Binding Cleft Research Articles

Molecular mechanism of Aspergillus fumigatus biofilm disruption by fungal and bacterial glycoside hydrolases

During infection, the fungal pathogen Aspergillus fumigatus forms biofilms that enhance its resistance to antimicrobials and host defenses. An integral component of the biofilm matrix is galactosaminogalactan (GAG), a cationic polymer of α-1,4-linked galactose and partially deacetylated N-acetylgalactosamine (GalNAc). Recent studies have shown that recombinant hydrolase domains from Sph3, an A. fumigatus glycoside hydrolase involved in GAG synthesis, and PelA, a multifunctional protein from Pseudomonas aeruginosa involved in Pel polysaccharide biosynthesis, can degrade GAG, disrupt A. fumigatus biofilms, and attenuate fungal virulence in a mouse model of invasive aspergillosis. The molecular mechanisms by which these enzymes disrupt biofilms have not been defined. We hypothesized that the hydrolase domains of Sph3 and PelA (Sph3h and PelAh, respectively) share structural and functional similarities given their ability to degrade GAG and disrupt A. fumigatus biofilms. MALDI-TOF enzymatic fingerprinting and NMR experiments revealed that both proteins are retaining endo-α-1,4-N-acetylgalactosaminidases with a minimal substrate size of seven residues. The crystal structure of PelAh was solved to 1.54 Å and structure alignment to Sph3h revealed that the enzymes share similar catalytic site residues. However, differences in the substrate-binding clefts result in distinct enzyme-substrate interactions. PelAh hydrolyzed partially deacetylated substrates better than Sph3h, a finding that agrees well with PelAh's highly electronegative binding cleft versus the neutral surface present in Sph3h Our insight into PelAh's structure and function necessitate the creation of a new glycoside hydrolase family, GH166, whose structural and mechanistic features, along with those of GH135 (Sph3), are reported here.

Crystallographic structure and molecular dynamics simulations of the major endoglucanase from Xanthomonas campestris pv. campestris shed light on its oligosaccharide products release pattern

Cellulases are essential enzymatic components for the transformation of plant biomass into fuels, renewable materials and green chemicals. Here, we determined the crystal structure, pattern of hydrolysis products release, and conducted molecular dynamics simulations of the major endoglucanase from the Xanthomonas campestris pv. campestris (XccCel5A). XccCel5A has a TIM barrel fold with the catalytic site centrally placed in a binding groove surrounded by aromatic side chains. Molecular dynamics simulations show that productive position of the substrate is secured by a network of hydrogen bonds in the four main subsites, which differ in details from homologous structures. Capillary zone electrophoresis and computational studies reveal XccCel5A can act both as endoglucanase and licheninase, but there are preferable arrangements of substrate regarding β-1,3 and β-1,4 bonds within the binding cleft which are related to the enzymatic efficiency.

Conformational coupling by trans-phosphorylation in calcium calmodulin dependent kinase II.

The calcium calmodulin-dependent protein kinase II (CaMKII) is a dodecameric holoenzyme important for encoding memory. Its activation, triggered by binding of calcium-calmodulin, persists autonomously after calmodulin dissociation. One (receiver) kinase captures and subsequently phosphorylates the regulatory domain peptide of a donor kinase forming a chained dimer as the first stage of autonomous activation. Protein dynamics simulations examined the conformational changes triggered by dimer formation and phosphorylation, aimed to provide a molecular rationale for human mutations that result in learning disabilities. Ensembles generated from X-ray crystal structures were characterized by network centrality and community analysis. Mutual information related collective motions to local fragment dynamics encoded with a structural alphabet. Implicit solvent tCONCOORD conformational ensembles revealed the dynamic architecture of inactive kinase domains was co-opted in the activated dimer but the network hub shifted from the nucleotide binding cleft to the captured peptide. Explicit solvent molecular dynamics (MD) showed nucleotide and substrate binding determinants formed coupled nodes in long-range signal relays between regulatory peptides in the dimer. Strain in the extended captured peptide was balanced by reduced flexibility of the receiver kinase C-lobe core. The relays were organized around a hydrophobic patch between the captured peptide and a key binding helix. The human mutations aligned along the relays. Thus, these mutations could disrupt the allosteric network alternatively, or in addition, to altered binding affinities. Non-binding protein sectors distant from the binding sites mediated the allosteric signalling; providing possible targets for inhibitor design. Phosphorylation of the peptide modulated the dielectric of its binding pocket to strengthen the patch, non-binding sectors, domain interface and temporal correlations between parallel relays. These results provide the molecular details underlying the reported positive kinase cooperativity to enrich the discussion on how autonomous activation by phosphorylation leads to long-term behavioural effects.

