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Comparative protein modeling by satisfaction of spatial restraints
Abstract
Approximately one third of known protein sequences are related to at least one known protein structure. As a result, an order of magnitude more sequences can be modeled by comparative modeling than there are experimentally determined protein structures. A large fraction of these models has an accuracy approaching that of a low resolution X-ray structure or a medium resolution nuclear magnetic resonance structure. The number of applications where homology modeling has been proven useful is growing rapidly.
... Field realistic pollen and nectar contamination can be on the order of a few μg per kg [1-3, 5, 6]. In honeybees, the acute LD 50 of imidacloprid varies between 5 and 500 ng/bee, probably as a result of detoxification capacity [12], and the acute oral LD 50 in bumblebees was determined to be 40 and 20 ng per bumblebee after 24 and 72 h of exposure, respectively [13]. Recent studies have indicated that imidacloprid causes different adverse effects on pollinators, with delayed and time-cumulative toxicity of imidacloprid observed in honeybees [14,15]. ...
... Then, we aligned the bumblebee SCAP to the 3JD8 structure using ClustalX 2.0 [49]. The alignment was then used as an input for Modeller 9.19 software [50] for 3D structure prediction. The validity of the resulting models was checked using PROCHECK software (G-score) [51], and the model with the ...
Determining the side effects of pesticides on pollinators is an important topic due to the increasing loss of pollinators. We aimed to determine the effects of chronic sublethal exposure of the neonicotinoid pesticide imidacloprid on the bumblebee Bombus terrestris under laboratory conditions. The analytical standard of imidacloprid in sugar solution was used for the treatment. Verification of pesticides using UHPLC-QqQ-MS/MS in the experimental bumblebees showed the presence of only two compounds, imidacloprid and imidacloprid-olefin, which were found in quantities of 0.57±0.22 and 1.95±0.43 ng/g, respectively. Thus, the level of the dangerous metabolite imidacloprid-olefin was 3.4-fold higher than that of imidacloprid. Label-free nanoLC-MS/MS quantitative proteomics of bumblebee heads enabled quantitative comparison of 2,883 proteins, and 206 proteins were significantly influenced by the imidacloprid treatment. The next analysis revealed that the highly downregulated markers are members of the terpenoid backbone biosynthesis pathway (KEGG: bter00900) and that imidacloprid treatment suppressed the entire mevalonate pathway, fatty acid synthesis and associated markers. The proteomics results indicate that the consequences of imidacloprid treatment are complex, and the marker changes are associated with metabolic and neurological diseases and olfaction disruption. This study provides important markers and can help to explain the widely held assumptions from biological observations.
Significance:
The major finding is that all markers of the mevalonate pathway were substantially downregulated due to the chronic imidacloprid exposure. The disbalance of mevalonate pathway has many important consequences. We suggest the mechanism associated with the novel toxicogenic effect of imidacloprid. The results are helpful to explain that imidacloprid impairs the cognitive functions and possesses the delayed and time cumulative effect.
... Figure 9. Schematic representation of the NanoBiT system. [38] δOR-CXCR4 FRET [43] D2R −SSTR5 FRET [67] δOR−CCR5 nd [43] D1R/D2R −H3 BRET [68] δOR−κOR Co-IP [44] D2R-OTR nd [69], [70] δOR−β2 Co-IP [45] D2R-GSHR1a nd [71] μOR−α2A FRET [46] D1R-GSHR1a Co-IP [72], [73] μOR-GRPR Co-IP [ ...
... The four-residue missing segment of intracellular loop 2 (ICL2) was ab initio modeled with MODELLER v9.18. [71] Co-crystallized risperidone and endolysin fusion protein were removed from the D2R structure. Energy minimization was then performed on added residues/loops and any clashing atoms in the AMBER14SB force-field. ...
... The cholesterol-StarD4 complex: The structure of apo-StarD4 was derived from the X-ray structure (PDB ID: 1jss) where residues 24 to 222 are resolved (8). To complete the structure, the missing residues K223 and A224 are added using Modeler 9.23 software, resulting in the conformation of StarD4 segment spanning residues 22-224, with acetylation on the N-terminus and carboxylation on the C-terminus (43). The initial structure of cholesterol bound (holo) StarD4 was obtained by docking the cholesterol into the hydrophobic pocket of apo-StarD4 using the Schrodinger Induced Fit Docking protocol (44)(45)(46). ...
Intracellular cholesterol trafficking, vital for cell function, is orchestrated in part by proteins with steroidogenic acute regulator-related lipid transfer (START) domains, such as StarD4 which is a sterol-specific transfer protein that contributes ~ 33% of non-vesicular sterol transport between plasma membrane (PM), the primary cholesterol reservoir, and the endoplasmic reticulum (ER). Comprising a single soluble START domain, StarD4 is known to target both sterol donor-, and acceptor-membranes, through interactions with anionic lipids. Experiments have illuminated the kinetics of this sterol transfer and shown it to be modulated by specific phosphatidylinositol phosphates (PIPs) on the target membrane. The distinct subtype distribution of PIPs in the membranes of cellular organelles serves as a guide directing StarD4 to particular cell components. To discover the molecular mechanism of membrane targeting by the recognition of the PIP2 subtype in the membrane, and how this affects the direction and kinetics of CHL transport (uptake vs release), we used molecular dynamics (MD) to simulate the processes. Analysis of the MD trajectories with machine learning and information theory methods revealed how StarD4 embeds in membrane containing different anionic lipids (PI(4,5)P2, PI(3,5)P2, and PS), how it recognizes the anionic lipid content by the different modes in which they bind to the protein, and how the protein responds to this recognition by adopting different orientations on the membrane and undergoing specific conformational changes. We identified the allosteric channels underlying these complex dynamics rearrangements by connecting the various PIP2-subtype-specific conformational states to different CHL binding modes in the pocket, which then relate to the dynamics of the gates that allow either CHL uptake into apo-StarD4, or its release from holo-StarD4. This reveals the crucial role of PIP2 subtypes in shaping functional StarD4 motifs responsible for organelle selectivity of the cholesterol trafficking, providing fundamental insights into cellular cholesterol regulation.
... The three-dimensional (3D) structures of six members of the P28 family were modelled in a stepwise procedure, using MODELLER version 9v1. This software implements homology modelling of proteins by satisfaction of spatial restraints [22]; 100 models for each P28 protein were generated with the help of Modeller. Bond and angle values are taken from CHARMM-22 force field. ...
The P28 family of proteins are 28 kDa proteins expressed on the surface of sexual stages-zygote, ookinete and young oocyst stages-of Plasmodium species when the parasite resides inside the mosquito midgut. Together with P25 proteins, P28 proteins protect the parasite from the harsh proteolytic environment prevailing inside the mosquito midgut. Vaccines against these proteins induce antibodies in vertebrate hosts that are capable of inhibiting parasite development in the mosquito midgut, thus preventing transmission of the parasite from the mosquito to another human host. These transmission-blocking vaccines are helpful in reducing the burden caused by malaria, which affects 300-600 million, and kills 1-3 million, people annually. The purpose of this study was to structurally characterise six members of the P28 family of ookinete surface proteins with the help of homology modelling, to compare these proteins in terms of transmission blocking and host parasite interactions, and to analyse phylogenetic relationships within the P28 family and with the P25 family. Our results indicate that all the members of the P28 family studied have four EGF domains arranged in triangular fashion with a very big C loop present in EGF domain IV, which could serve as a diagnostic feature of the P28 family as this loop is absent in the P25 family of ookinete surface proteins. The models of the P28 family of ookinete surface proteins obtained may help in understanding the biology of the parasite inside the mosquito midgut, and in designing transmission blocking vaccines against malaria in the absence of experimentally determined structures of these important surface proteins.
... The sequences were aligned in UGENE (Unipro) using the T-coffee algorithm with standard settings (Okonechnikov et al., 2012). Based on the alignment and the 6DHN structure, the D. melanogaster Gdh structure was predicted by MODELLER using UCSF Chimera (Sali, 1995;Pettersen et al., 2004). The best model (with the zDOPE score 1.12) was selected for further examination. ...
Glutamate dehydrogenases are enzymes that take part in both amino acid and energy metabolism. Their role is clear in many biological processes, from neuronal function to cancer development. The putative testis-specific Drosophila glutamate dehydrogenase, Bb8, is required for male fertility and the development of mitochondrial derivatives in spermatids. Testis-specific genes are less conserved and could gain new functions, thus raising a question whether Bb8 has retained its original enzymatic activity. We show that while Bb8 displays glutamate dehydrogenase activity, there are significant functional differences between the housekeeping Gdh and the testis-specific Bb8. Both human GLUD1 and GLUD2 can rescue the bb8 ms mutant phenotype, with superior performance by GLUD2. We also tested the role of three conserved amino acids observed in both Bb8 and GLUD2 in Gdh mutants, which showed their importance in the glutamate dehydrogenase function. The findings of our study indicate that Drosophila Bb8 and human GLUD2 could be novel examples of convergent molecular evolution. Furthermore, we investigated the importance of glutamate levels in mitochondrial homeostasis during spermatogenesis by ectopic expression of the mitochondrial glutamate transporter Aralar1, which caused mitochondrial abnormalities in fly spermatids. The data presented in our study offer evidence supporting the significant involvement of glutamate metabolism in sperm development.
... To avoid simulation artefacts due to missing residues, structures were rebuilt using Modeller 76 .Then 600 ns MD simulations were calculated using the GROMACS environment 50,77,78 . During the simulations, the temperature was controlled after the equilibration steps to be at 300 K and the density close to that of liquid water (1037 kg/m 3 ). ...
