 was conducted to identify functionally important residues and the involvements of these variants in the VirB6-VirB10 interaction have been assessed by BTH. To validate the residues identified and to quantify the effects of the variants on the interaction of VirB6 with VirB10, FRET experiments are currently ongoing. Second, the effects of these changes on VirB6 function are currently tested using a variety of in vivo assays with Agrobacterium tumefaciens model system (T-pilus, tumor and membrane protein complexes formation). Third, to characterize the interactions in vitro, these proteins will be overexpressed, purified and incorporated into nanodiscs allowing their structural analysis with SAXS, TEM and by diverse other biochemical methods.
Past research has provided information on the structures and functions of T4SS, on the effectors and on the mechanisms by which secreted proteins hijack the functions of cells during infection. However, many details, like the different interactions between proteins in these systems are still unknown. Understanding how these contribute to virulence is critical for the development of new antimicrobial therapies.
The rhomboid family of intramembrane serine proteases has a unique ability to catalyze proteolysis below the surface of the cell membrane, making them key players in numerous biological processes, such as parasitic host cell invasion and growth factor signalling. High-resolution X-ray crystal structures of the E. coli GlpG rhomboid indicate that the active site is sequestered away from the membrane environment by a series of transmembrane α-helices. It has been proposed that one such helix (TM5) can undergo a conformational change that exposes the active site, enabling it to function as a lateral gate for substrate entry and thus represents a key control point for proteolysis. While the effect of TM5 dynamics on proteolytic activity has been well documented, its ability to influence the structure and dynamics of rhomboid protease beyond the gate region has never been directly assessed. Here, NMR has been used for the first time to provide information on both the structure and dynamics of the rhomboid protease catalytic domain in its native solution state. In addition to facilitating substrate gating, increasing TM5 dynamics was found to induce structural changes to transmembrane helix 4 (TM4), which forms part of the active site and contains the catalytic serine along with several other functionally important residues. This perturbation resulted in a reduction in catalytic activity in some of the mutants tested, suggesting a dual role for TM5, both as a lateral gate and contributor to active site integrity. Curiously, the ability of both TM5 dynamics and rhomboid-micelle hydrophobic mismatch to disrupt activity was found to be dependent on the substrate used, suggesting an ability for rhomboids to discriminate target peptides through the preferential enhancement of active-site stability. Taken together, these results reveal two novel regulatory mechanisms for rhomboid protease in the form of TM5 dynamics and substrate-specific stabilization of the active site, with implications for the control of rhomboid activity and selectivity in-vivo.
The N-end rule pathway controls the half-life of proteins based on their N-terminal residue. Positively charged type 1 N-degrons are recognized by a negatively charged pocket on the Zn-finger named UBR-box. Here, we show that, while the UBR-box is rigid, the bound water molecules in the pocket provide the structural plasticity required to bind different positively charged amino acids. We demonstrate that the UBR-box is able to bind methylated arginine and lysine peptides with high affinity. Ultra-high resolution crystal structures of arginine, histidine and methylated arginine reveal water molecules as mediators of induced fit-like binding of N-degron peptides. Using a high-throughput assay, we measure the specificity for the second position in the N-degron peptide for binding to the UBR-box secondary pocket and reveal a strong preference for hydrophobic residues. Finally, we show that the V122L mutation present in Johansson-Blizzard syndrome patients changes the specificity for the second position due to occlusion of the secondary pocket.
The protein aggregates are toxic to the cells. A number of neurodegenerative diseases are resulted from the accumulation of mutant, misfolded protein aggregates. The key proteins that manage the misfolded proteins in the cells are the molecular chaperone Hsp70 and its co-chaperones. Hsp70 mediates protein refolding and also direct proteins into degradation, either by ubiquitin-proteasome system (UPS) or autophagy. However, disaggregation of proteins in mammalian cells is not well understood. For a long time, it was thought that animal cells lack disaggregation activity. Recently, disaggregation by a complex of human chaperones, assisted by a yeast small HSP, was observed in pure protein assays. In our study, we aim to demonstrate disaggregation activity in mammalian cells and to elucidate the mechanism of the core disaggregation machinery. We have found that Hsp70, Hsp110 and DNAJB1 are involved with disaggregation in cells. We hypothesize that DNAJB1 has an important role in mammalian disaggregation system. To elucidate the mechanism of DNAJB1, we have made certain mutants with abolished interaction with other chaperones and these are currently being examined. In addition, we will also address how disaggregation is related to the degradation via UPS and autophagy. Study of disaggregation system in cells will allow us to have a better grasp on the protective mechanism of cells against toxic aggregates.
Mitochondrial processing peptidase (MPP) is a heterodimeric metallopeptidase that cleaves the mitochondrial targeting signals from the majority of nuclear-encoded mitochondrial proteins. Mutations in both MPP and its substrates have been implicated in various neurodegenerative diseases, including Parkinson’s disease (PD) and non-progressive cerebellar ataxia (Valente et al. 2004; Jobling et al. 2015). One of these implicated substrates is PINK1 – a kinase whose mutations are known to cause early-onset autosomal PD. In healthy mitochondria, PINK1 is constitutively imported, cleaved first by MPP, and then retrotranslocated to the cytosol for proteasomal degradation. Upon mitochondrial damage and depolarization, PINK1’s import and subsequent processing are arrested. Instead, PINK1 accumulates on the outer mitochondrial membrane (OMM) where it triggers a mitophagic cascade. It was previously shown that knockdown of the catalytic MPP subunit also resulted in PINK1 OMM accumulation and mitophagy (Greene et al. 2012). In this regard, MPP acts as a key junction for PINK1 import, proteolysis, and overall mitochondrial quality control. However, both the MPP cleavage site on PINK1 and its binding conformation remain unknown. To gain insight into the proteolytic mechanisms concerning PINK1 and other disease-implicated substrates, we have begun to characterize the human MPP heterodimer. We demonstrate that the human MPP dimer can be successfully purified from a co-expression system in E. coli. We have also developed a proteomics-based method to monitor MPP activity, using a synthetic presequence from malate dehydrogenase as a positive control. Research into the PINK1 cleavage site and mechanism of cleavage are currently ongoing in our laboratory.
Nonribosomal peptide synthetases are elegant macromolecular machines that produce structurally and functionally diverse peptides, including siderophores, toxins, agriculturally-important compounds, and pharmaceutically-important compounds. The condensation domain is of particular importance to nonribosomal peptide synthesis because it catalyzes the assembly of peptides through amide bond formation between an incoming monomer and the growing peptide chain. However, even though it plays a central role in peptide synthesis, the reaction mechanism and specificity determinants of the condensation domain remain unclear. Here, we have developed chemical probes that covalently bind to an engineered cysteine residue located near the active site of the first condensation domain of the calcium-dependent antibiotic synthetase (CDA-C1), mimicking native acceptor substrate delivery to the site by carrier domains. Using mass spectrometry, we verified that the covalently-bound acceptor substrate analogues were competent for condensation only when the active site histidine is present, making the system faithful to assays previously published. We determined the crystal structure of the condensation domain in complex with two reaction-competent chemical probes, which suggested that the principal role of the active site histidine is to position the alpha amino group of the acceptor substrate for nucleophilic attack. In addition, the crystal structures lead to the identification of a mutation that altered the acceptor substrate specificity of CDA-C1. Further development of these chemical probes will lead to future studies interrogating other stages of the condensation domain reaction cycle, and can be developed for use in other systems with low binding affinity proteins.
Correlation between conformational dynamics and enzyme function has been well established for discrete enzyme systems, however, approaches for characterizing dynamical properties across diverse sequence homologs within a family and their correlation with enzyme activity remain challenging. Members of the pancreatic-type ribonuclease (RNase) superfamily share similarities in structure and fold, but display large variations in conformational dynamics and catalytic efficiencies, making them ideal model systems for probing the relationship between conformational motions and function. As a step towards determining the relationship between dynamics, catalytic mechanism and catalytic efficiency of various members of this broad vertebrate family, in this study, we have performed a systematic characterization of the intrinsic dynamics of over twenty RNases with experimentally solved structures over a wide range of time-scales by integrating molecular dynamics simulations and NMR 15N Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments. Our results show distinct patterns of dynamical variations between the canonical RNases clustered based on taxonomic groups. We show that conformational motions in the micro- to millisecond timescale of RNases sharing a common fold are significantly different from each other. Interestingly, sequences sharing similar conformational exchange in the catalytic timescale also share similar biological functions, suggesting that biological function, among other factors, may potentially impact dynamical properties affecting function. Further experiments are required to characterize this correlation between conserved dynamical characteristics and biological function.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a tightly regulated anion channel which mediates cAMP stimulated salt and fluid secretion and enables efficient mucociliary clearance in the airways. Most CF is caused by the deletion of phenylalanine (F) at position 508 which disrupts the folding, trafficking, gating and surface stability of the mutant. CFTR interactions with other proteins have been studied extensively and it is thought to form protein complexes that localize and stabilize it at the cell surface. Membrane lipids may also play a role in these processes but have received much less attention. We recently showed that wt-CFTR forms sub-resolution clusters that are homogeneously distributed on the plasma membrane of primary human airway epithelial cells. Our studies of CFTR protein dynamics revealed two dynamically distinct populations. One population exhibited small spatial scale transport dynamics and confinement that was dependent on membrane cholesterol suggestive of its localization in lipid rafts. A second population of CFTR molecules had larger spatial scale dynamics indicating CFTR lateral mobility outside the rafts. To better understand the importance of CFTR clustering for its membrane expression and stability and its possible role in CF, we have now compared the aggregation state and lateral mobility of wt-CFTR with F508del-CFTR rescued by treating cells with the small molecule folding corrector VX-809 or in combination with the potentiator VX-770. The distribution of wt- and F508del-CFTR was studied in live primary human bronchial epithelial cells using spatial image correlation spectroscopy (ICS). CFTR dynamics on the other hand were studied using a modified version of the k-space image correlation spectroscopy (kICS) analysis. Both analyses rely on the collection of high quality confocal time-series of GFP-tagged wt- or F508del-CFTR, expressed in the plasma membrane of live cells. To quantify the fraction of wt-CFTR and F508del-CFTR within cholesterol-rich membrane microdomains, and their mobility, transport and partitioning dynamics of these populations were compared under control conditions and following chronic treatment with the corrector VX-809 or in combination with the potentiator VX-770. ICS analysis showed that the aggregation state of wt-CFTR is 5-fold higher than F508del-CFTR despite treatments with the corrector or in combination with the potentiator. kICS analysis revealed that F508del-CFTR partitions into lipid rafts under control conditions, but the confinement is significantly weaker and a smaller fraction of the population displays confinement when compared with wt-CFTR. VX-809 correction does not restore the normal confined fraction or confined dynamics of F508del-CFTR, neither does the additional potentiation with VX-770, confirming that these treatments do not correct F508del-CFTR membrane behavior. The results show a clear dependence of CFTR distribution and dynamics on cholesterol and suggest that the reduction in F508del-CFTR confinement could be due to an alteration in its interaction with cholesterol which is not restored by CFTR modulators.