An Active, Ligand-Responsive Pulling Geometry Reports on Internal Signaling between Subdomains of the DnaK Nucleotide-Binding Domain in Single-Molecule Mechanical Experiments.

Single-molecule mechanical experiments have proven to be ideal tools for probing the energetics and mechanics of large proteins and domains. In this paper, we investigate the nucleotide-dependent unfolding mechanics of the nucleotide-binding domain (NBD) of the Hsp70 chaperone DnaK. The NBD binds ADP or ATP in the binding cleft formed by lobe I and lobe II, which consists of two subdomains each. When force is applied to the termini of the NBD, the observed unfolding forces are independent of the nucleotide state. In contrast, when force is applied across the nucleotide-binding pocket, the unfolding forces report specifically on the nucleotide-phosphate state. In this active, ligand-responsive pulling geometry, we observed a bifurcation of the unfolding pathway; the pathway proceeds either through a cooperative "coupled pathway" or through a noncooperative "uncoupled pathway". The partitioning between individual unfolding pathways can be effectively tuned by mutation or by the nucleotide exchange factor GrpE, i.e., by the factors affecting the strength of the lobe I-lobe II interactions within the native NBD. These experiments provide important insight into the molecular origin of the internal signaling between the subdomains of the nucleotide-binding domain of Hsp70 proteins and how signals are efficiently transferred inside the protein molecule.

The Structural Determinants Accounting for the Broad Substrate Specificity of the Quorum Quenching Lactonase GcL.

Quorum quenching lactonases are enzymes capable of hydrolyzing lactones, including N-acyl homoserine lactones (AHLs). AHLs are molecules known as signals in bacterial communication dubbed quorum sensing. Bacterial signal disruption by lactonases was previously reported to inhibit behavior regulated by quorum sensing, such as the expression of virulence factors and the formation of biofilms. Herein, we report the enzymatic and structural characterization of a novel lactonase representative from the metallo-β-lactamase superfamily, dubbed GcL. GcL is a broad spectrum and highly proficient lactonase, with kcat /KM values in the range of 104 to 106 m-1 s-1 . Analysis of free GcL structures and in complex with AHL substrates of different acyl chain length, namely, C4-AHL and 3-oxo-C12-AHL, allowed their respective binding modes to be elucidated. Structures reveal three subsites in the binding crevice: 1) the small subsite where chemistry is performed on the lactone ring; 2) a hydrophobic ring that accommodates the amide group of AHLs and small acyl chains; and 3) the outer, hydrophilic subsite that extends to the protein surface. Unexpectedly, the absence of structural accommodation for long substrate acyl chains seems to relate to the broad substrate specificity of the enzyme.