Newly synthesized peroxisomal proteins are recognized in the cytosol by the cycling receptor PEX5 and directed to a docking complex comprising PEX14 and PEX13 at the peroxisomal membrane. After cargo translocation, the unloaded PEX5 is recycled in an ATP-dependent manner. Receptor docking involves the WxxxF-motifs in the N-terminal domain (NTD) of PEX5 that are recognized by the N-terminal domain of PEX14. Here, we combine biochemical methods and NMR spectroscopy to identify a novel binding interface between human PEX5 and PEX14. The interaction involves the PEX5 C-terminal cargo-binding TPR domain and a conserved IPSWQI peptide motif in the C-terminal intrinsically disordered region of PEX14. The three-dimensional structure of the PEX14 IPSWQI peptide bound the PEX5 TPR domain, shows the PEX14 interaction is non-overlapping with PTS1 binding to the TPR domain. Notably, PEX14 IPSWQI motif binding to a hinge region in the TPR domain shows a more open supercoil of the TPR fold that resembles the apo conformation in the absence of PTS1 peptide. Mutation of binding site residues in PEX5 or PEX14 leads to a partial protein import defect and decrease of the steady-state-concentration of PEX5. This resembles the mutant phenotype of cells affected in receptor recycling, suggesting a role in this process.
... The structure of mouse apo-StarD4 was taken from the X-ray crystal structure (PDB ID: 1jss), which includes residues 24 to 222 of the protein . Lys223 and Ala224 were introduced using Modeller 9.23 software (Sali, 1995), resulting in the conformation of mouse StarD4 22-224 segment with N-terminus acetylation. StarD4 complexes with cholesterol (CHL) were obtained by docking the ligand in the hydrophobic pocket of StarD4 using Schrodinger's Induced Fit Docking protocol (Sherman et al., 2006a;Sherman et al., 2006b). ...
Complex mechanisms regulate the cellular distribution of cholesterol, a critical component of eukaryote membranes involved in regulation of membrane protein functions directly and through the physiochemical properties of membranes. StarD4, a member of the steroidogenic acute regulator-related lipid-transfer (StART) domain (StARD)-containing protein family, is a highly efficient sterol-specific transfer protein involved in cholesterol homeostasis. Its mechanism of cargo loading and release remains unknown despite recent insights into the key role of phosphatidylinositol phosphates in modulating its interactions with target membranes. We have used large-scale atomistic Molecular dynamics (MD) simulations to study how the dynamics of cholesterol bound to the StarD4 protein can affect interaction with target membranes, and cargo delivery. We identify the two major cholesterol (CHL) binding modes in the hydrophobic pocket of StarD4, one near S136&S147 (the Ser-mode), and another closer to the putative release gate located near W171, R92&Y117 (the Trp-mode). We show that conformational changes of StarD4 associated directly with the transition between these binding modes facilitate the opening of the gate. To understand the dynamics of this connection we apply a machine-learning algorithm for the detection of rare events in MD trajectories (RED), which reveals the structural motifs involved in the opening of a front gate and a back corridor in the StarD4 structure occurring together with the spontaneous transition of CHL from the Ser-mode of binding to the Trp-mode. Further analysis of MD trajectory data with the information-theory based NbIT method reveals the allosteric network connecting the CHL binding site to the functionally important structural components of the gate and corridor. Mutations of residues in the allosteric network are shown to affect the performance of the allosteric connection. These findings outline an allosteric mechanism which prepares the CHL-bound StarD4 to release and deliver the cargo when it is bound to the target membrane.
... software to construct the 3D structure of PxCSP1. 34 After querying the PDB95 database with the amino acid sequence of PxCSP1, crystal structure of the CSP from Mamestra brassicae (CSPMbraA6) in complex with 12-bromo-dodecanol (BrC12OH) (PDB ID: 1N8V, Chain A, Resolution = 1.39 Å) was selected as the template for homology modeling, 35 with sequence identity and R factor being 58% and 0.181, respectively. Then 100 3D models of PxCSP1 were produced and optimized by the automodel and loopmodel in Modeller9.10 ...
BACKGROUND
Insecticide resistance continuously poses a threat to agricultural production. Chemosensory protein‐mediated resistance is a new mechanism of insecticide resistance discovered in recent years. In‐depth research on resistance mediated by chemosensory proteins (CSPs) provides new insight into aid insecticide resistance management.
RESULTS
Chemosensory protein 1 in Plutella xylostella (PxCSP1) was overexpressed in the two indoxacarb‐resistant field populations and PxCSP1 has a high affinity with indoxacarb. PxCSP1 was upregulated when exposed to indoxacarb and the knockdown of this gene elevated sensitivity to indoxacarb, which demonstrate that PxCSP1 is involved in the indoxacarb resistance. Considering that CSPs may confer resistance in insects via binding or sequestering, we explored the binding mechanism of indoxacarb in PxCSP1‐mediated resistance. Using molecular dynamics simulations and site‐directed mutation, we found that indoxacarb forms a solid complex with PxCSP1 mainly through van der Waals interactions and electrostatic interactions. Between these, the electrostatic interaction provided by the Lys100 side chain in PxCSP1, and especially the hydrogen bonding between the NZ atom and the O of the carbamoyl carbonyl group of indoxacarb, are the key factors for the high affinity of PxCSP1 to indoxacarb.
CONCLUSIONS
The overexpression of PxCPS1 and its high affinity to indoxacarb is partially responsible for indoxacarb resistance in P. xylostella. Modification of indoxacarb's carbamoyl group has the potential to alleviate indoxacarb resistance in P. xylostella. These findings will contribute to solving chemosensory protein‐mediated indoxacarb resistance and provide a better understanding of the insecticide resistance mechanism. © 2023 Society of Chemical Industry.
... Their PDB codes are 3E9S, 3MJ5, 4M0W, 4OVZ, 4OW0, 5E6J and 5Y3E. Modeller (Sali, 1995) was used to perform homology modeling with the default parameters. Thirteen resulting models with the lowest RMSD from their templates were selected for further analysis. ...
The global COVID-19 coronavirus pandemic has infected over 109 million people, leading to over 2 million deaths up to date and still lacking of effective drugs for patient treatment. Here, we screened about 1.8 million small molecules against the main protease (M pro) and papain like protease (PL pro), two major proteases in severe acute respiratory syndrome-coronavirus 2 genome, and identified 1,851M pro inhibitors and 205 PL pro inhibitors with low nmol/L activity of the best hits. Among these inhibitors, eight small molecules showed dual inhibition effects on both M pro and PL pro, exhibiting potential as better candidates for COVID-19 treatment. The best inhibitors of each protease were tested in antiviral assay, with over 40% of M pro inhibitors and over 20% of PL pro inhibitors showing high potency in viral inhibition with low cytotoxicity. The X-ray crystal structure of SARS-CoV-2 M pro in complex with its potent inhibitor 4a was determined at 1.8 Å resolution. Together with docking assays, our results provide a comprehensive resource for future research on anti-SARS-CoV-2 drug development.
... The final version may differ from this version. and modeling was performed using Modeller 9.19 (Sali et al., 1995). Ten models were generated for each interface, of which the one with the lowest energy, calculated by Modeller as DOPE score (Shen and Sali, 2006), and the smallest percentage of amino acids in the disallowed region of the Ramachandran plot, obtained with Procheck (Laskowski et al., 1993), was selected for docking studies. ...
Alzheimer's disease is a multifactorial neurodegenerative disorder. Since cholinergic deficit is a major factor in this disease, two molecular targets for its treatment are the acetylcholinesterase (AChE) and the nicotinic acetylcholine receptors (nAChRs). Given that caffeine is a natural compound that behaves as an AChE inhibitor and as a partial agonist of nAChRs, the aim of this work was to synthetize more potent bifunctional caffeine analogs that modulate these two molecular targets. To this end, a theophylline structure was connected to a pyrrolidine structure through a methylene chain of different lengths (3 to 7 carbon atoms) to give compounds 7-11 All caffeine derivatives inhibited the AChE, of which compound 11 showed the strongest effect. Electrophysiological studies showed that all compounds behave as agonists of the muscle and the neuronal α7 nAChR with greater potency than caffeine. To explore whether the different analogs could affect the nAChR conformational state, the nAChR conformational-sensitive probe crystal violet (CrV) was used. Compounds 9 and 10 conduced the nAChR to a different conformational state comparable with a control nAChR desensitized state. Finally, molecular docking experiments showed that all derivatives interacted with both the catalytic and anionic sites of AChE and with the orthosteric binding site of the nAChR. Thus, the new synthetized compounds can inhibit the AChE and activate muscle and α7 nAChRs with greater potency than caffeine, which suggests that they could be useful leaders for the development of new therapies for the treatment of different neurologic diseases. SIGNIFICANCE STATEMENT: In this work we synthetized caffeine derivatives which can inhibit acetylcholinesterase and activate both muscle and α7 nicotinic acetylcholine receptors (nAChRs) with higher potency than caffeine. These analogs can be divided into two groups: a non-desensitizing and a desensitizing nAChR group. From the nAChR non-desensitizing group, we propose compound 11 as the most interesting analog for further studies since it inhibits acetylcholinesterase with the highest potency and activates the nAChRs in the picomolar range without inducing receptor desensitization.
... MODELLER was used for the creation of ten 5-LOX models. Further optimization of these models was performed via a conjugate gradient minimization scheme, followed by a restrained simulated annealing MD simulation (127). Based on the obtained results, a representative 5-LOX model was selected. ...