Several attempts to improve the resolution of crystals from a 150 kDa protein have proved only partially successful. These have included routine pre-crystallization optimizations such as modifying precipitant and protein concentration, screening for additives and detergents, and searching for completely different crystallization conditions. One newly found crystallization condition was optimized to the point where single crystals with a size ranging from 200 to 600 micrometers were obtained. Nevertheless, the crystals did not diffract. In cases such as this one, it has been shown that post-crystallization treatments can dramatically change the internal order of crystals leading to otherwise impossible-to-obtain datasets (Heras and Martin, 2005). Here we report a work flow that radically improved resolution by coupling dehydration and cryoprotection in a single step (based on Madhusudan et al. (1993) and Dobrianov et al. (2001)). This allowed the collection of several datasets with complete data up to 4.5 A. We suggest that this strategy could be applied for other cases of recalcitrant protein crystals.
Nonribosomal peptide synthetases (NRPSs) are large multi-domain enzymes that use amino acids and other substrates to generate biologically active peptides. NRPSs are organized as assembly lines of modules, each one responsible for the incorporation of one residue to a growing peptide. Some of these enzymes contain specialized tailoring domains, such as the ketoreductase domain (Kr Domain) found in the synthetases that produce the anti-cancer compound valinomycin and the toxin cereulide. Tailoring domains considerably widen the chemical space accessible to NRPS products via co-synthetic chemical modifications. In spite of the biochemical and biophysical data on modules containing Kr domains, it is still unknown which are the conformational changes and overall architecture required for their function. To tackle these questions, we propose a structural characterization of one of these modules. To date, we have grown crystals that diffract up to 4.0 Å. We aim to further improve this resolution by using chemical biology techniques to stall the sample in a single conformation, including vinylsulfonamide adenylate mechanism-based inhibitors, amino pantetheinyl mechanism-based inhibitors, non-hydrolyzable ATP analogs, NADPH analogs inhibitors and transition state analog inhibitors. The structural and functional characterisation of Kr-containing NRPSs contributes to the fundamental understanding of NRPSs necessary to potentiate their manipulation in future bioengineering experiments.
Galectins are small soluble lectins that bind beta-galactosides via their carbohydrate recognition domain (CRD). Their ability to dimerize is critical for the crosslinking of glycoprotein receptors and subsequent cellular signaling. This is particularly important in for their immunomodulatory role via the induction of T-cell apoptosis. Because galectins play a central role in many pathologies, including cancer, they represent valuable therapeutic targets for drugs or as biomarkers. At present, most inhibitors have been directed towards the CRD, a challenging task in terms of specificity given the high structural homology of the CRD among galectins. However, while the CRD β-galactoside binding site remains highly similar throughout galectin homologues, they display little sequence identity. This observation raises the possibility of targeting various galectins through the use of unusual ligands that would specifically bind galectins through in a carbohyrate-independent manner. Here, we report a non-carbohydrate ligands,. a porphyrin compounds functionalized with zinc ioins, that specifically binds human galectin-7 (hGal-7). The medical appeal and relevance of porphyrins as photosensitizers in cancer treatment has been amply demonstrated, especially in tumor imaging and photodynamic therapy, potentially providing a means to use these binding affinities and intrinsic physicochemical imaging properties as hGal-7 markers in cancerous tissue progression. We used a combination of fluorescence and NMR titration experiments to specifically define and map the low-micromolar, non-carbohydrate binding sites of porphyrins on the surface of hGal-7. We found that these porphyrin ligands offer limited selectivity with respect to charge and metal, and that their binding affinity to hGal-7(~20µM) is stronger than the previously characterized interactions mediated by glycan-binding residues in the CRD pocket, suggesting that the distinctively high porphyrin affinity to hGal-7 may be biologically significant. To our knowledge, these results highlight the first distinct and structurally characterized non-carbohydrate binding site on the surface of hGal-7, in addition to portraying the only structural characterization of porphyrin binding to human galectins to date.
Ribonucleases (RNases) are very ancient proteins present in all organisms studied so far. In addition to their conserved ribonucleolytic activities, they have been associated to a variety of biological functions such as anti-bactericidal, cytotoxic, angiogenic, immunosuppressive, anti-tumoral and/or anti-viral activities. In humans, RNase A superfamily members comprise eight rapidly evolving homologous enzymes with varying degrees of structural similarity and enzymatic activities. In this study, we are presenting the first crystal structure of the human RNase 6 in presence of a ligand, 5´-AMP. Its crystal structure is compared to RNase A and RNase 2, a closely related homologue of RNase 6. We have identified the phosphate binding residues and ionic binding architecture within the catalytic pocket of RNase 6. A new phosphate-binding site located within loop 4 and involving His67 and its effect on ligand binding were also uncovered.
We also present in this study the high-resolution crystal structure of RNase 4, along with its crystal structure in complex with the ligand 5´-AMP. We compare these structures with the structures of the RNase 4-dUP complex and other RNases and relate the differences to the selectivity of RNase 4 for uridine.
In addition to their crystal structures, biophysical properties of RNases 4 and 6 were analyzed via NMR experiments and isothermal titration calorimetry (ITC) with two different ligands: 3´-UMP and 5´-AMP. The results were compared to those of RNases A and 2.
Resolving the crystal structure of human RNases provides valuable insights into understanding their biological functions in humans, which may find applications in various fields such as drug design.
Nonribosomal peptide synthetases (NRPSs) are mutlimodular megaenzymes that produce natural peptides with antibiotic, anti-cancer, anti-viral, and many other properties. The core domains of NRPSs are the adenylation (A), peptidyl carrier protein (PCP), and condensation (C) domains. One set of these domains (module) catalyzes the activation, transport, and linking of a single monomer substrate, and each subsequent module systematically adds on a new monomer, to synthesize the final product. Linear gramicidin is a pentadecapeptide synthesized by an NRPS composed of four large polypeptides. The first polypeptide, LgrA, consists of seven domains; F-A-PCP-C-A-PCP-E, where F and E are formylation and epimerization domains, respectively. This study focuses on the purification and crystallization of two dimodular constructs of linear gramicidin; F-A-PCP-C-A and F-A-PCP-C-A-PCP to determine the first structure of a dimodular NRPS using X-ray crystallography, and provide insight into their catalytic cycle.
Besides insulin resistance, type II diabetes mellitus is characterized by a loss of β-cell mass as a result of the deposition of misfolded Islet Amyloid Polypeptide (IAPP) in the pancreatic islets. Recent studies have revealed that the amyloidogenic process mediates cell death whereas mature amyloid fibrils are poorly cytotoxic. Therefore, therapeutic strategies to prevent b-cell degeneration associated with amyloid deposition are not well developed. Besides, a few studies have reported that dendrimers can modulate amyloid formation from the amyloid-β peptide derivatives and inhibit its neurotoxicity. In this context, we evaluated the effects of multi-charged dendrimers on IAPP self-assembly and cytotoxicity. Densely functionalized dendritic scaffolds harboring 4 to 16 hydroxyl, amine, carboxylate or sulfonate units were designed. Whereas polyhydroxyl and polyamine dendrimers did not affect the kinetics of amyloid assembly, carboxylated dendrimers accelerated IAPP fibrilization proportionally to surface group density. Interestingly, as revealed by thioflavin T fluorescence, circular dichroism spectroscopy and atomic force microscopy, the G0 sulfonated dendrimers inhibited amyloid formation whereas G1 sulfonated dendritic scaffolds dramatically hastened IAPP self-assembly into long amyloid fibrils. G1 dendrimers decorated with carboxylate or sulfonate groups attenuated IAPP induced toxicity on pancreatic b-cells and the protective effect was proportional to the number of functional units attached. This study indicates that IAPP amyloidogenesis and cytotoxicity can be controlled with multi-charged dendrimers and offer novel mechanistic insights for the design of molecular identities to manipulate peptide self-assembly.
A wide range of proteins and polypeptides are known to self-assemble into amyloid fibrils that are related to degenerative diseases, including type II diabetes (T2D) and Alzheimer’s disease (AD). Given their involvement in numerous pathological states, several studies have focused on the process of fibrillization in order to understand and prevent amyloid formation. These studies have revealed that polypeptides can self-associate into amyloid fibrils by a variety of mechanisms that rely not only on the sequence but also on the conditions of assembly. In our study, we focused on the self-assembly of the islet amyloid polypeptide (IAPP), whose deposition is associated with T2D. The mechanism of IAPP fibrillization is often ascribed as a polymerization nucleation where monomeric peptides assemble into larger oligomeric aggregates until the formation of the so-called nucleus leading to rapid fibril growth. Until now the fibrillization kinetics of IAPP has been extensively studied with thioflavin-T (ThT), the most widespread technique used to monitor amyloid formation, although it is insensitive for oligomer formation. For this reason, we aim at developing a new detection method in order obtain new insight into the early steps of IAPP self-association. This method relies on a fluorescent dye, called fluorescein arsenical hairpin (FlAsH), which fluoresces when it binds to a tetra-cysteine motif. We first chemically synthesized an IAPP derivative where two non-native Cys were incorporated at its N-terminus (CC-IAPP). Thanks to this strategy, we were able to recreate the FlAsH tetra-Cys recognition motif upon association of the monomer units. Indeed, monomers are stacked along the fibril axis with the same orientation leading the Nter of each unit to get close. We initially evaluated if the addition of two non-native Cys at the Nter does not affect the conformational transition associated with the formation of fibrils by circular dichroism spectroscopy. Studies by atomic force microscopy allowed us to obtain information about the morphology of the fibrils and therefore to evaluate whether the addition of these two non-native Cys and / or the addition of the FlAsH molecule affect the supramolecular structure of the fibrils. Then, by evaluating the fluorescence spectra of FlAsH when bound to IAPP with or without the non-native Cys, we observed that the binding for the tetra-Cys motif was specific. Finally, we were able to monitor the fibrillization of CC-IAPP with a novel FlAsH based assay. Interestingly, we observed kinetics of assembly with a conformational FlAsH that are similar to the ones obtain by a ThT assay. These results suggest that there are no stable oligomers formed during the lag phase. Our study strengthens the hypothesis of a nucleated polymerization mechanism for the self-assembly of IAPP.