Structural basis of \u03b2TrCP1-associated GLI3 processing

Controlled ubiquitin-mediated protein degradation is essential for various cellular processes. GLI family regulates the transcriptional events of the sonic hedgehog pathway genes that are implicated in almost one fourth of human tumors. GLI3 phosphorylation by Ser/Thr kinases is a primary factor for their transcriptional activity that incurs the formation of both GLI3 repressor and activator forms. GLI3 processing is triggered in an ubiquitin-dependent manner via SCFβTrCP1 complex; however, structural characterization, mode of action based on sequence of phosphorylation signatures and induced conformational readjustments remain elusive. Here, through structural analysis and molecular dynamics simulation assays, we explored comparative binding pattern of GLI3 phosphopeptides against βTrCP1. A comprehensive and thorough analysis demarcated GLI3 presence in the binding cleft shared by inter-bladed binding grooves of β-propeller. Our results revealed the involvement of all seven WD40 repeats of βTrCP1 in GLI3 interaction. Conversely, GLI3 phosphorylation pattern at primary protein kinase A (PKA) sites and secondary casein kinase 1 (CK1) or glycogen synthase kinase 3 (GSK3) sites was carefully evaluated. Our results indicated that GLI3 processing depends on the 19 phosphorylation sites (849, 852, 855, 856, 860, 861, 864, 865, 868, 872, 873, 876, 877, 880, 899, 903, 906, 907 and 910 positions) by a cascade of PKA, GSK3β and CSKI kinases. The presence of a sequential phosphorylation in the binding induction of GLI3 and βTrCP1 may be a hallmark to authenticate GLI3 processing. We speculate that mechanistic information of the individual residual contributions through structure-guided approaches may be pivotal for the rational design of specific and more potent inhibitors against activated GLI3 with a special emphasis on the anticancer activity.

Food flavonoid ligand structure/estrogen receptor-α affinity relationships – toxicity or food functionality?

In silico molecular modelling is used to study interactions between flavonoid phytoestrogens and estrogen receptor (ER)α. Twenty flavonoids from foods were studied; e.g., genistein from soy, naringenin from grapefruit, phloretin from pears, chrysin from oyster mushrooms. These potential ligands' molecular attributes and their spatial arrangements that favour binding to the ligand binding cleft (LBC) of ERα are identified, and Docking Scores calculated. The Docking Score order is the same as the estrogenicity order for 8 of the flavonoids studied in detail. The number and position of flavonoid ring hydroxyls influence the Docking Scores which might relate to ERα′s bio-activity. Hydrophobic interactions between ligands and ERα are also important; the number of rotatable CC bonds in ligands likely affects the magnitude of hydrophobic interactions and ligand fit. Our findings suggest that flavonoids with diverse structural features could have different binding energies and binding affinities with ERα, which might confer different functionalities and toxicities.

Mechanisms of redundancy and specificity of the Aspergillus fumigatus Crh transglycosylases

Fungal cell wall synthesis is achieved by a balance of glycosyltransferase, hydrolase and transglycosylase activities. Transglycosylases strengthen the cell wall by forming a rigid network of crosslinks through mechanisms that remain to be explored. Here we study the function of the Aspergillus fumigatus family of five Crh transglycosylases. Although crh genes are dispensable for cell viability, simultaneous deletion of all genes renders cells sensitive to cell wall interfering compounds. In vitro biochemical assays and localisation studies demonstrate that this family of enzymes functions redundantly as transglycosylases for both chitin-glucan and chitin-chitin cell wall crosslinks. To understand the molecular basis of this acceptor promiscuity, we solved the crystal structure of A. fumigatus Crh5 (AfCrh5) in complex with a chitooligosaccharide at the resolution of 2.8 Å, revealing an extensive elongated binding cleft for the donor (−4 to −1) substrate and a short acceptor (+1 to +2) binding site. Together with mutagenesis, the structure suggests a “hydrolysis product assisted” molecular mechanism favouring transglycosylation over hydrolysis.

Allostery in Coagulation Factor VIIa Revealed by Ensemble Refinement of Crystallographic Structures

