Conformational Adaptation Research Articles

Exploring conformational landscapes and binding mechanisms of convergent evolution for the SARS-CoV-2 spike Omicron variant complexes with the ACE2 receptor using AlphaFold2-based structural ensembles and molecular dynamics simulations.

In this study, we combined AlphaFold-based approaches for atomistic modeling of multiple protein states and microsecond molecular simulations to accurately characterize conformational ensembles evolution and binding mechanisms of convergent evolution for the SARS-CoV-2 spike Omicron variants BA.1, BA.2, BA.2.75, BA.3, BA.4/BA.5 and BQ.1.1. We employed and validated several different adaptations of the AlphaFold methodology for modeling of conformational ensembles including the introduced randomized full sequence scanning for manipulation of sequence variations to systematically explore conformational dynamics of Omicron spike protein complexes with the ACE2 receptor. Microsecond atomistic molecular dynamics (MD) simulations provide a detailed characterization of the conformational landscapes and thermodynamic stability of the Omicron variant complexes. By integrating the predictions of conformational ensembles from different AlphaFold adaptations and applying statistical confidence metrics we can expand characterization of the conformational ensembles and identify functional protein conformations that determine the equilibrium dynamics for the Omicron spike complexes with the ACE2. Conformational ensembles of the Omicron RBD-ACE2 complexes obtained using AlphaFold-based approaches for modeling protein states and MD simulations are employed for accurate comparative prediction of the binding energetics revealing an excellent agreement with the experimental data. In particular, the results demonstrated that AlphaFold-generated extended conformational ensembles can produce accurate binding energies for the Omicron RBD-ACE2 complexes. The results of this study suggested complementarities and potential synergies between AlphaFold predictions of protein conformational ensembles and MD simulations showing that integrating information from both methods can potentially yield a more adequate characterization of the conformational landscapes for the Omicron RBD-ACE2 complexes. This study provides insights in the interplay between conformational dynamics and binding, showing that evolution of Omicron variants through acquisition of convergent mutational sites may leverage conformational adaptability and dynamic couplings between key binding energy hotspots to optimize ACE2 binding affinity and enable immune evasion.

Recognition of Actinides by Siderocalin.

Plain simulations and enhanced sampling unveil a novel siderocalin (Scn) recognition mode for An-Ent (where An = actinides and Ent = enterobactin) complexes and identify a "seesaw" relationship between actinide affinity to Ent and Scn recognition to an An-Ent complex. Electrostatic interactions predominantly govern competitive binding in both processes. Additionally, hydrolysis-induced negative charge, water expulsion-driven entropy, and Ent's conformational adaptability collectively enhance high-affinity recognition.

GAABind: a geometry-aware attention-based network for accurate protein-ligand binding pose and binding affinity prediction.

Protein-ligand interactions are increasingly profiled at high-throughput, playing a vital role in lead compound discovery and drug optimization. Accurate prediction of binding pose and binding affinity constitutes a pivotal challenge in advancing our computational understanding of protein-ligand interactions. However, inherent limitations still exist, including high computational cost for conformational search sampling in traditional molecular docking tools, and the unsatisfactory molecular representation learning and intermolecular interaction modeling in deep learning-based methods. Here we propose a geometry-aware attention-based deep learning model, GAABind, which effectively predicts the pocket-ligand binding pose and binding affinity within a multi-task learning framework. Specifically, GAABind comprehensively captures the geometric and topological properties of both binding pockets and ligands, and employs expressive molecular representation learning to model intramolecular interactions. Moreover, GAABind proficiently learns the intermolecular many-body interactions and simulates the dynamic conformational adaptations of the ligand during its interaction with the protein through meticulously designed networks. We trained GAABind on the PDBbindv2020 and evaluated it on the CASF2016 dataset; the results indicate that GAABind achieves state-of-the-art performance in binding pose prediction and shows comparable binding affinity prediction performance. Notably, GAABind achieves a success rate of 82.8% in binding pose prediction, and the Pearson correlation between predicted and experimental binding affinities reaches up to 0.803. Additionally, we assessed GAABind's performance on the severe acute respiratory syndrome coronavirus 2 main protease cross-docking dataset. In this evaluation, GAABind demonstrates a notable success rate of 76.5% in binding pose prediction and achieves the highest Pearson correlation coefficient in binding affinity prediction compared with all baseline methods.