Brain functions depend on the ability of neuronal cells to efficiently and accurately communicate for various processes such as memory and learning. Knowing that the abundance of the AMPA-type glutamate receptor (AMPAR) at synapses determines neurotransmission efficacy, there is consequently a critical need for research aimed at elucidating mechanisms regulating AMPARs at hippocampal synapses. Accordingly, recent evidences show that AMPAR are modified by ubiquitin (UB), a functionally relevant posttranslational modification that acts to regulate receptor trafficking, endocytosis and lysosomal degradation. In this manner, we recently reported that RNF167, the UB ligase enzyme who brings specificity to the ubiquitination process by recognizing the substrate protein, controls AMPAR membrane expression. However, our understanding the molecular characteristics and intrinsic properties of RNF167 are exceptionally limited. The main objective of this study is to identify and characterize UB-conjugating enzymes (UBE2) functionally interacting with RNF167 by implementing biochemical and biophysical assays. To reach this goal, we first optimized the expression and purification procedure. Then, we developed an in-vitro ubiquitination assay (in-UBn) to determine which E2 functionally pair with purified RNF167. Our result shows that our purified RNF167 is functionally active and that nine of the twenty-nine UBE2 sare functional pairing with RNF167. Also, by using GST-pulldown assay, we demonstrate the physical interaction between RNF167 and UBE2D1. Finally, the RNF167/UBE2 interactions are validated by Surface Plasmon Resonance (SPR) to gain key insights regarding the kinetics of interaction. Until now, our SPR experiment shows that the dissociation rate of UBE2D1/RNF167 is instantaneous and this is consistent with dissociation rate observed in the literature between UBE2s interacting with other UB ligases. Taken together, our results reveal functional interactions between RNF167 and UBE2s. Our research efforts provide significant insight into the fundamental mechanisms controlling AMPAR synaptic expression and glutamatergic neurotransmission at hippocampal synapses.
Lignolytic enzymes are a group of biocatalysts with potential applications in delignification and bioremediation. The enzymatic delignification process is a green chemistry alternative for the pretreatment of lignocellulosic material, providing a means for the efficient removal of lignin and the synthesis of biologically active compounds such as monolignols. Laccases (EC 188.8.131.52) are the most studied enzymes in delignification processes and Basidiomycete fungi are the main source. The aim of this study was to identify the gene sequence and protein structure of a protein band identified with a laccase activity from an enzymatic extract obtained by solid-state fermentation (SSF) of Dictyopanus pusillus. The enzymatic extract from D. pusillus was concentrated and purified by fast protein liquid chromatography (FPLC) using an anion exchanger. SDS-PAGE and native PAGE were used to determine the molecular weight and activity of the bands obtained. Tryptic digestion and Micro-HPLC-MS analyses were performed to identify peptides belonging to the protein band identified with laccase activity. From those peptides, degenerate primers were designed to amplify the coding gene sequence from D. pusillus. Two DNA sequences with high identities to lignolityc fungi were obtained and were used to desing of primers to elucidate the middle part of putative laccase gene. Upstream and downstream extrems were identified by TAIL-PCR and ORF finder tool was used to find the putative laccase gene domains. These results confirm the expression of a new laccase in D. pusillus, further allowing overproduction of this enzyme in a heterologous system.
Cilia (or flagella) are organelles attached to the surface of most human cells, and are responsible for cell motility and sensory function. The cilium is composed of approximately 600 proteins, and these proteins are transported to the correct assembly sites in the cilium by a process called intraflagellar transport (IFT). IFT is a bidirectional process involving IFT complex, cargos and motor proteins that move continuously along the axonemal microtubule doublets beneath the flagellar membrane. Individual IFT complexes or particles, composed of at least 20 individual proteins, are organized in a train consisting of consecutively connected particles. IFT is a highly dynamic process, with the trains moving at the speed of 2-4 μm per second.
Due to their logistical role, mutations in IFT proteins can cause ciliopathies (diseased related to the cilium). In human, common IFT-related ciliopathies are Bardet-Biedl syndrome, Meckel-Gruber syndrome, polycystic kidney disease, blindness and various developmental birth defects. These findings put IFT proteins at the forefront of research, however, detailed molecular mechanisms of IFT in cilia are poorly understood due to lack of structures of complete IFT complex and its high-ordered assembly IFT train.
My research project aims to understand the mechanism of IFT in cilia formation and maintenance, and contributes to the basic understanding of ciliopathies. The specific aims are:
1. Elucidation of the in vivo structure of IFT trains using correlative light and cryo-electron microscopy. I will obtain, for the first time the in situ structure of the anterograde and retrograde IFT trains. Ultimately, this structure will lead to an understanding of the molecular mechanisms of IFT processes, such as how the IFT complex interacts with cargo and how it remodels after dropping off its cargo.
2. To obtain high-resolution structures of the IFT complexes from both anterograde and retrograde trains by single particle cryo-electron microscopy. By combining information from both aims, I will produce the first high-resolution model of IFT complexes.
DNA damage repair is a central pillar of genomic integrity. All DNA repair pathways include DNA ligase carrying out a 3-step reaction. After self-adenylylation (step 1), ligase binds nicked DNA and transfers the AMP co-factor to the 3’OH (step 2). In step 3, AMP is released when the activated 3’OH attacks the 5’PO4, thereby creating a phosphodiester bond. Damaged DNA is not only produced from exogenous sources but also arises from normal cellular activities like genome replication. During replication, the lagging strand is synthesized as Okazaki fragments that require DNA ligase to function efficiently. The trimeric proliferating cell nuclear antigen (PCNA) orchestrates DNA ligase with nucleases and polymerases through their PCNA interacting peptide (PIP) motif. Despite a crystal structure of human ligase I in complex with nicked DNA (post-step 2), questions remain on ligase domain rearrangements throughout the ligation reaction and how this is influenced by PCNA.
The hyperthermophilic archaeon Sulfolobus solfataricus provides a convenient model system of the DNA repair machinery found in all three branches of life. Using a purification strategy that exploits the thermostability of the archaeal proteins, we purified S. solfataricus PCNA and ligase. DNA ligase was obtained in the post-step 1 state (self-adenylylated) and was able to ligate nicked DNA containing 5’PO4 and 3’OH at higher temperatures (60°C). PCNA was shown to interact with DNA ligase by co-elution from gel filtration. Ligation efficiency was stimulated by the addition of PCNA. To complete our understanding of DNA ligase structural rearrangements, we have initiated co-crystallization of ligase with nicked DNA. In parallel, the ligase-PCNA-DNA complex is being investigated by electron microscopy. By combining these two approaches, we will combine the atomic precision of X-ray crystallography with the ensemble domain dynamics of cryo-electron microscopy to obtain a comprehensive understanding of the final step of DNA damage repair.
Recent studies have revealed extensive remodeling of the protein-protein interaction (PPI) network (also known as interactome) mediated by alternative splicing, yet large-scale determination of isoform-specific PPIs remains a challenging task. Here, we present a domain-based method to computationally predict the isoform interactome from the reference interactome. Starting with experimentally determined interactions between reference proteins, we map known structural domains onto proteins and known domain-domain interactions onto PPIs, and construct the domain-resolved reference interactome. Next, we construct the isoform interactome by predicting that an alternative isoform loses an interaction if it loses the domain mediating the interaction. Finally, we apply the method to construct the human isoform interactome from the human reference interactome. We find that compared to proteins with identical interaction profiles with alternative isoforms of the same gene, proteins with different interaction profiles tend to be more divergent in terms of function, disease phenotype, and tissue expression, and tend to be more tissue specific, consistent with experimental studies. Our domain-based method for predicting the isoform interactome complements experimental efforts, and demonstrates that integrating structural domain information with PPI networks provides insights into the functional impact of alternative splicing.
Will Die Slowly (WDS) gene family was initially identified in this laboratory and was suggested to have anti-cell death and anti-senescence functions by phenotypic analysis of single or double mutants. wds2 and wds1/wds2 plants started to show signs of leaf senescence sooner than wds1 and wild type specimen did. The lack of remarkable differences between the phenotypes of WT, wds1, wds2 and wds1/wds2 plants indicates that WDS1 and WDS2 do not mediate cell death pathways that are responsible for shaping the architecture of the Arabidopsis plant. WDS1 is predicted to be located in the cytoplasm, to contain three possible ERK docking sites, to interact with a protein that appears to function in the repression of genes that may induce apoptosis via a mitochondria mediated pathway and also a protein that seems to be part of an ubiquitin ligase complex. Both WDS1 and WDS2 were shown to interact with ERK-type MAPKs.
Specific polypeptide sequences that auto-assemble into amyloid fibrils that are characterized by a cross-b-sheet quaternary structure appears attractive for nanomaterials development. These fibrils showed mechanical strength comparable to steel and silk while demonstrating structural plasticity. These properties contribute to the potential of amyloid nanofibrils as promising materials for biomedical and biotechnological applications. In this study, developed a novel biochemical strategy to functionalize amyloid nanofibrils, post-assembly. We investigate an enzymatic approach using a sortase to functionalize amyloid nanofibrils. Staphylococcus aureus sortase (SrtA) is a bacterial transpeptidase that covalently anchors surface proteins to the bacterial cell wall. This is accomplished by cleaving between threonine and glycine at an LPXTG recognition motif to generate an acyl-enzyme intermediate that reacts with a terminal amino group of pentaglycine on the cell wall. The amyloid scaffold was prepared by using peptide sequences derived from known amyloidogenic polypeptides, such as the islet amyloid polypeptide. Peptides were synthesized by solid phase synthesis and the amyloid formation was monitored/screened using thioflavin T (ThT) fluorescence. In order to follow the fibrillization conformational transitions, circular dichroism (CD) was employed whereas transmission electron microscopy (TEM) was used for morphological investigation. The SrtA is produced in-house using a recombinant system and the ligation approach was validated using synthetic peptides, high performance liquid chromatography (HPLC) and liquid chromatography–mass spectrometry (LC/MS). To verify our enzymatic-mediated ligation of pre-assembled amyloid structure, green fluorescence protein (GFP) was employed as a model and fused to the surface of fibrils. With this approach, amyloid scaffolds can similarly be decorated using multiple proteins (e.g. promoting cell growing, differentiation factors, and antimicrobial peptides). Overall, the ultimate goal of this project is to develop biocompatible scaffolds for applications in drug delivery and tissue repair/engineering.
G-quadruplexes (GQs) are 4-stranded DNA structures formed by tracts of stacked, Hoogsteen-hydrogen bonded guanosines. GQs are found in gene promoters and telomeres where they regulate gene transcription and telomere elongation. Though GQ structures are well-characterized, many aspects of their conformational dynamics are poorly understood. For example, when there are surplus guanosines in some of the tracts, they can slide with respect to one another, a process we term G-register (GR) exchange. These motions could in principle entropically stabilize the folded state, crucially beneﬁtting GQs as their stabilities are closely tied to biological function. We have developed a method for characterizing GR exchange where each isomer in the wild-type conformational ensemble is trapped by mutation and thermal denaturation data for the set of trapped mutants and wild-type are analyzed simultaneously. This yields GR isomer populations as a function of temperature, quantiﬁes conformational entropy and sheds light on correlated sliding motions of the G-tracts. We measured entropic stabilizations from GR exchange up to 14.3±1.6 J mol−1 K−1, with melting temperature increases up to 7.3 ± 1.6◦C. Furthermore, bioinformatic analysis suggests a majority of putative human GQ sequences are capable of GR exchange, pointing to the generality of this phenomenon.