A critical step in injury-induced initiation of blood coagulation is the formation of the complex between the trypsin-like protease coagulation factor VIIa (FVIIa) and its cofactor tissue factor (TF), which converts FVIIa from an intrinsically poor enzyme to an active protease capable of activating zymogens of downstream coagulation proteases. Unlike its constitutively active ancestor trypsin, FVIIa is allosterically activated (by TF). Here, ensemble refinement of crystallographic structures, which uses multiple copies of the entire structure as a means of representing structural flexibility, is applied to explore the impacts of inhibitor binding to trypsin and FVIIa, as well as cofactor binding to FVIIa. To assess the conformational flexibility and its role in allosteric pathways in these proteases, main-chain hydrogen bond networks are analyzed by calculating the hydrogen-bond propensity. Mapping pairwise propensity differences between relevant structures shows that binding of the inhibitor benzamidine to trypsin has a minor influence on the protease flexibility. For FVIIa, in contrast, the protease domain is “locked” into the catalytically competent trypsin-like configuration upon benzamidine binding as indicated by the stabilization of key structural features: the nonprime binding cleft and the oxyanion hole are stabilized, and the effect propagates from the active site region to the calcium-binding site and to the vicinity of the disulphide bridge connecting with the light chain. TF binding to FVIIa furthermore results in stabilization of the 170 loop, which in turn propagates an allosteric signal from the TF-binding region to the active site. Analyses of disulphide bridge energy and flexibility reflect the striking stability difference between the unregulated enzyme and the allosterically activated form after inhibitor or cofactor binding. The ensemble refinement analyses show directly, for the first time to our knowledge, whole-domain structural footprints of TF-induced allosteric networks present in x-ray crystallographic structures of FVIIa, which previously only have been hypothesized or indirectly inferred.

Hydration Modulates G‐Protein‐Coupled Receptor Signaling

Rhodopsin is the G‐protein‐coupled receptor (GPCR) responsible for scotopic vision in the retina. Up to this point, the role of water in the activation of GPCRs has remained largely unknown. Recently, however, nanosecond molecular dynamics simulations have revealed an influx of bulk water into rhodopsin during activation [1]. Utilizing rhodopsin as a model GPCR, we tested the hypothesis that rhodopsin activation is hydration mediated using osmotic stress techniques. We subjected rhodopsin within its native lipid membranes to varying osmotic pressures induced by different‐sized polyethylene glycol polymers. UV‐Visible spectroscopy of the photoactivated rhodopsin system reveals the fraction of protein in the active metarhodopsin‐II (MII) conformation, the receptor state capable of activating the G‐protein. We discovered high‐molecular weight osmolytes uniformly favored the closed, inactive metarhodopsin‐I conformation by dehydration of the protein interior. By contrast, small osmolytes penetrated into the transducin binding cleft and stabilized the active MII conformation until a quantifiable saturation point. A universal osmotic response occurred in the limit of increasing osmolyte size and maximal polymer exclusion from rhodopsin. By measuring the thermodynamic dependence of the metarhodopsin equilibrium on osmotic pressure, we determined that rhodopsin activation is coupled to a bulk influx of 80–100 water molecules into the protein core with a substantial increase in compressibility. We propose a new model for the functional role of water in GPCR signal transduction, in which a wet‐dry cycling mechanism amplifies the activation of G‐proteins. Our results necessitate a new understanding of GPCR activation, in which the influx of water plays a critical role in establishing the active receptor conformation.Support or Funding InformationThis work was supported by the NIH (EY026041 and EY012049) and the NSF (MCB 1817862).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Recruiting monomer for dimer formation: resolving the antagonistic mechanisms of novel immune check point inhibitors against Programmed Death Ligand-1 in cancer immunotherapy

ABSTRACTThe design of small molecule antagonists against Programmed Death Ligand-1 (PD-L1) has been the recent highlight of the immune checkpoint blockade therapy. This interventive approach has been potentiated by the development of BMS compounds; BMS-1001 and BMS-1166, which exert their therapeutic activities by inducing dimerisation of PD-L1; a molecular mechanism that has remained unclear. For the first time, we resolve the dynamical events that underlie the antagonistic mechanisms of BMS-1001 and BMS-1166 when bound to PD-L1 using an all-atom molecular dynamics (MD) simulations approach and free binding energy Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) calculations. Time-scale dynamical findings revealed that upon binding a PD-L1 monomer, the BMS-compounds gradually facilitated the ‘inbound’ motion of another PD-L1 monomer in the same conformational phase space up till dimer formation. Moreover, the non-liganded PD-L1 monomer exhibited the highest structural flexibility and atomistic motions relative to the BMS-liganded monomer as revealed by post-MD trajectory analyses using root mean square deviation (RMSD) and root mean square fluctuations (RMSF) parameters. Trajectory investigations into ligand motions also revealed that the BMS compounds exhibited mechanistic transitions from the monomeric binding site (monomer A) where they were initially bound, to the second monomeric site (monomer B) where they were strongly bound, followed by eventual high-affinity interactions at the tunnel-like binding cleft formed upon the dimerisation of both PD-L1 monomers. These findings present a model that describes the mechanism by which the BMS compounds induce PD-L1 dimerisation and could further enhance the design of highly selective and novel monomeric recruiters of PD-L1 in cancer immunotherapy.