The molecular basis for cellular function of intrinsically disordered protein regions.

Intrinsically disordered protein regions exist in a collection of dynamic interconverting conformations that lack a stable 3D structure. These regions are structurally heterogeneous, ubiquitous and found across all kingdoms of life. Despite the absence of a defined 3D structure, disordered regions are essential for cellular processes ranging from transcriptional control and cell signalling to subcellular organization. Through their conformational malleability and adaptability, disordered regions extend the repertoire of macromolecular interactions and are readily tunable by their structural and chemical context, making them ideal responders to regulatory cues. Recent work has led to major advances in understanding the link between protein sequence and conformational behaviour in disordered regions, yet the link between sequence and molecular function is less well defined. Here we consider the biochemical and biophysical foundations that underlie how and why disordered regions can engage in productive cellular functions, provide examples of emerging concepts and discuss how protein disorder contributes to intracellular information processing and regulation of cellular function.

Dynamic Metallosupramolecular Cages Containing 12 Adaptable Pockets for High-Order Guest Binding Beyond Biomimicry.

Molecular recognition lies at the heart of biological functions, which inspires lasting research in artificial host syntheses to mimic biomolecules that can recognize, process, and transport molecules with the highest level of complexity; nonetheless, the design principle and quantifying methodology of artificial hosts for multiple guests (≥4) remain a formidable task. Herein, we report two rhombic dodecahedral cages [(Zn/Fe)8Pd6-MOC-16], which embrace 12 adaptive pockets for multiguest binding with distinct conformational dynamics inherent in metal-center lability and are able to capture 4-24 guests to manifest a surprising complexity of binding scenarios. The exceptional high-order and hierarchical encapsulation phenomena suggest a wide host-guest dynamic-fit, enabling conformational adjustment and adaptation beyond the duality of induced-fit and conformational selection in protein interactions. A critical inspection of the host-guest binding events in solution has been performed by NMR and ESI-MS spectra, highlighting the importance of acquiring a reliable binding repertoire from different techniques and the uncertainty of quantifying the binding affinities of multiplying guests by an oversimplified method.

Comparative Analysis of Conformational Dynamics and Systematic Characterization of Cryptic Pockets in the SARS-CoV-2 Omicron BA.2, BA.2.75 and XBB.1 Spike Complexes with the ACE2 Host Receptor: Confluence of Binding and Structural Plasticity in Mediating Networks of Conserved Allosteric Sites.

In the current study, we explore coarse-grained simulations and atomistic molecular dynamics together with binding energetics scanning and cryptic pocket detection in a comparative examination of conformational landscapes and systematic characterization of allosteric binding sites in the SARS-CoV-2 Omicron BA.2, BA.2.75 and XBB.1 spike full-length trimer complexes with the host receptor ACE2. Microsecond simulations, Markov state models and mutational scanning of binding energies of the SARS-CoV-2 BA.2 and BA.2.75 receptor binding domain complexes revealed the increased thermodynamic stabilization of the BA.2.75 variant and significant dynamic differences between these Omicron variants. Molecular simulations of the SARS-CoV-2 Omicron spike full-length trimer complexes with the ACE2 receptor complemented atomistic studies and enabled an in-depth analysis of mutational and binding effects on conformational dynamic and functional adaptability of the Omicron variants. Despite considerable structural similarities, Omicron variants BA.2, BA.2.75 and XBB.1 can induce unique conformational dynamic signatures and specific distributions of the conformational states. Using conformational ensembles of the SARS-CoV-2 Omicron spike trimer complexes with ACE2, we conducted a comprehensive cryptic pocket screening to examine the role of Omicron mutations and ACE2 binding on the distribution and functional mechanisms of the emerging allosteric binding sites. This analysis captured all experimentally known allosteric sites and discovered networks of inter-connected and functionally relevant allosteric sites that are governed by variant-sensitive conformational adaptability of the SARS-CoV-2 spike structures. The results detailed how ACE2 binding and Omicron mutations in the BA.2, BA.2.75 and XBB.1 spike complexes modulate the distribution of conserved and druggable allosteric pockets harboring functionally important regions. The results are significant for understanding the functional roles of druggable cryptic pockets that can be used for allostery-mediated therapeutic intervention targeting conformational states of the Omicron variants.