Drug discovery and development is a very challenging, expensive and time-consuming process. Impressive technological advances in computer sciences and molecular biology have made it possible to use computer-aided drug design (CADD) methods in various stages of the drug discovery and development pipeline. Nowadays, CADD presents an efficacious and indispensable tool, widely used in medicinal chemistry, to lead rational drug design and synthesis of novel compounds. In this article, an overview of commonly used CADD approaches from hit identification to lead optimization was presented. Moreover, different aspects of design of multitarget ligands for neuropsychiatric and anti-inflammatory diseases were summarized. Apparently, designing multi-target directed ligands for treatment of various complex diseases may offer better efficacy, and fewer side effects. Antipsychotics that act through aminergic G protein-coupled receptors (GPCRs), especially Dopamine D2 and serotonin 5-HT2A receptors, are the best option for treatment of various symptoms associated with neuropsychiatric disorders. Furthermore, multi-target directed cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) inhibitors are also a successful approach to aid the discovery of new anti-inflammatory drugs with fewer side effects. Overall, employing CADD approaches in the process of rational drug design provides a great opportunity for future development, allowing rapid identification of compounds with the optimal polypharmacological profile.
... The PDB structure of the wild-type and mutant MmpRibK enzymes were modelled using Modeller. [42] The stereochemical quality of the modelled structures was estimated using programs PROCHECK and ERRAT. [14,15] All structural analyses were done using molecular visualization software Visual Molecular Dynamics (VMD) and UCSF Chimera. ...
Flavins play a central role in metabolism as molecules that catalyze a wide range of redox reactions in living organisms. Several variations in flavin biosynthesis exist among the domains of life, and their analysis has revealed many new structural and mechanistic insights till date. The cytidine triphosphate (CTP)‐dependent riboflavin kinase in archaea is one such example. Unlike most kinases that use adenosine triphosphate, archaeal riboflavin kinases utilize CTP to phosphorylate riboflavin and produce flavin mononucleotide. In this study, we present the characterization of a new mesophilic archaeal CTP‐utilizing riboflavin kinase homologue from Methanococcus maripaludis (MmpRibK), which is linked closely in sequence to the previously characterized thermophilic Methanocaldococcus jannaschii homologue. We reconstitute the activity of MmpRibK, determine its kinetic parameters and molecular factors that contribute to its unique properties, and finally establish the residues that improve its thermostability using computation and a series of experiments. Our work advances the molecular understanding of flavin biosynthesis in archaea by the characterization of the first mesophilic CTP‐dependent riboflavin kinase. Finally, it validates the role of salt bridges and rigidifying amino acid residues in imparting thermostability to this unique structural fold that characterizes archaeal riboflavin kinase enzymes, with implications in enzyme engineering and biotechnological applications.
... First, we generated a multiple sequence alignment (MSA) using MAFFT [54] including the sequences of the hABCC6, hABCC1, and hABCC5 proteins from multiple organisms (Table S1), retrieved from UniProtKB [55]. Based on the alignment of hABCC6 and rAbcc6 sequences with bAbcc1, homology models of hABCC6 and rAbcc6 were generated with Modeller 9v15 [56], using the inward-and outward-facing cryoEM structures of bAbcc1 as templates [22,23]. For hABCC6 and rAbcc6, 20 models were generated for both the inward-and the outward-facing states, by applying a slow refinement protocol and 20 cycles of simulated annealing as in our previous work on hABCC7 (CFTR) [57]. ...
Inactivating mutations in ABCC6 underlie the rare hereditary mineralization disorder pseudoxanthoma elasticum. ABCC6 is an ATP-binding cassette (ABC) integral membrane protein that mediates the release of ATP from hepatocytes into the bloodstream. The released ATP is extracellularly converted into pyrophosphate, a key mineralization inhibitor. Although ABCC6 is firmly linked to cellular ATP release, the molecular details of ABCC6-mediated ATP release remain elusive. Most of the currently available data support the hypothesis that ABCC6 is an ATP-dependent ATP efflux pump, an un-precedented function for an ABC transporter. This hypothesis implies the presence of an ATP-binding site in the substrate-binding cavity of ABCC6. We performed an extensive mutagenesis study using a new homology model based on recently published structures of its close homolog, bovine Abcc1, to characterize the substrate-binding cavity of ABCC6. Leukotriene C4 (LTC4), is a high-affinity substrate of ABCC1. We mutagenized fourteen amino acid residues in the rat ortholog of ABCC6, rAbcc6, that corresponded to the residues in ABCC1 found in the LTC4 binding cavity. Our functional characterization revealed that most of the amino acids in rAbcc6 corresponding to those found in the LTC4 binding pocket in bovine Abcc1 are not critical for ATP efflux. We conclude that the putative ATP binding site in the substrate-binding cavity of ABCC6/rAbcc6 is distinct from the bovine Abcc1 LTC4-binding site.
... umich .edu/I-TASSE R/) were used [69][70][71] . The prediction outputs were imported into Chimera 1.14 (http://www. ...
Thermostability and substrate specificity of proteases are major factors in their industrial applications. rEla is a novel recombinant cysteine protease obtained from a thermophilic bacterium, Cohnella sp.A01 (PTCC No: 1921). Herein, we were interested in recombinant production and characterization of the enzyme and finding the novel features in comparison with other well-studied cysteine proteases. The bioinformatics analysis showed that rEla is allosteric cysteine protease from DJ-1/ThiJ/PfpI superfamily. The enzyme was heterologously expressed and characterized and the recombinant enzyme molecular mass was 19.38 kD which seems to be smaller than most of the cysteine proteases. rEla exhibited acceptable activity in broad pH and temperature ranges. The optimum activity was observed at 50℃ and pH 8 and the enzyme showed remarkable stability by keeping 50% of residual activity after 100 days storage at room temperature. The enzyme Km and Vmax values were 21.93 mM, 8 U/ml, respectively. To the best of our knowledge, in comparison with the other characterized cysteine proteases, rEla is the only reported cysteine protease with collagen specificity. The enzymes activity increases up to 1.4 times in the presence of calcium ion (2 mM) suggesting it as the enzyme’s co-factor. When exposed to surfactants including Tween20, Tween80, Triton X-100 and SDS (1% and 4% v/v) the enzyme activity surprisingly increased up to 5 times.
... The modeled zein structure was hydrotreated for subsequent molecular docking. The initial structure was processed with AutoDockTools 1.5.6 (Sali, 1996), preserving the original charge of the protein and generating a pdbqt file for docking. The structure of the ligand ZEN was obtained from NCBI, hydrogenated, and optimized using the MOPAC program (Morris et al., 2009). ...
Hidden mycotoxins have been reported to be “protected” by macromolecular substances to escape routine determination, but release to free mycotoxins under gastrointestinal conditions. Nowadays, the hidden zearalenone (ZEN) that binding with macromolecular zein has been found in maize. However, the binding mechanism of ZEN with zein in maize has not been clarified. In this study, the formation of ZEN-zein complex was investigated applying ultrafiltration, multi-spectroscopic and molecular modeling techniques. The steady-state and transient fluorescence analysis suggested the ZEN could interact with zein to form the complex driven by hydrophobic force and hydrogen bonds, which is in accordance with the molecular modeling studies. The conformational changes of zein induced by binding with ZEN were revealed by Fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD). Elucidating the binding mechanism between zein and ZEN could help the development of detecting hidden ZEN and guarantee the safety of maize products.
... For the missing loop regions in the S1 subunit, we either built the homology model on the basis of SARS-CoV S structure (PDB: 6CRW) (27) through the SWISS-MODEL webserver (56), or built the loop manually according to the density in COOT (57). For the FP region, we first built the homology model by the Modeller tool within Chimera by using the MERS-CoV S structure (PDB: 6NB3) as template (2,58,59) and then used Rosetta to refine this region against the density map (60). Eventually, we used phenix.real_space_refine ...
The recent outbreaks of SARS-CoV-2 pose a global health emergency. The SARS-CoV-2 trimeric spike (S) glyco-protein interacts with the human ACE2 receptor to mediate viral entry into host cells. We report the cryo-EM structures of a tightly closed SARS-CoV-2 S trimer with packed fusion peptide and an ACE2-bound S trimer at 2.7-and 3.8-Å resolution, respectively. Accompanying ACE2 binding to the up receptor-binding domain (RBD), the associated ACE2-RBD exhibits continuous swing motions. Notably, the SARS-CoV-2 S trimer appears much more sensitive to the ACE2 receptor than the SARS-CoV S trimer regarding receptor-triggered transformation from the closed prefusion state to the fusion-prone open state, potentially contributing to the superior infectivity of SARS-CoV-2. We defined the RBD T470-T478 loop and Y505 as viral determinants for specific recognition of SARS-CoV-2 RBD by ACE2. Our findings depict the mechanism of ACE2-induced S trimer conformational transitions from the ground prefusion state toward the postfusion state, facilitating development of anti-SARS-CoV-2 vaccines and therapeutics.
... Three-dimensional variability analysis was performed in SPARX (27). The HT-AM pipeline was carried out by implementing the ALLOSMOD (29,71) package of MODELLER (72,73) in a streamlined pipeline. All graph theory and network-based analysis was performed using Python (Python Software Foundation; https://www.python.org/) ...
Significance
The HIV-1 Env “glycan shield” masks the surface of the protein from immune recognition, yet intrinsic heterogeneity defies a typical structure–function description. Using an integrated approach of cryo-EM, computational modeling, and mass spectrometry, we visualized the glycan shield structure in a new light. Our approach facilitated development of cryo-EM analysis methods and allowed for validation of models against experiment. Comparison of Env expressed in different cell lines revealed how subtle differences in composition impact glycan shield structure and affect the accessibility of epitopes on the surface, providing insights for vaccine design. Finally, time-resolved cryo-EM experiments uncovered how highly connected glycan clusters help stabilize the prefusion trimer, suggesting the glycan shield may function beyond immune evasion.