Structure and dynamics of Trichoderma harzianum Cel7B suggest molecular architecture adaptations required for a wide spectrum of activities on plant cell wall polysaccharides

Cellulases from glycoside hydrolase family 7 (GH7) play crucial roles in plant lignocellulose deconstruction by fungi, but structural information available for GH7 fungal endoglucanases is limited when compared to the number of known sequences in the family. Here, we report the X-ray structure of the glycosylated catalytic domain (CD) of Trichoderma harzianum endoglucanase, ThCel7B, solved and refined at 2.9 Å resolution. Additionally, our extensive molecular dynamics simulations of this enzyme in complex with a variety of oligosaccharides provide a better understanding of its promiscuous hydrolytic activities on plant cell wall polysaccharides. The simulations demonstrate the importance of the hydrogen bond between substrate O2 hydroxyl in the subsite −1 and a side chain of catalytic Glu196 which renders ThCel7B capable to catalytically cleave cello and xylooligosaccharides, but not mannooligosaccharides. Moreover, detailed structural analyses and MD simulations revealed an additional binding pocket, suitable for accommodation of oligosaccharide decorations and/or substrates with mixed glycoside bonds that abuts onto the binding cleft close to subsite +2.

ATXR5/6 Forms Alternative Protein Complexes with PCNA and the Nucleosome Core Particle

The proliferating cell nuclear antigen (PCNA) is a sliding clamp associated with DNA polymerases and serves as a binding platform for the recruitment of regulatory proteins linked to DNA damage repair, cell cycle regulation, and epigenetic signaling. The histone H3 lysine-27 (H3K27) mono-methyltransferase Arabidopsis trithorax-related protein 5/6 (ATXR5/6) associates with PCNA, and this interaction has been proposed to act as a key determinant controlling the reestablishment of H3K27 mono-methylation following replication. In this study, we provide biochemical evidence showing that PCNA inhibits ATXR6 enzymatic activity. The structure of the ATXR6 PCNA-interacting peptide (PIP) in complex with PCNA indicates that a trio of hydrophobic residues contributes to the binding of the enzyme to the sliding clamp. Finally, despite the presence of three PIP binding clefts, only two molecules of ATXR6 bind to PCNA likely enabling the recruitment of a third protein to the sliding clamp. Collectively, these results rule out the model wherein PCNA-bound ATXR6 actively reestablishes H3K27 mono-methylation following DNA replication and provides insights into the role of ATXR6 PIP motif in its interaction with PCNA.

Structure model of ferrochelatase from Salmonella Typhi elucidating metalation mechanism.

A homology model of ferrochelatase (HemH), the heme biosynthesis terminal step enzyme from Salmonella Typhi was generated to understand the mechanism of metal insertion into protoporphyrin IX for heme biosynthesis. The overall fold of membrane associated ferrochelatase (StFc) from S. Typhi is similar to human and Yeast ferrochelatase than Bacillus subtilis, and Bacillus anthracis. An insertion of 16 amino acid residues in helical switch having hydrophobic patch proposed to interact with membrane lipids and in opening and closing of heme binding cleft. The sequence analysis and the docking study revealed that the protoporphyrin binding site in StFc has a crucial replacement of Tyr/Met to Leu13 unique in comparison to other known structures, where Tyr13 observed in B. subtilis/B. anthracis while Met76 in human/yeast play important role in holding protoporphyrin in optimal orientation for metalation. A sitting-a-top (SAT) complex mechanism for metalation is proposed with His194 and Glu264 lie at the bottom and Leu13 on the top of the porphyrin ring. In addition, an entry and exit mechanism is also proposed for protoporphyrin binding into cavity by opening and closing of helical switch using molecular dynamics simulation studies of Apo and heme complexed model structure of S. Typhi HemH.