Drastic alterations in the loop structure around colchicine upon complex formation with an engineered lipocalin indicate a conformational selection mechanism.

Using Anticalin technology, a lipocalin protein dubbed Colchicalin, with the ability to bind the toxic plant alkaloid colchicine with picomolar affinity, has previously been engineered, thus offering a potential antidote in vivo and also allowing its sensitive detection in biological samples. To further analyze the mode of ligand recognition, the crystal structure of Colchicalin is now reported in its unliganded form and is compared with the colchicine complex. A superposition of the protein structures revealed major rearrangements in the four structurally variable loops of the engineered lipocalin. Notably, the binding pocket in the unbound protein is largely occupied by the inward-bent loop #3, in particular Ile97, as well as by the phenylalanine side chain at position 71 in loop #2. Upon binding of colchicine, a dramatic shift of loop #3 by up to 11.1 Å occurs, in combination with a side-chain flip of Phe71, thus liberating the necessary space within the ligand pocket. Interestingly, the proline residue at the neighboring position 72, which arose during the combinatorial engineering of Colchicalin, remained in a cis configuration in both structures. These findings provide a striking example of a conformational adaptation mechanism, which is a long-known phenomenon for antibodies in immunochemistry, during the recognition of a small ligand by an engineered lipocalin, thus illustrating the general similarity between the mode of antigen/ligand binding by immunoglobulins and lipocalins.

Visualization and characterization of the elusive MHC I intermediate states by x-ray crystallography and in silicostudies

Abstract Background: Studies have suggested that MHC class I (MHC I) molecules fluctuate rapidly between conformational states as they sample peptides for potential ligands. It is the molecular features of these intermediates that are recognized by tapasin and TAPBPR at the C-terminus of the groove. To date, however, MHC I intermediates remain elusive molecules due to their transient nature. Moreover, whether the N-terminus of the groove has a role in MHC I maturation, and if it also serves as a conformationally sensing region, is not well characterized. Methods: We designed the HLA-B8-restricted 20mer (RA) 6FAKKKYCL and (RA) 6FVKKKYCL peptides and corresponding 8mer FAKKKYCL and FVKKKYCL controls. We determined the x-ray crystal structures of HLA-B8E76C presenting these four peptides. Molecular dynamics (MD) simulations of the complexes were also performed. Results: The structures of FA and FV 20mers revealed significant conformational heterogeneity at the N-terminus of the groove. Long stretches of N-terminal MHC I residues were missing in the electron density maps creating an unstructured and widely open-ended groove. Our structures also showed highly unusual features in MHC I and peptides at the N-terminus of the groove, and in MHC I-peptide interaction. MD simulations showed that these complexes have varying degrees of mobility in a manner consistent with our structures. We suggest that our 20mer structures represent conformational substates explored by MHC I during peptide selection. Conclusion: Our study advances our understanding of peptide-induced conformational adaptation and functional dynamics in MHC I, and brings into focus the N-terminus of the groove as an important region in mechanisms of high-affinity peptide selection. NIAID, National Institutes of Health (R01 AI114467, R01 AI108546, and R21 AI173863)