... 18 αbungarotoxin is a neurotoxin that acts as a nAChR antagonist, directly competing with acetylcholine, 38 and has high sequence similarity with the Y674-R685 region of the S protein of SARS-CoV-2 ( Figure S1). Twenty models were generated for each complex, and the one with the lowest Modeller objective function 39 (Figures 2 and S6) was used as the starting point for MD simulations (see the Supporting Information for more details). Three replicates, each 300 ns long, were performed for each complex to investigate the peptide-receptor conformational behaviour and possible induced-fit effects. ...
Changeux et al. recently suggested that the SARS-CoV-2 spike (S) protein may interact with nicotinic acetylcholine receptors (nAChRs). Such interactions may be involved in pathology and infectivity. Here, we use molecular simulations of validated atomically detailed structures of nAChRs, and of the S protein, to investigate this "nicotinic hypothesis". We examine the binding of the Y674-R685 loop of the S protein to three nAChRs, namely the human α4β2 and α7 subtypes and the muscle-like αβγδ receptor from Tetronarce californica . Our results indicate that Y674-R685 has affinity for nAChRs and the region responsible for binding contains the PRRA motif, a four-residue insertion not found in other SARS-like coronaviruses. In particular, R682 has a key role in the stabilisation of the complexes as it forms interactions with loops A, B and C in the receptors binding pocket. The conformational behaviour of the bound Y674-R685 region is highly dependent on the receptor subtype, adopting extended conformations in the α4β2 and α7 complexes and more compact ones when bound to the muscle-like receptor. In the α4β2 and αβγδ complexes, the interaction of Y674-R685 with the receptors forces the loop C region to adopt an open conformation similar to other known nAChR antagonists. In contrast, in the α7 complex, Y674-R685 penetrates deeply into the binding pocket where it forms interactions with the residues lining the aromatic box, namely with TrpB, TyrC1 and TyrC2. Estimates of binding energy suggest that Y674-R685, forms stable complexes with all three nAChR subtypes, but has highest affinity for the muscle-type receptor. Analyses of the simulations of the full-length S protein show that the Y674-R685 region is accessible for binding, and suggest a potential binding orientation of the S protein with nAChRs.
... For the missing loop regions in S1 subunit, we either built the homology (Emsley and Cowtan, 2004). For the FP region, we first built the homology model by Modeller tool within Chimera by using MERS-CoV S structure (PDB: 6NB3) as template (Pettersen et al., 2004;Sali, 1995;Walls et al., 2019), then used Rosetta to refine this region against the density map (DiMaio et al., 2015). Eventually, we used phenix.real_space_refine ...
The recent outbreaks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its rapid international spread pose a global health emergency. The trimeric spike (S) glycoprotein interacts with its receptor human ACE2 to mediate viral entry into host-cells. Here we present cryo-EM structures of an uncharacterized tightly closed SARS-CoV-2 S-trimer and the ACE2-bound-S-trimer at 2.7-angstrom and 3.8-angstrom-resolution, respectively. The tightly closed S-trimer with inactivated fusion peptide may represent the ground prefusion state. ACE2 binding to the up receptor-binding domain (RBD) within S-trimer triggers continuous swing-motions of ACE2-RBD, resulting in conformational dynamics of S1 subunits. Noteworthy, SARS-CoV-2 S-trimer appears much more sensitive to ACE2-receptor than SARS-CoV S-trimer in terms of receptor-triggered transformation from the closed prefusion state to the fusion-prone open state, potentially contributing to the superior infectivity of SARS-CoV-2. We defined the RBD T470-T478 loop and residue Y505 as viral determinants for specific recognition of SARS-CoV-2 RBD by ACE2, and provided structural basis of the spike D614G-mutation induced enhanced infectivity. Our findings offer a thorough picture on the mechanism of ACE2-induced conformational transitions of S-trimer from ground prefusion state towards postfusion state, thereby providing important information for development of vaccines and therapeutics aimed to block receptor binding.
... While consistently successful in RNA-Puzzles, FARFAR modeling has involved problem-specific expert intuition, such as guesses of ligand-binding sites based on inspection of sequence alignments, as well as algorithmic extensions created ''on-the-fly'' to explore novel ideas inspired by the targets, including different approaches to model A-form helices or tertiary contacts (Cruz et al., 2012;Miao et al., 2015Miao et al., , 2017. As in early days of protein structure prediction (Sali, 1995;Simons et al., 1997), many of these steps have not been well documented, automated, or systematically benchmarked, so the performance and current limitations of FARFAR remain uncertain. Indeed, the original FARFAR study as well as subsequent studies (Das et al., 2010;Ding et al., 2008;Laing and Schlick, 2010;Popenda et al., 2012) suggested that FARFAR would have difficulty sampling native-like folds for RNA molecules larger than 12 nt. ...
Predicting RNA three-dimensional structures from sequence could accelerate understanding of the growing number of RNA molecules being discovered across biology. Rosetta's Fragment Assembly of RNA with Full-Atom Refinement (FARFAR) has shown promise in community-wide blind RNA-Puzzle trials, but lack of a systematic and automated benchmark has left unclear what limits FARFAR performance. Here, we benchmark FARFAR2, an algorithm integrating RNA-Puzzle-inspired innovations with updated fragment libraries and helix modeling. In 16 of 21 RNA-Puzzles revisited without experimental data or expert intervention, FARFAR2 recovers native-like structures more accurate than models submitted during the RNA-Puzzles trials. Remaining bottlenecks include conformational sampling for >80-nucleotide problems and scoring function limitations more generally. Supporting these conclusions, preregistered blind models for adenovirus VA-I RNA and five riboswitch complexes predicted native-like folds with 3- to 14 Å root-mean-square deviation accuracies. We present a FARFAR2 webserver and three large model archives (FARFAR2-Classics, FARFAR2-Motifs, and FARFAR2-Puzzles) to guide future applications and advances.
... Although previous work has shown that the C5 influenza-virus-binding peptide has a high affinity to hemagglutinin, we needed to confirm that C5 could still interact with hemagglutinin after being fused with the CsgA protein. To such ends, we first used MODELLER [22,23] to build the homology models of CsgA-C5 and Glide [24] to get the complex of the monomer CsgA-C5 and hemagglutinin (PDB ID: 1HGG). Molecular dynamics simulations of the interaction between a CsgA-C5 fusion monomer and hemagglutinin by GROMACS [25] indicated that these two proteins interact strongly: the bound complex structure was stable even after 800 ns (Figure 2a); the binding energy was calculated using the molecular mechanics/generalized born surface area (MM/GBSA) method, [26] and the ΔG bind value was about −62 ± 22 kcal mol −1 , which is similar to the binding energy between biotin analogous and avidin. ...
Waterborne viruses frequently cause disease outbreaks and existing strategies to remove such viral pathogens often involve harsh or energy‐consuming water treatment processes. Here, a simple, efficient, and environmentally friendly approach is reported to achieve highly selective disinfection of specific viruses with living engineered biofilm materials. As a proof‐of‐concept, Escherichia coli biofilm matrix protein CsgA was initially genetically fused with the influenza‐virus‐binding peptide (C5). The resultant engineered living biofilms could correspondingly capture virus particles directly from aqueous solutions, disinfecting samples to a level below the limit‐of‐detection for a qPCR‐based detection assay. By exploiting the surface‐adherence properties of biofilms, it is further shown that polypropylene filler materials colonized by the CsgA‐C5 biofilms can be utilized to disinfect river water samples with influenza titers as high as 1 × 10⁷ PFU L⁻¹. Additionally, a suicide gene circuit is designed and applied in the engineered strain that strictly limits the growth of bacterial, therefore providing a viable route to reduce potential risks confronted with the use of genetically modified organisms. The study thus illustrates that engineered biofilms can be harvested for the disinfection of pathogens from environmental water samples in a controlled manner and highlights the unique biology‐only properties of living substances for material applications.
... The constraint-based approach is based on distance geometry [105] and already used in homology modeling [106], where one uses a template protein with an as high as possible sequence similarity to the target protein to model the structure of the target protein. It can also be used for fitting structures into images got from cryo-EM [107], applied to structure determination either from distances between protons, yielded by NMR measurements [108] or from distances derived from SAXS profile data [109,110]. ...
The structure of proteins is essential for its function. The determination of protein structures is possible by experimental or predicted by computational methods, but also a combination of both approaches is possible. Here, first an overview about experimental structure determination methods with their pros and cons is given. Then we describe how mass spectrometry is useful for semi-experimental integrative protein structure determination. We review the methodology and describe software programs supporting such integrated protein structure prediction approaches, making use of distance constraints got from mass spectrometry cross-linking experiments
... 370 371 Structural analysis 372 Multiple sequence alignment was performed using Clustal Omega (Madeira et al., 2019). The substrate-bound 373 structure of a UbiA homolog from A. pernix (PDB: 4OD5) was displayed on Chimera (Pettersen et al., 2004) 374 and the C. elegans sequence was threaded by homology using Modeller (Sali, Blundell, 1993, Webb, Sali, 375 2016. 376 ...
Parasitic helminths use two benzoquinones as electron carriers in the electron transport chain. In aerobic environments they use ubiquinone (UQ) but in the anaerobic environment of the host, they require rhodoquinone (RQ) and greatly increase RQ levels. The switch to RQ synthesis is driven by substrate selection by the polyprenyltransferase COQ-2 but the mechanisms underlying this substrate choice are unknown. We found that helminths make two coq-2 isoforms, coq-2a and coq-2e, by alternative splicing. COQ-2a is homologous to COQ2 from other eukaryotes but the COQ-2e-specific exon is only found in species that make RQ and its inclusion changes the enzyme core. We show COQ-2e is required for RQ synthesis and for survival in cyanide in C. elegans. Crucially, we see a switch from COQ-2a to COQ-2e as parasites transition into anaerobic environments. We conclude that under anaerobic conditions helminths switch from UQ to RQ synthesis via alternative splicing of coq-2.