Aprotinin interacts with substrate-binding site of human dipeptidyl peptidase III

Human dipeptidyl peptidase III (hDPP III) is a zinc-exopeptidase of the family M49 involved in final steps of intracellular protein degradation and in cytoprotective pathway Keap1-Nrf2. Biochemical and structural properties of this enzyme have been extensively investigated, but the knowledge on its contacts with other proteins is scarce. Previously, polypeptide aprotinin was shown to be a competitive inhibitor of hDPP III hydrolytic activity. In this study, aprotinin was first investigated as a potential substrate of hDPP III, but no degradation products were demonstrated by MALDI-TOF mass spectrometry. Subsequently, molecular details of the protein–protein interaction between aprotinin and hDPP III were studied by molecular modeling. Docking and long molecular dynamics (MD) simulations have shown that aprotinin interacts by its canonical binding epitope with the substrate binding cleft of hDPP III. Thereby, free N-terminus of aprotinin is distant from the active-site zinc. Enzyme-inhibitor complex is stabilized by intermolecular hydrogen bonding network, electrostatic and hydrophobic interactions which mostly involve constituent amino acid residues of the hDPP III substrate binding subsites S1, S1', S2, S2' and S3'. This is the first study that gives insight into aprotinin binding to a metallopeptidase.Communicated by Ramaswamy H. Sarma

A novel bifunctional xylanase/cellulase TcXyn10A from Thermoascus crustaceus JCM12803

Efficient utilization of cellulose and xylan is of importance in the bioethanol industry. In this study, a novel bifunctional xylanase/cellulase gene, Tcxyn10a, was cloned from Thermoascus crustaceus JCM12803, and the gene product was successfully overexpressed in Pichia pastoris GS115. The recombinant protein was then purified and characterized. The pH and temperature optima of TcXyn10A were determined to be 5.0 and 65-70 °C, respectively. The enzyme retained stable under acid to alkaline conditions (pH 3.0-11.0) or after 1-h treatment at 60 °C. The specific activities of TcXyn10A towards beechwood xylan, wheat arabinoxylan, sodium carboxymethyl cellulose and lichenan were (1 480±26) U/mg, (2 055±28) U/mg, (7.4±0.2) U/mg and (10.9±0.4) U/mg, respectively. Homologous modeling and molecular docking analyses indicated that the bifunctional TcXyn10A has a single catalytic domain, in which the substrate xylan and cellulose shared the same binding cleft. This study provides a valuable material for the study of structure and function relationship of bifunctional enzymes.

Antigenic Peptide Prediction From E6 and E7 Oncoproteins of HPV Types 16 and 18 for Therapeutic Vaccine Design Using Immunoinformatics and MD Simulation Analysis.

Human papillomavirus (HPV) induced cervical cancer is the second most common cause of death, after breast cancer, in females. Three prophylactic vaccines by Merck Sharp & Dohme (MSD) and GlaxoSmithKline (GSK) have been confirmed to prevent high-risk HPV strains but these vaccines have been shown to be effective only in girls who have not been exposed to HPV previously. The constitutively expressed HPV oncoproteins E6 and E7 are usually used as target antigens for HPV therapeutic vaccines. These early (E) proteins are involved, for example, in maintaining the malignant phenotype of the cells. In this study, we predicted antigenic peptides of HPV types 16 and 18, encoded by E6 and E7 genes, using an immunoinformatics approach. To further evaluate the immunogenic potential of the predicted peptides, we studied their ability to bind to class I major histocompatibility complex (MHC-I) molecules in a computational docking study that was supported by molecular dynamics (MD) simulations and estimation of the free energies of binding of the peptides at the MHC-I binding cleft. Some of the predicted peptides exhibited comparable binding free energies and/or pattern of binding to experimentally verified MHC-I-binding epitopes that we used as references in MD simulations. Such peptides with good predicted affinity may serve as candidate epitopes for the development of therapeutic HPV peptide vaccines.