Glycosaminoglycans (GAGs) are a class of linear, non-branched polysaccharides which can be found in large numbers along cell membranes as well as in the extracellular matrix. Variants of these GAGs containing large numbers of sulfate moieties have been found in extracellular deposits associated with many different amyloid-linked diseases. Furthermore, these polysaccharides have been shown to significantly influence the formation of amyloid fibril assembly of a wide variety of polypeptides and proteins responsible for protein misfolding diseases. Perhaps even more remarkable yet is that GAGs are capable of inducing an amyloid structure in an otherwise non-amyloidogenic peptide. While it has been demonstrated in the past that many non-amyloidogenic proteins can form amyloid structure under harsh conditions (acidic pH and near-boiling temperatures), GAGs are able to induce a fibril structure in physiologically-relevant conditions. The mechanism by which GAGs exert this enhanced self-assembly effect is still unclear, and actively debated. We used a model system consisting of a low-molecular weight heparin (LMWH) and the highly soluble, non-amyloidogenic PACAP27. PACAP27 is a non-pathogenic neurohormone consisting of a 27-residue sequence, stable in solution, and does not readily form amyloid-like fibrils on its own. Upon addition of LMWH, PACAP27 rapidly adopts a helical structure as shown by circular dichroism. After a certain lag-time, these helical structures are replaced with β-sheets. The result is a self-assembly of the peptide into amyloid fibrils, as evidenced by transmission electron microscopy, atomic force microscopy, and fluorescence using the amyloid-sensitive fluorophore Thioflavin-T. The importance of this transient helical conformation to the amyloidogenic pathway was evaluated using two synthetic variants with reducing helical folding propensity. These conformationally restricted peptides retained their ability to form amyloid, despite increased resistance to form α-helices. Lag-phases during aggregation kinetic experiments of the conformationally restricted peptides were greatly reduced as well, strongly suggesting that the initial GAG-induced α-helical structures are not a requirement for the natively disordered peptide to assemble into cross-β-sheet fibrils.
Cystic fibrosis (CF) is the most common lethal genetic disease in the Caucasian population with an incidence of approximately 1 in 2500. More than 2000 mutations have been identified in the CF transmembrane conductance regulator (CFTR), affecting translation, folding/processing and/or channel function. The most common CF mutation is the deletion of the phenylalanine residue at position 508 (F508del), which is present in at least one allele of ~80-90% of CF patients. In addition, ~30% of all CF patients have one CFTR allele containing a rare mutation (hereafter referred to as CFTR2 mutations), while ~10% of all patients are heterozygous for two rare CF-causing mutations. The second most common of these is G551D-CFTR, with an incidence of ~4%. G551D represents one mutation with an exclusive gating defect, showing an expression level comparable to WT-CFTR, but negligible channel function.
A recently approved drug, ivacaftor (VX770), developed by Vertex Pharmaceuticals and marketed as Kalydeco, has proven successful in ameliorating the respiratory phenotype of CF patients carrying at least one copy of G551D-CFTR, however F508del patients show little benefit, an effect which could be explained by a biochemical and functional downregulation of this particular mutated channel under extended ivacaftor treatment. Moreover, it has been shown that for some CFTR2 mutations a similar side-effect as F508del is observed upon long term exposure to pharmacologically relevant levels of ivacaftor.
We started by addressing the uncertainty in the scientific literature with respect to the EC50 of VX-770, as well as the concentration required for G551D patients to exhibit clinical benefit. Van Goor et al. had previously reported an EC50 for G551D expressed in FRT cells as 100+/-47nM, while in G551D/F508del HBE cells, he reports 236+/-200nM. We questioned whether the quality of these data could be improved. Titration of VX770 in CR-HNE yielded an EC50 of ~0.63 mM (+/-0.07), two-fold higher than previously published. This EC50 justified the examination of the effect of VX770 in the concentration range of 1 nM to 1 mM during chronic treatment, a concentration range which is most certainly attained in the plasma and lungs of CF patients undergoing treatment.
We next tested a panel of CFTR2 mutations and found that a substantial number show biochemical and functional downregulation under extended exposure to VX770. In order to check if this is a generalized effect, we tested non-downregulating potentiators which had previously been published to be safe for use with F508del-CFTR, looking at both biochemical and functional expression. We observed that non-downregulating potentiators of F508del have CFTR2 mutation-specific destabilizing effects and show no discernible pattern in their propensity to downregulate CFTR2 mutants.
On the basis of these observations, we argue that each CFTR2 gating mutation must be screened against a panel of potentiators with varying mechanisms of action in order to assess the mutation specific downregulation susceptibility and to select the most suitable potentiator for each mutation.
Cilia and flagella are slender, microscopic, hair-like structures that extend from the surface of eukaryotic cell. They are not only important in lower eukaryotes for motility of the cell but also essential in higher eukaryotes for such as sperm motility, generation of fluid flow in trachea and brain ventricles, and determination of left/right asymmetry. Therefore, defects in cilia cause numerous diseases and developmental disorders such as primary cilia dyskinesia and polycystic kidney disease. Cilia and flagella share a canonical architecture composed of nine outer doublet microtubules (doublets), either surrounding two central microtubule singlets in the case of motile cilia (9+2), or without a central singlet for non-motile cilia (9+0). The doublet is very stable and robust structure and serves as a scaffold for axonemal proteins like dyneins and radial spokes, which are docked to doublets periodically. Using cryo-electron tomography, intact 9+2 structure was resolved at 20-30Å resolution. Inside doublets, periodic structures called microtubule inner proteins (MIPs) were characterized (Nicastro et al., 2006; Nicastro et al., 2011; Pigino et al., 2012). However, the biological role of MIPs has not been understood mainly because of no existing high-resolution structure of MIPs interacting with the microtubule lattice of the doublet.
Here, we aimed to get high resolution structure of doublet tubulin lattice and MIPs to gain insight of function of the MIPs. For this aim, we purified split doublet from ciliate Tetrahymena and performed structural analysis by single particle analysis cryo-electron microscopy. Compared with previous studies using intact axonemal structure (> 200 nm), split doublet (~50 nm) allowed us to get thinner ice embedding leading higher signal to noise ratio. Further, by using latest FEI cryo-electron microscope Titan Krios equipped with direct electron detector, we were able to get higher quality of EM images. Combined with better image processing, we improved the resolution of doublet significantly from previous studies. Based on our high resolution structure of doublet, molecular interactions with MIPs and tubulin lattice will be discussed.
Ankyrin B (AnkB) is a bacterial protein that plays an essential role in the intracellular proliferation of Legionella pneumophila, the causative agent of Legionnaires’ disease. It collects proteins to target for degradation into free amino acids, generating a source of carbon and energy, and preventing a starvation response. AnkB contains two eukaryotic-like domains, the combination of which have never been found in the same eukaryotic protein. The N-terminal F-box domain allows mimicry of host F-box proteins for interaction with the host’s ubiquitination pathway via Skp1 of the SCF (Skp1-Cullin-F-box) E3 ubiquitin ligase complex. The C-terminal ankyrin repeats allow AnkB to selectively bind targets for degradation. Here, we report the crystal structure of full length AnkB in complex with host Skp1. We found that AnkB contains an enlarged the substrate-binding site with three ankyrin repeats rather than the two expected based on its sequence. Structural analyses and mutational studies have identified key residues responsible for decorating the Legionella-containing vacuole with ubiquitin and for the replication of the pathogen during infection. Our study provides the first structural insights into the structural mimicry that allows the bacterial virulence factor, AnkB, to associate with the host ubiquitination complex and select substrates.
Introduction: Sarcomeres are large assembles of contractile and structural proteins. The inter-sarcomere dynamics within a myofibril is not well understood, which limits our understanding on how muscle cells produce force. In this study, we addressed the biophysical processes underlying the interaction among sarcomeres in series using isolated skeletal muscle myofibrils. We designed an experimental system that uses microfluidic perfusion to control individual sarcomeres within a myofibril, allowing for the first time direct investigation of inter-sarcomere dynamics. Methods: Isolated rabbit psoas myofibrils were attached between pre-calibrated micro-needles and tested at three initial average sarcomere lengths (SLi): (a) between 2.4-2.65μm, (b) between 2.65-2.9μm, and (c) above 2.9μm. Microperfusion was used to locally control individual sarcomeres or groups of sarcomeres within a myofibril while the remaining sarcomeres were maintained deactivated. Results: Microperfusion of a solution containing calcium produced shortening of one target sarcomere within a myofibril. The force produced by this sarcomere displaced all adjacent sarcomeres within the myofibril towards the activation point. The spread of activation was reduced from sarcomere to sarcomere along the myofibril extension. The magnitude of this effect was a function of the initial SLi [(a) 1.01 ±0.03μm, (b) 1.17 ±0.03μm, (c) 1.28 ±0.037μm]. The displacement of adjacent sarcomeres due to local activation of one sarcomere was further increased when the myofibril was tested in conditions of high stiffness (1.079 ±0.034μm) in comparison with normal conditions (0.918 ±0.037μm). Sarcomeres produced similar active forces (21.07 ±0.5568nN/μm2) within a myofibril at a fixed SLi. Conclusion: Force produced by the local contraction of one sarcomere spreads within a myofibril. The magnitude of this effect is dependent on SLi, suggesting a length dependent effect for the inter-sarcomere dynamics.
A specialized basal lamina (sBL) mediates the adhesion of dental associated epithelial cells.