... Tupaia chinensis (XP_014444914.1), and Homo sapiens mutated (NX_Q92508-1) and used for sequence alignment using ClustalW [34,35]. The structural model of human Piezo1 proteins including the mutations/insertions was performed with Modeller using the cryo-electron microscopy structural coordinates of the full-length mouse Piezo1 (mPiezo1) (PDB code 3JAC) as the reference [16,[36][37][38]. Positioning of the mutations, structural differences, and detailed analysis of the helices were performed with Pymol [39]. ...
Hereditary Xerocytosis (HX) is an autosomal dominantly inherited congenital hemolytic anemia associated with erythrocyte dehydration due to decreased intracellular potassium content resulting in increased mean corpuscular hemoglobin concentration. The affected members of HX families show compensated anemia with splenomegaly, hemosiderosis, and perinatal edema but are in large part transfusion independent. Functional studies show a link between mutations in mechanosensitive ion channel, encoded by PIEZO1 gene and the HX. We identified new PIEZO1 variants that are likely pathogenic in three phenotypically characterized multi-generational HX Brazilian families. Interestingly, one missense variant of the PIEZO1 gene identified, p.E2494V was associated in trans with the previously reported most frequent pathogenic duplication p.E2496ELE. The three-dimensional structure of the human protein modeled using structural coordinates of the mouse Piezo1 solved by cryo-electron microscopy (Cryo-ME) showed that the two identified variants, p.M2007L and p.T2014I, are localized to an important mechanosensitive transmembrane domain suggesting a conformational mechanism for altered channel's gating. The p.E2496ELE variant identified alters the extension of helix α1 bringing it much closer to the beam affecting the position of it structure at the end of the pore.
... Missing residue Q73 and missing atoms of residues K31, Q73, K94, K97, Q109, Q110, D136, K187, K218, Q229, T245, R255, Q288, K306, K449 were added with Modeller 9.3. [69] For docking and MD simulations with CYP1A2, the crystal structure from PDB ID:2 HI4 was used. [70] To obtain proteinbinding poses for MF and TE to start MD simulations from, they were docked into the protein templates (using the PLANTS docking software, version 1.2 [71] and the ChemPLP scoring function [72] ) and equilibrated in MD simulations in which the heme group was described in its resting state (i.e.,w ith af erryl-oxygen dummy atom). ...
The regio‐ (and stereo‐)selectivity and specific activity of cytochrome P450s are determined by the accessibility of potential sites of metabolism (SOMs) of the bound substrate relative to the heme, and the activation barrier of the regioselective oxidation reaction(s). The accessibility of potential SOMs depends on the relative binding free energy (ΔΔGbind) of the catalytically active substrate‐binding poses, and the probability of the substrate to adopt a transition‐state geometry. An established experimental method to measure activation energies of enzymatic reactions is the analysis of reaction rate constants at different temperatures and the construction of Arrhenius plots. This is a challenge for multistep P450‐catalyzed processes that involve redox partners. We introduce a modified Arrhenius approach to overcome the limitations in studying P450 selectivity, which can be applied in multiproduct enzyme catalysis. Our approach gives combined information on relative activation energies, ΔΔGbind values, and collision entropies, yielding direct insight into the basis of selectivity in substrate conversion.
... ngen.mpg.de/#/tools / hhpre d), which uses the Modeller program [62] to model the homology model. We built the homology model of OR2M3 using the X-ray structure of the M1 muscarinic receptor as a template (5CXV.pdb) ...
Sulfur-containing compounds within a physiological relevant, natural odor space, such as the key food odorants, typically constitute the group of volatiles with the lowest odor thresholds. The observation that certain metals, such as copper, potentiate the smell of sulfur-containing, metal-coordinating odorants led to the hypothesis that their cognate receptors are metalloproteins. However, experimental evidence is sparse—so far, only one human odorant receptor, OR2T11, and a few mouse receptors, have been reported to be activated by sulfur-containing odorants in a copper-dependent way, while the activation of other receptors by sulfur-containing odorants did not depend on the presence of metals. Here we identified an evolutionary conserved putative copper interaction motif CC/CSSH, comprising two copper-binding sites in TMH5 and TMH6, together with the binding pocket for 3-mercapto-2-methylpentan-1-ol in the narrowly tuned human receptor OR2M3. To characterize the copper-binding motif, we combined homology modeling, docking studies, site-directed mutagenesis, and functional expression of recombinant ORs in a cell-based, real-time luminescence assay. Ligand activation of OR2M3 was potentiated in the presence of copper. This effect of copper was mimicked by ionic and colloidal silver. In two broadly tuned receptors, OR1A1 and OR2W1, which did not reveal a putative copper interaction motif, activation by their most potent, sulfur-containing key food odorants did not depend on the presence of copper. Our results suggest a highly conserved putative copper-binding motif to be necessary for a copper-modulated and thiol-specific function of members from three subfamilies of family 2 ORs.
... The 3.9 Å resolution crystal structure of the human a4b2 nicotinic receptor (PDB code: 5KXI) (Morales- Perez et al., 2016) was used as the starting point for this work. All the missing loops were modelled with the program MODELLER 9v17 (Sali, 1995) and all the nonnatural linkers inserted to promote crystallisation were removed (e.g. the EA linker inserted between F559-S560 in the a4 subunits and between Q420-S421 in the b2 subunits) (Morales- Perez et al., 2016). We recognise that even small loops pose particular challenges for MD simulations (Liao et al., 2018). ...
Nicotinic acetylcholine receptors (nAChRs) modulate synaptic transmission in the nervous system. These receptors have emerged as therapeutic targets in drug discovery for treating several conditions, including Alzheimer's disease, pain, and nicotine addiction. In this in silico study, we use a combination of equilibrium and nonequilibrium molecular dynamics simulations to map dynamic and structural changes induced by nicotine in the human α4β2 nAChR. They reveal a striking pattern of communication between the extracellular binding pockets and the transmembrane domains (TMDs) and show the sequence of conformational changes associated with the initial steps in this process. We propose a general mechanism for signal transduction for Cys-loop receptors: the mechanistic steps for communication proceed firstly through loop C in the principal subunit, and are subsequently transmitted, gradually and cumulatively, to loop F of the complementary subunit, and then to the TMDs through the M2-M3 linker.
... Progress still needs to be made before larger proteins can be analyzed. Comparative protein modeling uses previously solved protein structures to predict the structures of proteins with similar amino acid sequences (Sali, 1995). This method appears to achieve a reasonable degree of accuracy but relies on the assumption that although there is an enormous amount of distinct proteins to be found in nature, there is a relatively limited amount of different possible folds (Marti-Renom et al., 2000). ...
Protein folding is a complex, multisystem process characterized by heavy molecular and cellular footprints. Chaperone machinery enables proper protein folding and stable conformation. Other pathways concomitant with the protein folding process include transcription, translation, post-translational modifications, degradation through the ubiquitin-proteasome system, and autophagy. As such, the folding process can go awry in several different ways. The pathogenic basis behind most neurodegenerative diseases is that the disruption of protein homeostasis (i.e. proteostasis) at any level will eventually lead to protein misfolding. Misfolded proteins often aggregate and accumulate to trigger neurotoxicity through cellular stress pathways and consequently cause neurodegenerative diseases. The manifestation of a disease is usually dependent on the specific brain region that the neurotoxicity affects. Neurodegenerative diseases are age-associated, and their incidence is expected to rise as humans continue to live longer and pursue a greater life expectancy. We presently review the sequelae of protein misfolding and aggregation, as well as the role of these phenomena in several neurodegenerative diseases including Alzheimer’s disease, Huntington’s disease, amyotrophic lateral sclerosis, Parkinson’s disease, transmissible spongiform encephalopathies, and spinocerebellar ataxia. Strategies for treatment and therapy are also conferred with respect to impairing, inhibiting, or reversing protein misfolding.
Usher syndrome (USH) is a retinal autosomal recessive genetic disorder, characterized by congenital severe-to-profound sensorineural hearing loss, retinitis pigmentosa (RP), and rarely vestibular dysfunction. A transmembrane inner ear gene TMIE causing autosomal recessive usher syndrome hearing loss, which may open up interesting perspectives into the function of this protein in inner ear. This disease is linked with mutations in TMIE gene. In this study delineates the pathogenic association, miss-fold aggregation, and conformational paradigm of a missense variant (c.242G>A) resulting into (p.Arg81His) in TMIE gene segregating usher syndrome through a molecular dynamics simulations approach. The transmembrane inner ear expressed protein assumes a critical role as its helices actively engage in binding with specific target DNA base pairs. The alteration observed in the mutant protein, characterized by an outward repositioning of the proximal helical portion, which is attributed to the absence of preceding beta-hairpins in the C-terminal region. This structural modification results in the loss of hydrogen bonds, exposure of hydrophobic residues to the solvent, and a consequential transformation of helices into loops, ultimately leading to functional impairment in the TMIE protein. These notable modifications in the stability and conformation of the mutant protein were verified through essential dynamics analysis, revealing that a point mutation induces distinct overall motions and correlations between proteins, ultimately resulting in usher syndrome. The current study provides insilico evidences of Usher syndrome hearing loss disease as protein folding disorder. The energy calculation also revealed that there is a difference of -251.211Kj/mol which also indicates that the SNP has significantly decreased the stability of protein consequently folding into Usher syndrome. This study contributes molecular insights into the structural correlation between the TMIE protein and usher syndrome. The docking analysis highlight various interaction between wild and mutant structure emphasizing key residues involved in hydrogen and hydrophobic interaction.