Mechanistic basis of vitamin B12 and cobinamide salvaging by the Vibrio species

Biosynthesis of vitamin B12, which occurs through salvaging pathway or de novo synthesis, is essential for the survival and growth of bacteria. While the mechanism is known for many bacteria, it is elusive yet for diarrhea causing pathogenic bacteria Vibrio cholerae or the other Vibrio species. Sequence analysis using genome databases delineated that majority of the Vibrio species including V. cholerae contain genes required for salvaging cobalamin/cobinamide in aerobic pathway while lack the genes required for de novo synthesis of B12. Fluorescence quenching study showed that VcBtuF, the PBP of putative ABC transporter BtuF-CD of V. cholerae O395 binds cyanocobalamin and dicyanocobinamide with micromolar dissociation constants (Kd). Productive internalization of these nutrients has been established through growth assay. The crystal structure of cyanocobalamin bound VcBtuF has shown that although interactions between cyanocobalamin and VcBtuF are largely similar to E. coli BtuF, VcBtuF possesses a wider binding pocket. MD simulations indicated that in contrast to EcBtuF that executes ‘open-close’ movement, inter-lobe twisting is prevalent in VcBtuF. Although H70, located at the entrance of the substrate binding cleft of VcBtuF, executes swinging motion, it cannot act as ‘closed gate’ to retain cyanocobalamin or cobinamide in the pocket like corresponding residue W66 of EcBtuF. Rather, VcBtuF shows a distinctive phenomenon of heme binding with comparable affinity to B12. Soret shift of heme upon binding with VcBtuF pointed towards involvement of H70 in heme recognition. This may lead to a restricted B12 or cobinamide binding during abundance of heme in the periplasmic space.

Structural Basis of Membrane Protein Chaperoning through the Mitochondrial Intermembrane Space

SummaryThe exchange of metabolites between the mitochondrial matrix and the cytosol depends on β-barrel channels in the outer membrane and α-helical carrier proteins in the inner membrane. The essential translocase of the inner membrane (TIM) chaperones escort these proteins through the intermembrane space, but the structural and mechanistic details remain elusive. We have used an integrated structural biology approach to reveal the functional principle of TIM chaperones. Multiple clamp-like binding sites hold the mitochondrial membrane proteins in a translocation-competent elongated form, thus mimicking characteristics of co-translational membrane insertion. The bound preprotein undergoes conformational dynamics within the chaperone binding clefts, pointing to a multitude of dynamic local binding events. Mutations in these binding sites cause cell death or growth defects associated with impairment of carrier and β-barrel protein biogenesis. Our work reveals how a single mitochondrial “transfer-chaperone” system is able to guide α-helical and β-barrel membrane proteins in a “nascent chain-like” conformation through a ribosome-free compartment.

Conserved epitopes in variants of amastin protein of Trypanosoma cruzi for vaccine design: A bioinformatics approach

Chagas disease caused by protozoan parasite Trypanosoma cruzi is endemic disease in South and Central American countries but due to migrating human populations it has shown emergence in Europe, North America and Australia. With only two drugs, benznidazole and nifurtimox for its treatment there is need for newer therapies. In the current study, we have tried to analyse the potential of amastin, a major surface protein as a vaccine target using bioinformatics tools. Using 282 variants of this protein available in NCBI protein database we have found out five conserved potential Tc cell and two TH cell epitopes. These epitopes are conserved in more than 90% of the cohort of sequences used in the study. The epitopes showed binding to the peptide binding cleft of HLA-A02 and HLA-DR molecules. With coverage of pan world populations and being non-toxic and non-allergic these epitopes could be used for future vaccine applications.