The sBL is compositionally distinct from typical basal laminae because it does not contain
collagens type IV and VII, is enriched in laminin-332, and contains three novel protein
constituents known as amelotin (AMTN), odontogenic ameloblast-associated (ODAM), and
secretory calcium-binding phosphoprotein proline-glutamine rich 1 (SCPPPQ1), which have
no known homology. The objective of this study was to better understand the nature of the
sBL and the interactions of its constituents by combining molecular and structural
approaches. Fluorescence and colloidal gold immunolabeling showed that AMTN, ODAM
and SCPPPQ1 co-localize in the sBL. Quantitative analysis of the relative position of gold
particles on the sBL demonstrates that the distribution of ODAM is skewed towards the cell
while that of AMTN and SCPPPQ1 is more towards the tooth surface. Bacterial two hybrid
analysis and co-immunoprecipitation, gel filtration of purified proteins and transmission
electron and atomic force microscopies highlight the propensity of AMTN, ODAM, and
SCPPPQ1 to interact with and among themselves, as well as to form supramolecular
aggregates. These data suggest that AMTN, ODAM and SCPPPQ1 participate in structuring
an extracellular matrix with the distinctive capacity to attach to epithelial cells to mineralized
surfaces. This unique feature is particularly relevant for the adhesion of gingival epithelial
cells to the tooth surface, which form a protective seal that is the first line of defense against
Heme proteins are important for many biological process such as cell respiration, transcription, and antioxidant defense. Heme is synthesized in an eight-step process that finalizes with the insertion of ferrous ion (Fe2+) into the porphyrin ring. This reaction is catalyzed by ferrochelatase (FECH), but no heme acceptor that interacts directly or indirectly with FECH has yet been described. Interestingly, cytochrome c peroxidase (Ccp1) is a heme protein that is synthesized in fermenting yeast before heme accumulation. Thus, apoCcp1 may be a heme acceptor of FECH, but these proteins appear to be localized on different sides of the inner mitochondrial membrane. Pet9, a highly abundant ATP/ADP transporter in the inner membrane has additionally been characterized as a heme transporter and was found to be both a Ccp1 and FECH interactor in pulldown studies. My aim is to characterize in detail the interaction between Ccp1, Pet9 and FECH and, as a starting point, I have optimized the expression of GST-apoCcp1 and of the GST control in BL21(DE3) E. coli cells. I added purified recombinant GST-apoCcp1 as bait or GST as a control to lysates of the mitochondrial-enriched (P10) fraction from 1-day BY4741 yeast cells. To solubilize the inner membrane, 20 mM N-octylglucoside was also added to the P10 lysates. Pet9 and mitochondrial matrix proteins such as aconitase and succinate dehydrogenase were detected in the GST-apoCcp1 pulldowns, which confirms successful solubilization of the inner mitochondrial membrane. However, extensive cleavage of GST-apoCcp1 in the P10 lysates was detected by SDS-PAGE, which reduces the sensitivity of the pulldown assays. Addition of freshly prepared PMSF, a serine protease inhibitor, to the P10 fractions significantly reduced cleavage of GST-apoCcp1. This and other strategies to optimize pulldown assays will be discussed.
The Ser/Thr protein kinase MARK2 also known as Par1b belongs to the highly conserved group of PAR proteins which are associated with regulation of cell polarity and partitioning through animal kingdom. The protein has been subjected to several X-ray crystallographic studies, but none of them has yielded a precise and complete description of the protein structure and dynamics especially pertaining to functional aspects. In the current study, we represented the native structures of human MARK2 by modeling and simulation for both the inactive and active conformations, based on available incomplete crystal structures in Protein Data Bank and considering the sequence of human genome as the reference sequence. We also considered the structural and dynamical features of the protein active state, in presence of Mg-ATP. To explore the interconversion motions of enzyme through the activation process, the intermediate conformations were interpolated between the inactive and active structures by using the Yale morph server. Together, the results of this study may shed light on the structural bases of some proved and alleged functions of MARK2. None of the MARK2 wild type inactive crystal structures represent the position of activation segment. Thus, the contribution of this loop to the formation of inactive state is not clear. Here, we showed how this structure occludes the active site of enzyme and assumes a relatively stable position in this conformation. We also presented a detailed description of major structural deviations in protein structure through activation process and presented a framework on how these deviations might be affected from the phosphorylation of Thr208 or existence of the UBA domain. The results also suggest the reason for the necessity of UBA domain inhibitory function based on structural similarities to other related kinases and confirmed the alleged mild auto-inhibitory role of UBA domain.
The molecular basis of idiopathic ventricular fibrillation (IVF) is poorly understood. Previously, we identified crucial role of DPP6, a Kv4.3-associating β-subunit, in Purkinje fiber Ito and gene variants causing DPP6 gain-of-function in IVF. This study investigated the molecular basis of Kv4.3-DPP6 interaction, about which little is known. A computational model of the Kv4.3-DPP6 interaction (using AutoDock Vina) predicted that the N-terminus and some charged regions of DPP6 might interact with the N-terminus and 3 positively charged residues of Kv4.3. The predictions were verified in a patch-clamp study of wildtype and engineered mutant proteins expressed in HEK cells. Wildtype Kv4.3 showed substantial current enhancement upon co-expression with DPP6 after 48 hrs, as previously reported. Deletion of 5 amino acids in the Kv4.3 N-terminus decreased the peak-current density compared to wildtype Kv4.3 but did not prevent current-enhancement by wildtype DPP6, suggesting that this region of Kv4.3 N-terminus is not essential for interaction with DPP6. Neutralization of the 3 positively charged amino acids in the Kv4.3 N-terminus by alanine mutation enhanced the DPP6-induced current-increase. On the other side, deletion of the N-terminal region of DPP6 prevented its current-enhancing effect on Kv4.3. Neutralization of either positively or negatively charged regions in DPP6 significantly attenuated its ability to increase current density when co-expressed with wildtype Kv4.3. We conclude that Kv4.3 interacts with DPP6 via docking involving the DPP6 N-terminus, and that crucial charged amino acids on both Kv4.3 and DPP6 strongly affect the interaction. However, the opposite effects of charge neutralization in the Kv4.3 and DPP6 N-termini argue against a simple electrostatic interaction. These results provide insights into the biochemical anatomy of IVF and may help to develop effective molecularly-targeted blockers for patients with IVF-inducing DPP6 gain-of-function.
The enzyme aminoglycoside phosphotransferase (APH) inactivates aminoglycoside antibiotics by phosphorylation, thereby conferring bacterial resistance. APH inhibitors could potentially re-sensitize resistant bacteria, and are therefore of clinical interest. Enzyme kinetic studies of APH suggest that the substrate ATP is required to bind first, followed by aminoglycoside, and that product phospho-aminoglycoside dissociates rapidly while ADP dissociates slowly. However the molecular basis of this mechanism is not at all obvious from X-ray crystal structures of the enzyme and its complexes. We therefore proposed using ITC to study the kinetic and mechanism of the APH inhibition of aminoglycoside. Isothermal titration calorimetry (ITC) kinetic assay study measured an apparent enthalpy for the reaction of -9.98Kcal/mol. The Km and Kcat values were determined by single injection method and continue injection method, with values of 24.4uM, and 1.99s-1 respectively, agreed with the reference values. The inhibitor, ADP, binding affinity KiADP was determined for the first time via a continue injection ITC assay as KiADP = 2.35uM. We will investigate the full inhibition kinetic and mechanism in our future study.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a well-known glycolytic enzyme, exhibits various moonlighting functions that change with its oligomeric state. The tetramer is a NAD+-dependent dehydrogenase, which binds its cofactor with negative cooperativity, and separations of 3.4-3.7 Å between the catalytic Cys152 and its H-bond acceptor, His179, reveals subunit asymmetry in crystal structures of holo-GAPDH (NAD+-loaded). Loss of NAD+ increases the total protein volume, the number of internal waters, and the tendency to dissociate into monomers and aggregate presumably because NAD+ binding stabilizes the S-loops (residues 181-205) in the tetramer, which enhances interactions between subunits O-R and subunits P-Q. Molecular dynamics (MD) simulations reveal that the NAD+ binding domain (NBD) exhibits marginally higher root-mean-square fluctuation (RMSF) and a slightly more open conformation in the apo- vs holo-tetramer. Principal component 1 (PC1), derived from PCA of the MD data, shows that displacements of the two NBDs are similar in the apo- but not the holo-dimer, indicating that NAD+ binding also induces asymmetry in dimeric GAPDH. Cross-correlation analysis (CCA) uncovers similar collective backbone motions in the apo- and holo-tetramer and dimer but all residues undergo highly anti-correlated motions in the apo-monomer, consistent with its relatively low stability. In contrast, mainly S-loop motion is anti-correlated in the holo-monomer but, as exposed by PC1, this motion possesses 4-fold higher amplitude than in the apo-monomer. The high intrinsic disorder of its S-loop, revealed by both CCA and PC1, may promote oligomerization of the holo-monomer, which will compete with NAD+ release. Also, as seen in crystal structures of the apo- and holo-tetramer, the increased Cys152-His179 separation of 1.2 Å on NAD+ removal may serve to deactivate and protect the catalytic Cys152. These subtle changes at the active site combined with differential NBD and S-loop flexibility likely dictate the functions of the apo- and holo-GAPDH oligomers.
Xylanases catalyze the hydrolysis of xylan, an abundant carbon and energy source with important commercial ramifications. Despite tremendous efforts devoted to the catalytic improvement of xylanases, success remains limited due to relatively poor understanding of their molecular properties. Previous reports suggested the potential role of atomic-scale residue dynamics in modulating the catalytic activity of GH11 xylanases; however, dynamics in these studies was probed on timescales orders of magnitude faster than the catalytic time frame. Here, we used NMR titration, chemical shift projection analysis (CHESPA) and relaxation dispersion experiments (15N-CPMG) in combination with computational simulations to probe conformational motions occurring on the catalytically relevant millisecond time frame in xylanase B2 (XlnB2) and its catalytically impaired mutant E87A from Streptomyces lividans 66. Our results show distinct dynamical properties for the apo and ligand-bound states of the enzymes. The apo form of XlnB2 experiences conformational exchange for residues in the fingers and palm regions of the catalytic cleft while the catalytically impaired E87A variant only displays millisecond dynamics in the fingers, demonstrating the long-range effect of mutation on flexibility. Ligand binding induces enhanced conformational exchange of residues interacting with the ligand in the fingers and thumb loop regions, emphasizing the potential role of residue motions in the fingers and thumb loop regions for recognition, positioning, and/or stabilization of ligands in XlnB2. To the best of our knowledge, this work represents the first experimental characterization of millisecond dynamics in a GH11 xylanase family member. These results offer new insights into the potential role of conformational exchange in GH11 xylanases, providing essential dynamic information to help improve protein engineering and design applications.
A major goal of molecular docking is to predict the experimentally observed binding mode between a biomolecule, i.e. a polymer of amino acids, DNA or RNA, and a ligand (e.g. small-molecules, peptides or nucleic acids). However, there is no current molecular docking methods that properly account for the configurational entropy of the predicted complexes. It has been previously shown that the estimation of configurational entropy a posteriori significantly enhances the precision of popular scoring functions and may favour the prediction of binding poses of synthetic drugs.
Our research group develops FlexAID, a competitive ligand and biomolecule docking method that includes ligand flexibility, biomolecule side chains and large scale backbone flexibility. Here we introduce the methods used to implement FlexAID’s newest feature that allows its scoring function to estimate the conformational entropy from the occupancy of the observed binding modes. FlexAID has been redesigned with the addition of a density-based clustering algorithm, FastOPTICS, to automatically recognize and classify the different binding modes. FlexAID redefines the static binding mode usually predicted in molecular docking as a dynamic collection of poses scored altogether. The higher accuracy of FlexAID on dynamic target, the addition of novel features, i.e. the conformational entropy, its accessibility and its easy-to-use graphical user interface place FlexAID in an interesting position to tackle biologically and pharmacologically relevant situations currently ignored by other methods.