Bats are reservoir hosts for many zoonotic viruses. Despite this, relatively little is known about the diversity and abundance of viruses within individual bats, and hence the frequency of virus co-infection and spillover among them. We characterize the mammal-associated viruses in 149 individual bats sampled from Yunnan province, China, using an unbiased meta-transcriptomics approach. This reveals a high frequency of virus co-infection (simultaneous infection of bat individuals by multiple viral species) and spillover among the animals studied, which may in turn facilitate virus recombination and reassortment. Of note, we identify five viral species that are likely to be pathogenic to humans or livestock, based on phylogenetic relatedness to known pathogens or in vitro receptor binding assays. This includes a novel recombinant SARS-like coronavirus that is closely related to both SARS-CoV and SARS-CoV-2. In vitro assays indicate that this recombinant virus can utilize the human ACE2 receptor such that it is likely to be of increased emergence risk. Our study highlights the common occurrence of co-infection and spillover of bat viruses and their implications for virus emergence.
Complex mechanisms regulate the cellular distribution of cholesterol, a critical component of eukaryote membranes involved in regulation of membrane protein functions directly and through the physiochemical properties of membranes. StarD4, a member of the steroidogenic acute regulator-related lipid-transfer (StART) domain (StARD)-containing protein family, is a highly efficient sterol-specific transfer protein involved in cholesterol homeostasis. Its mechanism of cargo loading and release remains unknown despite recent insights into the key role of phosphatidylinositol phosphates in modulating its interactions with target membranes. We have used large-scale atomistic Molecular dynamics (MD) simulations to study how the dynamics of cholesterol bound to the StarD4 protein can affect interaction with target membranes, and cargo delivery. We identify the two major cholesterol (CHL) binding modes in the hydrophobic pocket of StarD4, one near S136&S147 (the Ser-mode), and another closer to the putative release gate located near W171, R92&Y117 (the Trp-mode). We show that conformational changes of StarD4 associated directly with the transition between these binding modes facilitate the opening of the gate. To understand the dynamics of this connection we apply a machine-learning algorithm for the detection of rare events in MD trajectories (RED), which reveals the structural motifs involved in the opening of a front gate and a back corridor in the StarD4 structure occurring together with the spontaneous transition of CHL from the Ser-mode of binding to the Trp-mode. Further analysis of MD trajectory data with the information-theory based NbIT method reveals the allosteric network connecting the CHL binding site to the functionally important structural components of the gate and corridor. Mutations of residues in the allosteric network are shown to affect the performance of the allosteric connection. These findings outline an allosteric mechanism which prepares the CHL-bound StarD4 to release and deliver the cargo when it is bound to the target membrane.
Extreme environments, such as Antarctic habitats, present major challenges for many biological processes. Antarctic icefishes (Crynotothenioidea) represent a compelling system to investigate the molecular basis of adaptation to cold temperatures. Here we explore how the sub-zero habitats of Antarctic icefishes have impacted rhodopsin (RH1) function, the temperature-sensitive dim-light visual pigment found in rod photoreceptors. Using likelihood-models and ancestral reconstruction, we find that accelerated evolutionary rates in icefish RH1 underlie unique amino acid mutations absent from other deep-dwelling fishes, introduced before (S160A) and during (V259M) the onset of modern polar conditions. Functional assays reveal that these mutations red-shift rhodopsin spectral absorbance, consistent with spectral irradiance under sea-ice. These mutations also lower the activation energy associated with retinal release of the light-activated RH1, and accelerate its return to the dark-state, likely compensating for a cold-induced decrease in kinetic rates. These are adaptations in key properties of rhodopsin that mediate rod sensitivity and visual performance in the cold dark seas of the Antarctic.
Plant diseases cause a huge impact on food security and are of global concern. While application of agrochemicals is a common approach in the control of plant diseases currently, growing drug resistance and the impact of off-target effects of these compounds pose major challenges. The identification of pathogenicity-related virulence mechanisms and development of new chemicals that target these processes are urgently needed. One such virulence mechanism is the detoxification of reactive oxygen species (ROS) generated by host plants upon attack by pathogens. The machinery of ROS detoxification might therefore serve as a drug target for preventing plant diseases, but few anti-ROS-scavenging drugs have been developed. Here, we show that in the model system Botrytis cinerea secretion of the cytochrome c-peroxidase, BcCcp1 removes plant-produced H2O2 and promotes pathogen invasion. The peroxidase secretion is modulated by a Tom1-like protein, BcTol1, through physical interaction. We show that BcTol1 is regulated at different levels to enhance the secretion of BcCcp1 during the early infection stage. Inactivation of either BcTol1 or BcCcp1 leads to dramatically reduced virulence of B. cinerea. We identify two BcTol1-targeting small molecules that not only prevent B. cinerea invasion but also have effective activity against a wide range of plant fungal pathogens without detectable effect on the hosts. These findings reveal a conserved mechanism of ROS detoxification in fungi and provide a class of potential fungicides to control diverse plant diseases. The approach described here has wide implications for further drug discovery in related fields.
Connexins (Cxs) are a family of vertebrate proteins constituents of gap junction channels (GJCs) that connect the cytoplasm of adjacent cells by the end-to-end docking of two Cx hemichannels. The intercellular transfer through GJCs occurs by passive diffusion allowing the exchange of water, ions, and small molecules. Despite the broad interest to understand, at the molecular level, the functional state of Cx-based channels, there are still many unanswered questions regarding structure-function relationships, perm-selectivity, and gating mechanisms. In particular, the ordering, structure, and dynamics of water inside Cx GJCs and hemichannels remains largely unexplored. In this work, we describe the identification and characterization of a believed novel water pockettermed the IC pocketlocated in-between the four transmembrane helices of each human Cx26 (hCx26) monomer at the intracellular (IC) side. Using molecular dynamics (MD) simulations to characterize hCx26 internal water structure and dynamics, six IC pockets were identified per hemichannel. A detailed characterization of the dynamics and ordering of water including conformational variability of residues forming the IC pockets, together with multiple sequence alignments, allowed us to propose a functional role for this cavity. An in vitro assessment of tracer uptake suggests that the IC pocket residue Arg-143 plays an essential role on the modulation of the hCx26 hemichannel permeability. Methodology and Results Modeling and simulation Figure 1: Overview of the IC pocket. (A) View from the intracellular side of the hCx26 hemichannel. (B) View from inside the pore of the hCx26 hemichannel showing only three monomers for clarity. IC pocket definition The IC pocket region was geometrically approximated by a sphere of radius 6Å, centered at the geometric center of the amino acid residues. A water molecule was considered inside the pocket if its position vector was located within this region. Sequence alignments and structural projection of the IC pocket conservation Figure 2: Multiple sequence alignment of selected representatives of human Cx subfamilies (α, β, γ, δ, ζ and). Columns surrounded by a red rectangle represent aligned residues from other subfamilies that match those located in the IC pocket of the hCx26. Trajectory and water dynamics analyses Figure 3: Monomer of human Cx26, water pocket, and involves residues. (A) Cartoon representation of one hCx26 monomer from a side view. (B) Selected snapshot from the MD simulation depicting relevant residues. Figure 4: Water dynamics inside the IC pocket of each hCx26 monomer. (A) Water occupancy depicted as a box plot. (B) Survival probabilities P (t) for water molecules. (C) Correlation time C 2,û (t) forû dipole. (D) Correlation time C 2,û (t) forû OH. Figure 5: Angular orientation and the energy of Arg-143 dipole on Cx26 hemichannels. Angular orientation of Arg-143 dipole on Cx26 hemichannels trajectory under 0v (A),-1v (B), and +1v (C). Energy of Arg-143 dipole on Cx26 hemichannels trayectory under 0v (D),-1v (E), and +1v (F). Assessment of hemichannel permeability Hemichannel function was assessed through time-lapse imaging of ethidium (Eth) bromide (314 Dalton, +1) uptake. Figure 6: Arg-143 plays an essential role on Cx26 hemichannels activity. (A) Time course of Eth uptake in a Ca 2+-free solution to promote hemichannel opening. HeLa parental cells with MOCK transfection are depicted in gray. (B) Rate of Eth uptake extracted from the slopes of the curves showed in (A). (C) Relationship between the levels of Eth uptake and connexin level expression as a function of the GFP fluorescence intensity, under divalent cation-free solution. Conclusions We have identified, to our knowledge, a novel water pocket, termed IC pocket, in the intracellular side of each monomer of the Cx26 hemichannel. When studying the composition and dynamics of the amino acid residues comprising the IC pocket we found that Phe-29 and Arg-143 establish strong interactions with water molecules within the IC pocket. Arg-143 changes its dipole moment involved in different voltage conditions, showing two states energetically favorable when we apply difference of electrical potential. We characterized in detail the dynamics, ordering, and orientation of water molecules inside these pockets, finding a clear nonbulk-like behavior mainly because of interactions between water molecules and the protein interface. Our in vitro experiments demonstrate that Arg-143 plays an essential role on the hCx26 hemichannel activity. These findings might provide new insights into the molecular mechanism on gating of hCx26 hemichannels as well as novel potential drug design strategies for Cx-related diseases.
Gap junction (GJ) channels are formed by two hemichannels (HC), each contributed by the cells taking part in this direct cell-cell communication conduit. In this work, we compare two connexin proteins (Cx), human Cx26 and mouse Cx39, in the two possible conformations, HC and GJ. The simulation of these molecular systems requires the use of too many computational resources for all-atom (AA) models. However, we have built coarse-grained (CG) models of Cx26 and Cx39 in HC and GJ conformations. We performed molecular dynamics (MD) simulations with each of these CG models. All simulations were conducted for a time of 1 µs and several analyzes have been performed on the resulting trajectories. We focus our analyses on pore radius measurements to study the mechanism of opening and closing of GJ and HC systems. Our preliminary studies show the main regions of connexin involved in the mechanism of gating, the N-terminal (NTH), parahelix region (PH), and extracellular loops (E1 and E2). These findings might provide new insights into the molecular mechanism of gating of HC and GJ as well as novel potential drug design strategies for Cx-related diseases, such as deafness, and skin and skeletal muscle diseases.
italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">Background.