FlexAID is available as source code, pre-compiled executables or through the NRGsuite, a PyMOL integrated user interface allowing the user to use FlexAID in an interactive, intuitive and visual manner — http://bcb.med.usherbrooke.ca/flexaid
A computationally-guided semi-rational protein design approach will be used to improve the enzymatic selectivity and catalytic efficiency of the lipase B from Pseudozyma antarctica (CalB) to synthesize methyl salicylate. This fatty acid ester is a flavoring and fragrance compound with significant relevance in the biotechnological industry. CalB is one the most widely used lipases for the enzymatic hydrolysis and synthesis of esters, offering potential for the biological production of flavoring agents. However, the relatively confined organization of its active site precludes the recognition of more complex substrates. To overcome this limitation, in silico docking analyses of the best clones obtained from a previous mutant library generated in the Doucet lab will be undertaken. This will allow identification of the most significant amino acid residues involved in methyl salicylate precursor binding and recognition. These “hot spots” will be subjected to combinatorial mutagenesis to synthesize a ‘second generation’ library of CalB variants, which will further be screened for the desired activity. Finally, up scaling production of the most efficient variants will be tested for the proper industrial enzymatic synthesis of this flavor.
Bone Sialoprotein (BSP) is a matricellular protein that is abundantly expressed in mineralized tissues, and that plays an important role in nucleation of hydroxyapatite, primary mineralization, metastasis to the bone and osteoclastogenesis. This member of the SIBLING family contains two polyglutamic-acid (poly [E])-rich domains that are believed to participate in nucleation of mineral and binding to hydroxyapatite. In rat, the first poly [E] domain occupies residues 62–69, and the second residues 139–148. Substitution of the second poly [E] domain by poly-alanine (poly [A]) reduces the ability of recombinant BSP to bind to mineral in vivo. In ongoing studies, we have further shown that mutation of the second poly [E] domain may have an effect on the progression of MC3T3-E1 osteogenic cell cultures and/or may inhibit mineral deposition. To better understand the functional structural biology of BSP, we have applied Bacterial Two Hybrid (BTH) analysis to assess how substitution of poly [E] domains by poly [A] affects self-interaction. An enzymatic colorimetric assay using ortho-nitrophenyl-β-galactoside as substrate was used to quantify levels of interaction for wild type (WT) BSP and for variants of poly [E] domains. The quantitative results of this assay showed that WT-BSP has strong propensity for self-interaction. Variant of the first poly [E] domain only, had no significant effect while that of both first and second poly [E] domains reduced the propensity of interaction by about half. On the other hand, mutations of the second poly [E] sequence only, caused an almost complete loss of self-interaction capacity. These results further highlight the importance of this domain, and complement its observed functional importance for in vivo binding to mineral and for the in vitro progression of MC3T3-E1 cells. Supported by NSERC, CIHR, RSBO and CDMC-create.
Tankyrase-1 (TNKS1 or PARP-5a) is a member of the Poly (ADP-ribose) polymerase (PARP) superfamily. PARPs use NAD+ as a substrate to posttranslationally modify themselves (automodification) and target proteins (heteromodification) with poly (ADP-ribose) (PAR). PAR is an important signaling molecule involved in many cellular processes such as DNA damage repair, telomere maintenance, metabolic regulation, and WNT signaling. Canonical WNT signaling is a tightly regulated cellular pathway crucial for embryonic development, cell migration, and adult tissue homeostasis. Inappropriate activation of WNT signaling is attributed to various disease states, in particular, colorectal cancer. Inhibition of TNKS1 (and related TNKS2), and subsequent suppression of the WNT signaling pathway, has recently emerged as a promising therapeutic treatment. Currently, TNKS inhibitors target the C-terminal catalytic (CAT) domain common among all PARP family members. While PARP family members share a common CAT domain, the regulatory domains of individual family members differ substantially and are critical for determining cellular function. The domain architecture of TNKS1 is characterized by a low complexity N-terminal histidine-proline-serine rich (HPS) domain of unknown function, an ankyrin repeat (ARC) domain that serves as a binding platform, a sterile-α motif (SAM) domain, and a C-terminal CAT domain. SAM domains are often implicated in homo- and hetero- oligomerization and are known to display a diverse functional profile. TNKS1 oligomerization is believed to facilitate assembly of highly ordered complexes such as the multi-protein “destruction complex” pertinent for WNT signaling. Given the therapeutic potential of TNKS inhibition, we sought to elucidate the functions of the unique regulatory domains of TNKS1, particularly the SAM domain. The structure of TNKS1 SAM oligomer, its role in regulating TNKS1 catalytic activity, and its cellular function has not been explored. Here we present two crystal structures of the TNKS1 SAM domain: (i) the polymer form of the wild-type (WT) SAM domain and (ii) an interface mutation (V1056H) of the TNKS1 SAM domain polymer. Using structure-guided mutagenesis, we demonstrate both partial and complete disruption of the TNKS1 SAM domain polymer. Using functional analysis, we furthermore show that TNKS1 oligomerization has a major impact on TNKS1 automodification, cellular distribution, association with interacting proteins, and function in WNT signaling.
NRPSs are multifunctional enzymes that synthesize biopeptides with a broad range of activities such as antifungal, antibacterial, antiviral and include compounds like penicillin, vancomycin, cyclosporin, etc. In recent years, various studies comprising structures of different domains of NRPSs, initiation modules with a tailoring domain (formylation) and a termination module with a chain-releasing domain (Te) have led to remarkable insights into the catalytic cycle of NRPSs. However, a comprehensive mechanistic understanding of this assembly-line synthesis machinery remains elusive in the absence of any structural information on intact NRPSs. It is important to determine multiple structures of this macromolecular assembly line to delineate the details of its synthetic cycle.
Here, we investigated the overall structural architecture of a dimodular NRPS using single particle electron microscopy. The study revealed the flexible nature of the modules even when mechanism-based inhibitors were used to lock them in a specific conformation. Our work delineates the structural properties of the NRPS and represents an important step, providing the first 3D views of intact NRPSs.
One of the key elements for proper directed evolution of proteins is the cyclic use of mutagenesis and selection processes, giving rise to libraries containing millions of mutants. However, analyzing such an important number of mutants is not a trivial task, as the identification of active variants among millions of possibilities quickly becomes exhaustive and inefficient. Here we describe a semi-rational combinatorial approach supported by virtual docking to generate smaller and smarter libraries. Because of its ability to perform the synthesis of esters in organic media, lipase B from Pseudozyma antarctica (CalB) was used as an industrially-relevant model system. Since CalB displays very low activity towards bulky substrates, the main goal of this project was aimed at the development of CalB variants with enhanced synthetic activity towards bulky substrates. Substrate-imprinted docking was used to uncover target positions involved with the stabilization of the enzyme-substrate complex, identifying “hot spots” that are most likely to yield active improvements for desired ligands. The Iterative Saturation Mutagenesis strategy was employed to sequentially incorporate favorable mutations, further increasing our chances of selecting improved variants with a concomitant reduction in screening effort. We tested a limited number of 164 mutants that explored 6 residue positions in the active-site cavity of CalB. For a single round of mutagenesis and selection against 2 different substrates, a number of variants showed up to 5-fold increase in activity relative to WT CalB. These results represent the first stage in the development of additional CalB variants with improved activity towards bulky esters.
Mutations in the PINK1- and Parkin-encoding genes cause autosomal recessive form of Parkinson’s disease. PINK1 is a protein kinase, best known for its role in signaling mitochondrial damage and consequently initiating mitochondrial repair or autophagy mechanisms. Upon mitochondrial damage, PINK1 accumulates on the outer membrane (OMM) of the mitochondria, and recruits the E3 ubiquitin ligase Parkin in a kinase activity-dependent fashion. Parkin then ubiquitinates multiple OMM proteins and signals mitochondria for autophagic destruction. In recent years, the mechanism for Parkin’s recruitment and activation has been a subject of extensive study in the context of PINK1’s kinase activity. The most recent evidence suggests that the role of PINK1 is to phosphorylate both Parkin (on its ubiquitin-like domain) and ubiquitin on Ser65 for a complete activation of Parkin. This makes PINK1 the first known ubiquitin kinase. However the underlying molecular mechanisms underlying Parkin’s activation are unknown. Here, I have developed a PINK1 production system to characterize the kinetics of Parkin and ubiquitin phosphorylation and its consequences for the activation of Parkin. After optimization, I found that the PINK1 ortholog from the insect species Tribolium castaneum (TcPINK1) expresses well in E. coli, can be purified in solution, and produces an active kinase. This recombinant PINK1 was essential to elucidate the role of phosphorylated ubiquitin (pUb) as an enhancer for Parkin phosphorylation by PINK1, hence establishing a role of pUb in the mitochondrial quality control pathway. Nuclear magnetic resonance studies and phosphorylation assays combined with mutagenesis reveal the residues in Parkin that are critical for the interactions with PINK1. This work sets the stage for more detailed investigations of the structural features of PINK1 that allow the enzyme to recognize ubiquitin domains and phosphorylate specifically Ser65.
In eukaryotes, the SET1 family of methyltransferases carries out the methylation of Lysine 4 on Histone H3 (H3K4). Alone, these enzymes exhibit low enzymatic activity and require the presence of additional regulatory proteins, which include RbBP5, Ash2L, WDR5 and DPY-30, to stimulate their catalytic activity. While previous structural studies established the structural basis underlying the interaction between RbBP5, Ash2L and WDR5, the molecular underpinnings controlling the formation of the Ash2L/DPY-30 complex have remained largely unexplored. Here we report the crystal structure of the Ash2L/DPY-30 complex solved at 2.2Å. The structure shows that Ash2L C-terminus folds in two distinct domains that include a b-sandwich composed of 12 b-strands and a long a-helix located at the C-terminal of the protein. This amphipathic a-helix makes several hydrophobic interactions with a dimer of DPY-30. Mutational analysis of hydrophobic residues located at the interface between DPY-30 and Ash2L shows that the interactions between these two proteins are critical for hematopoietic differentiation and histone H3K4 tri-methylation in murine erythroleukemia cells. Interestingly, close inspection of the Ash2L/DPY-30 revealed a large positive fourier located on the surface of the complex that is analogous to a lipid. Binding assays revealed that the Ash2L/DPY-30 preferentially binds to cardiolipin suggesting that lipids may play a role in epigenetic signaling in modulating histone H3K4 methylation. Overall, our findings show that the interaction between Ash2L and DPY-30 is critical for hematopoietic differentiation and highlight the potential role of lipids in regulating the SET1 family of methyltransferases
Leishmania parasite infection can lead to two main pathologies, a cutaneous form causing self-healing skin lesions, or a lethal visceral form causing hepatosplenomegaly and internal organ failure. Close to one billion people are at risk of infection and 1.3 million new cases arise each year, almost exclusively in third world countries. The A2 virulence factor has been found to promote Leishmania donovani survival in the visceral organs. The mechanism by which it contributes to visceral organ infection remains unknown. Here we present biochemical data supporting the role of A2 as an ATP-independent chaperone-like protein. Through gel filtration and Blue-Native PAGE studies, we observed the formation of a dynamic quaternary structure. We further confirmed our observations using negative staining electron microscopy. The transition from its sand-fly vector to a human host is associated with a shift in temperature from 25 C to 37 C and higher during fever caused by chronic infection. We propose that through its chaperone-like activity, A2 protects the infecting parasites by preventing the formation of toxic denatured protein aggregates when exposed to elevated temperatures inside the host. This protective role of A2 is supported by in vitro and in vivo chaperone assays. Hence, A2 has been considered as a target for the generation of a mutant live attenuated vaccine. Preliminary studies with A2 deletion mutants in human macrophages and animal models are under way.