Much of the recent success in protein structure prediction has been a result of accurate protein contact prediction—a binary classification problem. Dozens of methods, built from various types of machine learning and deep learning algorithms, have been published over the last two decades for predicting contacts. Recently, many groups, including Google DeepMind, have demonstrated that reformulating the problem as a multi-class classification problem is a more promising direction to pursue. As an alternative approach, we recently proposed real-valued distance predictions, formulating the problem as a regression problem. The nuances of protein 3D structures make this formulation appropriate, allowing predictions to reflect inter-residue distances in nature. Despite these promises, the accurate prediction of real-valued distances remains relatively unexplored; possibly due to classification being better suited to machine and deep learning algorithms.
Methods.
Can regression methods be designed to predict real-valued distances as precise as binary contacts? To investigate this, we propose multiple novel methods of input label engineering, which is different from feature engineering, with the goal of optimizing the distribution of distances to cater to the loss function of the deep-learning model. Since an important utility of predicted contacts or distances is to build three-dimensional models, we also tested if predicted distances can reconstruct more accurate models than contacts.
Results.
Our results demonstrate, for the first time, that deep learning methods for real-valued protein distance prediction can deliver distances as precise as binary classification methods. When using an optimal distance transformation function on the standard PSICOV dataset consisting of 150 representative proteins, the precision of ‘top-all’ long-range contacts improves from 60.9% to 61.4% when predicting real-valued distances instead of contacts. When building three-dimensional models we observed an average TM-score increase from 0.61 to 0.72, highlighting the advantage of predicting real-valued distances.
Computational methods play a key role for investigating allosteric mechanisms in proteins, with the potential of generating valuable insights for innovative drug design. Here we present the SenseNet (“Structure ENSEmble NETworks”) framework for analysis of protein structure networks, which differs from established network models by focusing on interaction timelines obtained by molecular dynamics simulations. This approach is evaluated by predicting allosteric residues reported by NMR experiments in the PDZ2 domain of hPTP1e, a reference system for which previous computational predictions have shown considerable variance. We applied two models based on the mutual information between interaction timelines to estimate the conformational influence of each residue on its local environment. In terms of accuracy our prediction model is comparable to the top performing model published for this system, but by contrast benefits from its independence from NMR structures. Our results are complementary to experimental data and the consensus of previous predictions, demonstrating the potential of our new analysis tool SenseNet. Biochemical interpretation of our model suggests that allosteric residues in the PDZ2 domain form two distinct clusters of contiguous sidechain surfaces. SenseNet is provided as a plugin for the network analysis software Cytoscape, allowing for ease of future application and contributing to a system of compatible tools bridging the fields of system and structural biology.
Author Summary
Regulation and signal transduction processes in proteins are often correlated to structural changes induced by ligand binding, which can lead to suppression or enhancement of protein function. A common method to investigate such changes are numerical simulations of protein dynamics. We developed the analysis software SenseNet for predicting how protein dynamics and function is affected by e.g. ligand binding events based on molecular dynamics simulations. Our model estimates which structural elements of the protein confer the most information about their local environment, reasoning that these elements are essential for signal propagation. Applying this method on the PDZ2 domain of the hPTP1e protein, we were able to accurately predict structure elements with known signaling roles as determined by previous experiments. Integrating these experimental data with the consensus of other computational models and our predictions, we find two separate pathways which may transmit information through the PDZ2 protein structure. In addition to deepening our insight into the behavior of this particular protein, these results demonstrate the usefulness of our methods for other systems, such as potential drug targets. To make this analysis available to a broad audience, we implemented it as a plugin for the popular network analysis software Cytoscape.
Changeux et al. recently suggested that the SARS-CoV-2 spike protein may interact with nicotinic acetylcholine receptors (nAChRs), and that such interactions may be involved in pathology and infectivity. This hypothesis is based on the fact that the SARS-CoV-2 spike protein contains a sequence motif similar to known nAChR antagonists. Here, we use molecular simulations of validated atomically detailed structures of nAChRs, and of the spike, to investigate the possible binding of the Y674-R685 region of the spike to nAChRs. We examine the binding of the Y674-R685 loop to three nAChRs, namely the human α4β2 and α7 subtypes and the muscle-like αβγδ receptor from Tetronarce californica. Our results predict that Y674-R685 has affinity for nAChRs. The region of the spike responsible for binding contains a PRRA motif, a four-residue insertion not found in other SARS-like coronaviruses. The conformational behaviour of the bound Y674-R685 is highly dependent on the receptor subtype: it adopts extended conformations in the α4β2 and α7 complexes, but is more compact when bound to the muscle-like receptor. In the α4β2 and αβγδ complexes, the interaction of Y674-R685 with the receptors forces the loop C region to adopt an open conformation, similar to other known nAChR antagonists. In contrast, in the α7 complex, Y674-R685 penetrates deeply into the binding pocket where it forms interactions with the residues lining the aromatic box, namely with TrpB, TyrC1 and TyrC2. Estimates of binding energy suggest that Y674-R685 forms stable complexes with all three nAChR subtypes. Analyses of simulations of the glycosylated spike show that the Y674-R685 region is accessible for binding. We suggest a potential binding orientation of the spike protein with nAChRs, in which they are in a non-parallel arrangement to one another.
Dense surface glycosylation on the HIV-1 envelope (Env) protein acts as a shield from the adaptive immune system. However, the molecular complexity and flexibility of glycans make experimental studies a challenge. Here we have integrated high-throughput atomistic modeling of fully glycosylated HIV-1 Env with graph theory to capture immunologically important features of the shield topology. This is the first complete all-atom model of HIV-1 Env SOSIP glycan shield that includes both oligomannose and complex glycans, providing physiologically relevant insights of the glycan shield. This integrated approach including quantitative comparison with cryo-electron microscopy data provides hitherto unexplored details of the native shield architecture and its difference from the high-mannose glycoform. We have also derived a measure to quantify the shielding effect over the antigenic protein surface that defines regions of relative vulnerability and resilience of the shield and can be harnessed for rational immunogen design.
Background . Neuropeptides exert their activity through binding to G-protein coupled receptors (GPCRs). GPCRs are well-known drug targets in the pharmaceutical industry and are currently discussed as targets to control pest insects. Here we investigate the neuropeptide adipokinetic hormone (AKH) system of the desert locust Schistocerca gregaria . The desert locust is known for its high reproduction, and for forming devastating swarms consisting of billions of individual insects. It is also known that S. gregaria produces three different AKHs as ligands but has only one AKH receptor. The AKH system is known to be essential for metabolic regulation, which is necessary for reproduction and flight activity.
Methods. Nuclear magnetic resonance techniques (NMR) in a dodecylphosphocholin (DPC) micelle solution were used to determine the structure of the three AKHs. The primary sequence of the S. gregaria AKH receptor (AKHR) was used to construct a 3D molecular model. Next, the 3 AKHs were individually docked to the receptor, and dynamic simulation of the whole ligand-receptor complex in a model membrane was performed.
Results . Although the three endogenous AKHs of S. gregaria have quite different amino acids sequences and chain length (two octa- and one decapeptide), NMR experiments assigned a turn structure in DPC micelle solution for all. The GPCR-ModSim program identified human kappa opioid receptor (hk-OR) to be the best template after which the S. gregaria AKHR was modeled. All three AKHs were found to have the same binding site on this receptor, interact with similar residues of the receptor and have comparable binding constants. Molecular switches were also identified; the movement of the receptor could be visually shown when ligands (AKHs) were docked and the receptor was activated.
Conclusions . The study proposes a model of binding of the three endogenous ligands to the one existing AKH receptor in the desert locust and paves the way to use such a model for the design of peptide analogs and finally, peptide mimetics, in the search for novel species-specific insecticides based on receptor-ligand interaction.
Norovirus is the major cause of epidemic nonbacterial gastroenteritis worldwide. Lack of structural information on infection and replication mechanisms hampers the development of effective vaccines and remedies. Here, using cryo-electron microscopy, we show that the capsid structure of murine noroviruses changes in response to aqueous conditions. By twisting the flexible hinge connecting two domains, the protruding (P) domain reversibly rises off the shell (S) domain in solutions of higher pH, but rests on the S domain in solutions of lower pH. Metal ions help to stabilize the latter conformation in this process. Furthermore, in the resting conformation, the cellular receptor CD300lf is readily accessible, and thus infection efficiency is significantly enhanced. Two P domain conformations were also found in the human norovirus GII.3 capsid. These results provide new insights into the infection mechanisms of the non-envelope viruses that function in dramatic environmental changes such as the digestive tract.
Significance Statement
The capsid structures of caliciviruses have been reported to be classified into two different types, according to species and genotype. One is the rising P domain type as shown in human norovirus GII.10 and rabbit hemorrhagic disease virus, where the P domain rises from the S domain surface. The other is the resting P domain type as shown in human norovirus GI.1, sapovirus and San Miguel sea lion virus, where the P domain rests upon the S domain. Here, we demonstrate that the P domain of the murine norovirus infectious particles changes reversibly between the rising and resting P domain types in response to aqueous conditions. Our findings provide new insights into the mechanisms of viral infection of caliciviruses.