One of the primary etiological factors causing leukemia is triggered by the mutations of the myeloid lymphoma leukemia(MLL) gene which results in the mis-regulation of developmental genes and stimulation of white blood cell proliferation. Recent evidence has shown that the oncogenic potential of MLL mutations is dependent on the presence of the wild-type form of the protein and more specifically on its ability to methylate histone H3 on Lys-4 (H3K4); a post-translational modification (PTM) known to stimulate gene expression. H3K4 methylation by MLL1 is allosterically regulated by a four subunit complex composed of WDR5-RbBP5-Ash2L-DPY-30 (WRAD) and disruption of this complex abrogates developmental gene expression and white blood cell proliferation. Previous structural studies demonstrated that an amphipathic a-helix located on the C-terminus of Ash2L makes several hydrophobic interactions with DPY-30 dimerization docking (D/D) domain and that disruption of these contacts impair erythroid cells terminal differentiation. Moreover, binding assays using peptide arrays demonstrated that mutations of key residues on Ash2L a-helix conferred stronger binding to DPY-30 D/D, raising the possibility that we can develop inhibitory peptides designed to block the interaction between Ash2L and DPY-30. To understand the structural basis underlying the gain of binding of these mutants, the Ash2L mutants and DPY-30 were purified using immobilized metal affinity chromatography (IMAC). The complexes were reconstituted and further purified by size-exclusion chromatography. Preliminary screening identified two conditions yielding crystals for two specific Ash2L mutants/DPY-30 complexes. The crystals diffract to 2Å and belong to P4 and P3 space groups. Ultimately, the crystal structure of these complexes will provide a glimpse into the first inhibitor bound DPY-30 complex and may provide new therapeutic avenues for the treatment of leukemia caused by MLL1 mutations.
IFIT1 and IFIT1B are effectors of the mammalian antiviral response that prevent propagation of virus infection by selectively inhibiting translation of viral mRNA. They rely on their ability to compete with the translation initiation factor eIF4F to specifically recognize foreign capped mRNAs, while remaining inactive against host mRNAs marked as ‘self’ by ribose 2´-O methylation at the first cap proximal nucleotide (N1, also called Cap1 methylation). In an attempt to evade IFIT1/IFIT1B-mediated restriction, many viruses have acquired the means to produce Cap1-modified mRNA. To gain insight into the IFIT1 mechanisms of self vs non-self discernment, we determined several crystals structures of RNA-bound human IFIT1, including a 1.6 Å complex with capped RNA.
The structures show that IFIT1 forms a water-filled, positively-charged, RNA-binding tunnel with a separate hydrophobic extension for cap binding. IFIT1 unexpectedly engages the cap in multiple conformations (syn- and anti-), relying mainly on stacking and water-mediated interactions that give rise to a plastic and relatively non-specific mode of binding. Cap-proximal nucleotides encircled by the tunnel provide affinity to compete with eIF4F, and allow IFIT1 to select against Cap1 methylated mRNA. Gel-shift binding assays confirm that Cap1 interferes with IFIT1 recognition, but in an RNA-dependent manner, while translation assays indicate that Cap1 methylation alone is not sufficient to prevent mRNA recognition at high IFIT1 concentrations. Structural and functional analysis show that 2´-O methylation at N2 (Cap2), another abundant mRNA modification, is also occluded, thus revealing a novel and potentially synergistic role for it in distinguishing self from non-self. Surprisingly, despite a conserved mechanism for mRNA binding, we find that IFIT1 and IFIT1B differ in their ability to sense the Cap2 methylation status of mRNA. Our structural and biochemical analysis defines the molecular basis for IFIT1 translational inhibition of capped viral RNA.
The MmpL family of proteins translocates complex (glyco) lipids and siderophores across the cell envelope of mycobacteria and closely related Corynebacteriaceae and plays important roles in the biogenesis of the outer membrane of these organisms. Despite their significance in the physiology and virulence of Mycobacterium tuberculosis, and from the perspective of developing novel antituberculosis agents, little is known about their structure and mechanism of translocation. In this study, the essential mycobacterial mycolic acid transporter, MmpL3, and its orthologue in Corynebacterium glutamicum, CmpL1, were investigated as prototypical MmpL proteins to gain insight into the transmembrane topology, tertiary and quaternary structures, and functional regions of this transporter family. The combined genetic, biochemical, and biophysical studies indicate that MmpL3 and CmpL1 are structurally similar to Gram negative resistance-nodulation and division efflux pumps. They harbor 12 transmembrane segments interrupted by two large soluble periplasmic domains and function as homotrimers to export long-chain (C22−C90) mycolic acids, possibly in their acetylated form, esterified to trehalose. The mapping of a number of functional residues within the middle region of the transmembrane domain of MmpL3 shows a striking overlap with mutations associated with resistance to MmpL3 inhibitors. The results suggest that structurally diverse inhibitors of MmpL3 all target the proton translocation path of the transporter and that multi-drug resistance to these inhibitors is enabled by conformational changes in MmpL3.
KEYWORDS: Mycobacterium, tuberculosis, MmpL3, acyltrehaloses, mycolic acids, lipid translocation
Mutations in the parkin and PINK1 genes are responsible for a common inherited form of Parkinson's disease (PD) with an early onset. The gene products E3 ubiquitin ligase parkin and kinase PINK1 are involved in autophagy of damaged mitochondria termed mitophagy. In this pathway, PINK1 phosphorylates parkin and ubiquitin, thus activating parkin ligase activity. Upon recruitment to mitochondria, activated parkin ubiquitinates various mitochondrial substrates leading to autophagy of the damaged organelles. Parkin contains a ubiquitin-like (Ubl) domain at the N-terminus and four RING-like domains at the C-terminus. We previously reported the structure of full-length parkin in autoinhibited conformation. Autoinhibited parkin is activated by phosphorylation at the UBl domain by PINK1 and binding to phospho-ubiquitin, both releasing Ubl domain from the E2 binding site on parkin. Parkin Ubl domain also binds SH3 domain of Endophilin A1, a BAR domain brain specific protein involved in endocytosis. The binding affinity is comparable to proline-rich domains (PRDs) from well-established SH3 partners. Parkin structure reveals that Ubl uses similar surfaces for binding to the RING1 domain of parkin and SH3 domain of Endophilin A1. This could explain why SH3 to full-length parkin is not as high as the truncated Ubl domain, and that conditions that promote phosphorylation enhance the interaction between full-length proteins at nerve terminals. Here, we report that phosphorylated Ubl also binds to SH3 domain with similar affinity in vitro. Moreover, phosphorylation of parkin, its binding to phospho-ubiquitin, and parkin mutants that release Ubl domain increase the binding of full-length parkin to the SH3 domain of Endophilin A1. Current work is directed towards studying the effect of this interaction on parkin activity in vitro and in cells. The findings may identify the link between synaptic vesicles endocytosis and mitophagy.
In today’s world, the importance of recycling has become more prominent. Sustainable urbanism now promotes the use of recycled materials and favors methods that maximize energy savings. Nature itself has elegantly designed ways to maximize its efficiency and its use of resources.
Many important substrate salvaging or recycling pathways are commonly found in primary metabolism, such as for nucleosides, uracil and coenzyme B1. However, there are only rare examples of such mechanism in secondary metabolism. Substrate salvage allows the recycling of metabolites derived from regular cellular catabolism back to the general biosynthetic pathway. Herein, we provide the first biochemical and structural evidence for flavoenzyme-mediated substrate recycling in secondary metabolism.
In this study, we report a shunt product recycling pathway in the biosynthesis of caerulomycin A, a 2,2-bipyridine-containing natural product under development as a potent novel immunosuppressive agent. This pathway is mediated by flavine-dependent oxidase CrmK. Biochemical characterizations demonstrate that CrmK plays an unexpectedly important role in caerulomycin A biosynthesis by catalyzing the conversion of an alcohol shunt product to an aldehyde and then to a carboxylate, thus recycling or salvaging the shunt product into the main pathway of caerulomycin A. Moreover, the crystal structures of CrmK in apo form and in complex with the substrate CRM P and site-directed mutagenesis studies provide insights into the catalytic mechanisms and the molecular basis of CrmK in shunt product recycling.
Natural product biosynthetic gene clusters (BGCs) are well known to often carry additional genes for resistance, transport, regulation, or carrying out natural protection group chemistry. Now the functional characterization of CrmK in caerulomycin biosynthesis adds a recycling mechanism that may operate in secondary metabolism. The majority of antibiotic BGCs carry genes with unknown or unassigned function, and this work indicates that a salvage/recycling role could be a hypothesis to test for some of such genes. With the exponential growth of characterization of BGCs for secondary metabolites, it was expected that more and more enzymes with a salvaging/recycling function would be found in the future and explored for the yield improvement of secondary metabolites.
Many powerful bioactive compounds are produced by microorganisms in their constant war for survival. Among the molecular tools used to produce these compounds are the polyketide synthases (PKS) which consists of multiple domains functioning in an assembly line fashion, incorporating malonyl groups from malonyl-CoenzymeA into a full polyketide. The final polyketide is then cyclized or hydrolyzed off the synthase by the terminal domain, the Thioesterase (TE) domain. TE domains act both as cogs in the mechanism and as gate keepers controlling the stereochemistry of the final product, as they are very selective and will only cyclize the correct polyketide. However, fungal TE domains appear to be very stereo-tolerant and can cyclize both D- and L-configured products. They still retain their function as gatekeepers though, as they can either cyclize benzoic or phenylacetic substrate but not both. The cause of this new selectivity is not known but is most likely related to structural differences in the substrate binding pocket of the TE domains. Therefore further study is required to determine what structural feature is responsible for this selectivity in these pharmaceutically very important synthases.
Here, we investigate the structure of the TE domains of the non-reducing PKSs of the fungal Radicicol (RAD-TE) and Dehydrocurvularin (DHC-TE) biosynthetic pathways as they represent both the benzoic (RAD-TE) and phenylacetic (DHC-TE) variant of the TE domain. Both proteins were heterologously expressed and purified from E. coli. Crystals were generated under several conditions for structure elucidation by x-ray crystallography.