The dense arrangement of N-glycans masking antigenic surfaces on the HIV-1 envelope (Env) protein acts as a shield from the adaptive immune system. The molecular complexity of glycan modifications and their inherent dynamic heterogeneity on a protein surface make experimental studies of glycoprotein structures a challenge. Here we have integrated a high-throughput atomistic modeling with graph-theory based method to capture the native glycan shield topological network and identify concerted behavior of these glycans. This is the first time that a complete computational model of an HIV-1 Env trimeric SOSIP structure has been generated with a native glycosylation pattern including both oligomannose and complex glycans, thus obtaining results which are immunologically more relevant. Important global and local feature differences due to the native-like glycosylation pattern have been identified, that stem from the charged sialic acid tips, fucose rings at the base, and different branching patterns of the complex glycans. Analyses of network attributes have aided in detailed description of the shield in a biological context. We have also derived a measure to quantify the shielding effect based on the number of glycan heavy atoms encountered over the antigenic protein surface that can define regions of relative vulnerability and resilience on the shield, and can be harnessed for potential immunogen design.
Limited information on sex pheromone recognition by tea moths, Ectropis obliqua Prout (Lepidoptera: Geometridae), exists for this economically important pest of tea (Camellia sinensis L.; Theaceae). Pheromone binding proteins (PBPs), a sub-family of odorant-binding proteins, control transportation of pheromone molecules that may contribute to the discrimination of sex pheromone components. It has been reported previously that EoblPBP1 gene (a pheromone binding protein of E. obliqua) is highly expressed in antennae of the male moth. Based on this information, a reliable model of EoblPBP1 was constructed by homology modeling using the enantiomers of Z3Z9-6,7-epo-18:Hy docked into the hydrophobic cavity of the model. Docking results suggested similar binding affinities of this enantiomer to EoblPBP1. However, electroantennogram and field trapping experiments of E. obliqua males revealed that response to Z3Z9-(6S,7R)-epo-18:Hy was significantly greater than the opposite configuration, and suggested enantiomeric discrimination could occur on sex pheromone receptors of this species of tea moth.
Background . Neuropeptides exert their activity through binding to G-protein coupled receptors (GPCRs). GPCRs are well-known drug targets in the pharmaceutical industry and are currently discussed as targets to control pest insects. Here we investigate the neuropeptide adipokinetic hormone (AKH) system of the desert locust Schistocerca gregaria . The desert locust is known for its high reproduction, and for forming devastating swarms consisting of billions of individual insects. It is also known that S. gregaria produces three different AKHs as ligands but has only one AKH receptor. The AKH system is known to be essential for metabolic regulation, which is necessary for reproduction and flight activity.
Methods. Nuclear magnetic resonance techniques (NMR) in a dodecylphosphocholin (DPC) micelle solution were used to determine the structure of the three AKHs. The primary sequence of the S. gregaria AKH receptor (AKHR) was used to construct a 3D molecular model. Next, the 3 AKHs were individually docked to the receptor, and dynamic simulation of the whole ligand-receptor complex in a model membrane was performed.
Results . Although the three endogenous AKHs of S. gregaria have quite different amino acids sequences and chain length (two octa- and one decapeptide), NMR experiments assigned a turn structure in DPC micelle solution for all. The GPCR-ModSim program identified human kappa opioid receptor (hk-OR) to be the best template after which the S. gregaria AKHR was modeled. All three AKHs were found to have the same binding site on this receptor, interact with similar residues of the receptor and have comparable binding constants. Molecular switches were also identified; the movement of the receptor could be visually shown when ligands (AKHs) were docked and the receptor was activated.
Conclusions . The study proposes a model of binding of the three endogenous ligands to the one existing AKH receptor in the desert locust and paves the way to use such a model for the design of peptide analogs and finally, peptide mimetics, in the search for novel species-specific insecticides based on receptor-ligand interaction.
The human fibroblast growth factor-2 (FGF-2) highly expressed in tumors is an important factor to promote tumor angiogenesis and lymphangiogensis. A disulfide-stabilized diabody (ds-Diabody) could specifically target FGF-2 and show its advantages in inhibition of tumor angiogenesis and growth. It is very important for antibody drugs to confirm the fine epitope. Here, theoretical structure models of FGF-2 and antibody were built by homology modeling. The amino acid residues in the interaction interface of antigen and antibody were analyzed by molecular docking. The potential epitope was predicted by homology modeling and molecular docking of antigen-antibody and site-directed mutation assays of alanine scanning. The predicted epitope was verified by antigen mutagenesis and enzyme-linked immunosorbent assay (ELISA). The epitope mapping assay showed that the epitope of ds-Diabody against FGF-2 was defined by the discontinuous sites including six amino acid residues (P23, Q65, R69, G70, Y82 and R118). The results showed that the epitope was localized in the interaction interface of FGF-2 and ds-Diabody. The fine epitope mapping provided the important information for understanding the inhibition activity of ds-Diabody against FGF-2 and helping in the further development of ds-Diabody against FGF-2 as a potentially promising antibody drug for future cancer therapy.
Determining the side effects of pesticides on pollinators is an important topic due to the increasing loss of pollinators. We aimed to determine the effects of chronic sublethal exposure of the neonicotinoid pesticide imidacloprid on the bumblebee Bombus terrestris under laboratory conditions. The analytical standard of imidacloprid in sugar solution was used for the treatment. Verification of pesticides using UHPLC-QqQ-MS/MS in the experimental bumblebees showed the presence of only two compounds, imidacloprid and imidacloprid-olefin, which were found in quantities of 0.57±0.22 and 1.95±0.43 ng/g, respectively. Thus, the level of the dangerous metabolite imidacloprid-olefin was 3.4-fold higher than that of imidacloprid. Label-free nanoLC-MS/MS quantitative proteomics of bumblebee heads enabled quantitative comparison of 2,883 proteins, and 206 proteins were significantly influenced by the imidacloprid treatment. The next analysis revealed that the highly downregulated markers are members of the terpenoid backbone biosynthesis pathway (KEGG: bter00900) and that imidacloprid treatment suppressed the entire mevalonate pathway, fatty acid synthesis and associated markers. The proteomics results indicate that the consequences of imidacloprid treatment are complex, and the marker changes are associated with metabolic and neurological diseases and olfaction disruption. This study provides important markers and can help to explain the widely held assumptions from biological observations.
Significance:
The major finding is that all markers of the mevalonate pathway were substantially downregulated due to the chronic imidacloprid exposure. The disbalance of mevalonate pathway has many important consequences. We suggest the mechanism associated with the novel toxicogenic effect of imidacloprid. The results are helpful to explain that imidacloprid impairs the cognitive functions and possesses the delayed and time cumulative effect.
A protein sequence with at lease 40% identity to a known structure can now be modelled automatically, with an accuracy approaching that o fa low-resolution X-ray structure or a medium-resolution nuclear magnetic resonance structure. In general, these models have goods stereochemistry and an overall structural accuracy that is as high as the similarity between the template and the actual structure being predicted. As a result, the number of sequences that can be modelled is an order of magnitude larger then the number of experimentally determined protein structures. In addition, evaluation techniques are available that can estimated errors in different regions of the model. Thus, the number of applications where homology modelling is proving useful is growing rapidly.
We describe a database of protein structure alignments as well as methods and tools that use this database to improve comparative protein modeling. The current version of the database contains 105 alignments of similar proteins or protein segments. The database comprises 416 entries, 78,495 residues, 1,233 equivalent entry pairs, and 230,396 pairs of equivalent alignment positions. At present, the main application of the database is to improve comparative modeling by satisfaction of spatial restraints implemented in the program MODELLER (Sali A, Blundell TL, 1993, J Mol Biol 234:779-815). To illustrate the usefulness of the database, the restraints on the conformation of a disulfide bridge provided by an equivalent disulfide bridge in a related structure are derived from the alignments; the prediction success of the disulfide dihedral angle classes is increased to approximately 80%, compared to approximately 55% for modeling that relies on the stereochemistry of disulfide bridges alone. The second example of the use of the database is the derivation of the probability density function for comparative modeling of the cis/trans isomerism of the proline residues; the prediction success is increased from 0% to 82.9% for cis-proline and from 93.3% to 96.2% for trans-proline. The database is available via electronic mail.
We describe a comparative protein modelling method designed to find the most probable structure for a sequence given its alignment with related structures. The three-dimensional (3D) model is obtained by optimally satisfying spatial restraints derived from the alignment and expressed as probability density functions (pdfs) for the features restrained. For example, the probabilities for main-chain conformations of a modelled residue may be restrained by its residue type, main-chain conformation of an equivalent residue in a related protein, and the local similarity between the two sequences. Several such pdfs are obtained from the correlations between structural features in 17 families of homologous proteins which have been aligned on the basis of their 3D structures. The pdfs restrain C alpha-C alpha distances, main-chain N-O distances, main-chain and side-chain dihedral angles. A smoothing procedure is used in the derivation of these relationships to minimize the problem of a sparse database. The 3D model of a protein is obtained by optimization of the molecular pdf such that the model violates the input restraints as little as possible. The molecular pdf is derived as a combination of pdfs restraining individual spatial features of the whole molecule. The optimization procedure is a variable target function method that applies the conjugate gradients algorithm to positions of all non-hydrogen atoms. The method is automated and is illustrated by the modelling of trypsin from two other serine proteinases.
We evaluate 3D models of human nucleoside diphosphate kinase, mouse cellular retinoic acid binding protein I, and human eosinophil neurotoxin that were calculated by MODELLER, a program for comparative protein modeling by satisfaction of spatial restraints. The models have good stereochemistry and are at least as similar to the crystallographic structures as the closest template structures. The largest errors occur in the regions that were not aligned correctly or where the template structures are not similar to the correct structure. These regions correspond predominantly to exposed loops, insertions of any length, and non-conserved side chains. When a template structure with more than 40% sequence identity to the target protein is available, the model is likely to have about 90% of the mainchain atoms modeled with an rms deviation from the X-ray structure of approximately 1 A, in large part because the templates are likely to be that similar to the X-ray structure of the target. This rms deviation is comparable to the overall differences between refined NMR and X-ray crystallography structures of the same protein.