Despite the importance of RNA in many biological processes, there is still a great need for additional structural information on many RNAs to improve our understanding of their function and better exploit them for biomedical applications. Over the past several years, our laboratory has focused on the Neurospora VS ribozyme. In contrast to other small ribozymes that target unstructured single-stranded RNAs, the VS ribozyme specifically recognizes and cleaves a stem-loop substrate. This unique aspect of its mechanism makes it an attractive system for structural and engineering studies.
Although the VS ribozyme has been extensively characterized since its discovery more than 25 years ago, it is only very recently that three-dimensional structures of the complete ribozyme became available. Our laboratory has successfully used a divide-and-conquer approach for structural characterization, which consists of determining NMR structures of individual subdomains with the ultimate goal of building a structure of the full VS ribozyme based on the structures of these individual subdomains. I will present a summary of our research progress and the advantages of NMR spectroscopy in the characterization of the high-resolution structure and dynamics of RNA. In addition, I will describe our progress in the engineering of VS-derived ribozymes for specific cleavage of non-natural substrates. These structural and engineering studies impact not only on our understanding of the VS ribozyme and its potential use for biomedical and biotechnological applications, but also help delineate fundamental principles of RNA structure and engineering.
For proteins with a single well-defined native state, protein three-dimensional structure is a major determinant of sequence evolution at the residue level. On the other hand, many proteins adopt multiple, distinct native structures under different conditions (“conformational switches”), yet the impact of such native state switching on protein evolution is not known at the residue level. Here, we performed a proteome-wide analysis of how protein structure impacts sequence evolution at the residue level for protein conformational switches in S. cerevisiae. We observed a strong linear relationship between residue evolutionary rate and residue burial for conformational switches. In addition, we found that conformational switches evolve significantly and consistently more slowly at the residue level than proteins with a single native state, even after controlling for degree of residue burial. Next, we focused on proteins that switch conformations upon molecular binding. We found that interfacial residues in these conformational switches evolve more slowly than interfacial residues in proteins with a single native state, and that the bound conformation is a better predictor for residue evolutionary rate than the unbound conformation. Our findings suggest that for conformational switches, the necessity to encode multiple distinct native structures under different conditions impose strong evolutionary constraints on the entire protein, rather than just a few key residues. Our results provide new insights into the structure-evolution relationship and deeper understanding of the evolutionary design principles of protein conformational switches.
Mutations in the Parkin and PINK1 genes cause familial forms of Parkinson’s disease (PD). Parkin and PINK1 work in a mitochondrial quality control pathway essential to prevent neurodegeneration. The kinase PINK1 senses damaged mitochondria by accumulating at depolarized membranes, where it phosphorylates ubiquitin. Phospho-ubiquitin (pUb) then recruits and activates the E3 ubiquitin ligase Parkin, which in turn ubiquitinates outer mitochondrial membrane (OMM) proteins, marking them for proteasomal degradation and recruiting the autophagy machinery.
We along with others have previously shown that Parkin adopts an auto-inhibited conformation (Trempe et al. 2013). The release of inhibition is initiated by pUb binding to the RING1 domain of Parkin, which allosterically displaces its Ubl domain (Sauvé et al., 2015). This promotes phosphorylation of the Ubl at Ser65 by PINK1, which increases ubiquitin ligase activity. However, the molecular mechanisms underlying the conformational changes and dictating substrate specificity on mitochondria remain unclear.
Here, we dissect the steps of Parkin activation through a combination of biophysical measurements, mitochondrial ubiquitination and cellular mitophagy assays. Mutation of Arg275, a residue implicated in PD, decouples pUb binding from Ubl release and is rescued by a mutation in the Ubl-binding site. Similarly, mutation of Trp403, which anchors the Repressor Element of Parkin, rescues the phospho-dead mutant S65A, supporting our hypothesis that S65 phosphorylation releases the REP and enables E2-binding. These rescue experiments pave the way for novel therapeutic approaches that could restore activity of impaired Parkin or PINK1. Finally, we propose that Parkin substrate specificity is conferred by the proximity of a subset of OMM proteins such as mitofusin to pUb chains on damaged mitochondria. This cluster suggests a mechanism for cross-talk between mitochondrial fusion/fission and damage control.
Immunity in higher organisms is divided into the innate and the adaptive immune systems. Whereas the adaptive immune system is highly specific and acquired through one’s exposure to different pathogens, innate immunity is present from birth and is used to distinguish ‘self’ from ‘nonself’ in a more general manner. Innate immune responses are initiated by the recognition of pathogen associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). There are three types of PRRs: membrane-bound, cytoplasmic and secreted. My interest lies in intracellular PRRs, more specifically in inflammatory Caspases.
Caspases are a family of multi-domain endoproteases that play critical roles in cell death and inflammation. Caspase activation requires processing by cleavage of the catalytic domain. Once activated, they use N-terminal accessory protein-protein interactions domains such as CARD domains or Death domains to activate downstream factors leading to inflammation or apoptosis. In mammals, Caspases-3, -6, -7, -8 and -9 are implicated in apoptosis, and mutations in these genes can lead to diseases such as Alzheimer’s and cancer. Caspase-1, -4, -5 and -12, on the other hand, are inflammatory Caspases essential in innate immunity. Recently, human Caspase-4 and -5, and their murine ortholog Caspase-11, were shown to directly bind lipopolysaccharides (LPS) to initiate an inflammatory response. Introduction of LPS into the cytoplasm of human monocytes and epithelial cells resulted in cell death that depended on Caspase-4/5 expression. Deletion analysis revealed that the interaction between Caspase-4/11 and LPS is confined to their CARD domains, which is a novel finding since previously CARD domains were known to mediate only protein- protein interactions. Interestingly, fusion of the Caspase-11 CARD domain onto Caspase- 1 also sensitized cells to LPS, confirming further the unusual activation of Caspase-4/11. Once bound to LPS, Caspase-4/5/11 oligomerize to form a non-canonical inflammasome that leads to direct or Caspase-1 mediated release of crucial inflammatory cytokines, including IL-1β and IL-18.
Understanding the molecular basis for the interaction of LPS with CARD domains and how this triggers formation of a non-canonical inflammasome will be essential for the development of small molecule inhibitors as potential treatments of septic shock and uncontrolled inflammatory responses. My project will use X-ray crystallography and other biophysical approaches to obtain an atomic level characterization of the interaction between LPS and Caspase-4/5.
Non-ribosomal peptide synthetases (NRPSs) are large multimodular mega-enzymes that catalyze peptide bonds resulting in the formation of biologically interesting compounds such as bleomycin (anti-tumour). Each module contains three integral domains including the adenylation domain (A) which transports the substrate to the peptidyl carrier protein (PCP) arm. The PCP domain of the current module and the PCP arm of the previous module will then transport their covalently bound substrates to the condensation domain (C) where the peptide bond is catalyzed. The growing peptide chain will eventually be transported to the C domain of the next module where elongation continues. This process continues until the final product is released at the termination domain (TE).
In some cases, the C domain is replaced with a cyclization domain (Cy) in non-canonical NRPSs. These specialized domains are responsible for two reactions where the first involves the catalysis of the peptide bond. The latter reaction involves forming 5-membered heterocyclic rings with cysteine, threonine and serine side chains resulting in oxazole and thiazole ring formation found in anti-tumour compounds such as epothilone. The function of the Cy domain has not been fully elucidated but a PhD student in our lab has managed to grow crystals of the Cy domain from the bacillamide synthetase and solve the first structure of its kind. The problem is that this single crystal took 5 months to grow. I am working with a truncated construct where the last 16 residues of the C-terminal are eliminated in hopes of growing crystals faster. These crystals would then be amenable for co-crystallization experiments where we can introduce molecule analogues and study substrate specificity using a chemical tethering technique that the same PhD in the lab has developed.
Furthermore, along with the Cy domain there are also two other components that are important in the bacillamide biosynthetic pathway. This includes BmdA, involved in a decarboxylation reaction converting tryptophan to tryptamine, which is an important substrate for the final condensation reaction in the NRPS. An oxidase (BmdC) is also involved to catalyze a double bond in the oxazole and thiazole rings that are formed by the Cy domain. Crystallizing this oxidase will give important structural information that will ultimately give insight on its function within the biosynthetic pathway.
Nonribosomal peptide synthetases (NRPSs) are true macromolecular machines, using modular assembly-line logic, a complex catalytic cycle, moving parts and many active sites to make their bio-active products. Substrates are tethered to a prosthetic phosphopantetheinyl arm attached to a peptidyl carrier protein (PCP) domain to facilitate shuttling between active sites and modules. Modules, in turn, must act in concert to coordinate passing along the growing peptide to continue synthesis. Previously, we have determined a series of crystal structures of the initiation module of an antibiotic-producing NRPS, linear gramicidin synthetase. This module includes the specialized tailoring formylation domain, and we captured states that represent every major step of the assembly-line synthesis in the initiation module. To complete the catalytic cycle of the first PCP domain, we present the structure of a construct that includes the full initiation module and the condensation domain of the adjacent module. The structure shows the conformation required for the donation of formyl-valine in peptide bond formation with the second substrate, glycine. This is the first structure of a PCP domain at the donor site of a C domain, and also provides novel insight into the movements and interactions neighbouring modules undergo in the synthetic cycle. Together our published and unpublished work presents a holistic view of the function of this elegant NRPS initiation module.
Enzymes are increasingly being used in pharmaceutical and industrial environments, particularly as greener and more efficient alternatives to chemical catalysts. However, engineering new enzyme reactions is an arduous and inefficient process, mainly because the predictable outcome of protein engineering on 3D structure, function and dynamics remains elusive. Recent experimental evidence suggests that conformational exchange may be involved in promoting catalysis in many enzyme systems, but the mechanisms underlying this atomic flexibility remain unclear. It is still unknown whether sequence and/or structure are evolutionarily conserved to promote flexibility events linked to biological function among protein homologs. Understanding phenomena underlying protein dynamics is thus an important step in facilitating protein engineering. In order to tackle these interrogations, we have used NMR to characterize the millisecond timescale conformational exchange in various members of the ribonuclease A superfamily. While these enzymes display very similar structure, their evolutionary distance and diversified biological activities complicate flexibility-function analyses. To solve this issue, we have investigated mammalian homologs of human ribonuclease 3 (Eosinophil Cationic Protein, ECP), comparing the human enzyme with its close ECP homologs from Macaca fascicularis, Pongo pygmaeus and Pongo abelii. Our findings show that conformational exchange in the monkey enzymes strongly resembles that of their human counterpart, providing insights into the effects of sequence and phylogenetic diversity on protein dynamics. In parallel, antibacterial assays have been performed on these proteins, and we have found that the more the protein sequence diverges from the common ancestor, the more potent its antibacterial activity is. We noted that this coincides with a slowing down of the global conformational exchange rate (kex) within each protein. Further experiments are required to establish the interdependence that could exist between the function of these proteins and their atomic flexibility.