Coarse-Grained Molecular Simulations and Ensemble-Based Mutational Profiling of Protein Stability in the Different Functional Forms of the SARS-CoV-2 Spike Trimers: Balancing Stability and Adaptability in BA.1, BA.2 and BA.2.75 Variants

Evolutionary and functional studies have suggested that the emergence of Omicron variants can be determined by multiple fitness tradeoffs including immune escape, binding affinity, conformational plasticity, protein stability, and allosteric modulation. In this study, we embarked on a systematic comparative analysis of the conformational dynamics, electrostatics, protein stability, and allostery in the different functional states of spike trimers for BA.1, BA.2, and BA.2.75 variants. Using efficient and accurate coarse-grained simulations and atomistic reconstruction of the ensembles, we examined the conformational dynamics of the spike trimers that agree with the recent functional studies, suggesting that BA.2.75 trimers are the most stable among these variants. A systematic mutational scanning of the inter-protomer interfaces in the spike trimers revealed a group of conserved structural stability hotspots that play a key role in the modulation of functional dynamics and are also involved in the inter-protomer couplings through local contacts and interaction networks with the Omicron mutational sites. The results of mutational scanning provided evidence that BA.2.75 trimers are more stable than BA.2 and comparable in stability to the BA.1 variant. Using dynamic network modeling of the S Omicron BA.1, BA.2, and BA.2.75 trimers, we showed that the key network mediators of allosteric interactions are associated with the major stability hotspots that are interconnected along potential communication pathways. The network analysis of the BA.1, BA.2, and BA.2.75 trimers suggested that the increased thermodynamic stability of the BA.2.75 variant may be linked with the organization and modularity of the residue interaction network that allows for allosteric communications between structural stability hotspots and Omicron mutational sites. This study provided a plausible rationale for a mechanism in which Omicron mutations may evolve by targeting vulnerable sites of conformational adaptability to elicit immune escape while maintaining their control on balancing protein stability and functional fitness through robust allosteric communications with the stability hotspots.

A Competition between Relative Stability and Binding Energy in Caffeine Phenyl-Glucose Aggregates: Implications in Biological Mechanisms.

Hydrogen bonds and stacking interactions are pivotal in biological mechanisms, although their proper characterisation within a molecular complex remains a difficult task. We used quantum mechanical calculations to characterise the complex between caffeine and phenyl-β-D-glucopyranoside, in which several functional groups of the sugar derivative compete with each other to attract caffeine. Calculations at different levels of theory (M06-2X/6-311++G(d,p) and B3LYP-ED=GD3BJ/def2TZVP) agree to predict several structures similar in stability (relative energy) but with different affinity (binding energy). These computational results were experimentally verified by laser infrared spectroscopy, through which the caffeine·phenyl-β-D-glucopyranoside complex was identified in an isolated environment, produced under supersonic expansion conditions. The experimental observations correlate with the computational results. Caffeine shows intermolecular interaction preferences that combine both hydrogen bonding and stacking interactions. This dual behaviour had already been observed with phenol, and now with phenyl-β-D-glucopyranoside, it is confirmed and maximised. In fact, the size of the complex's counterparts affects the maximisation of the intermolecular bond strength because of the conformational adaptability given by the stacking interaction. Comparison with the binding of caffeine within the orthosteric site of the A2A adenosine receptor shows that the more strongly bound caffeine·phenyl-β-D-glucopyranoside conformer mimics the interactions occurring within the receptor.

Single Molecule Imaging with Liquid Phase Electron Microscopy

AbstractFew single‐molecule experiments have enabled the direct imaging of functional biomacromolecules in real‐time in their native liquid environments, resolving their conformational adaptations, transient interactions, and intermediate states. Liquid phase electron microscopy (LP‐EM), due to its unique combination of spatial and temporal resolution, has shown to be a promising tool. Recent experiments have enabled successful imaging of intact structures of organic molecules and biological systems with an ordinary electron microscope. Adapting image processing methods and quantitative data analysis from single particle experiments based on the optical microscope, quantifying motion and relaxation of these interacting molecules allows the experimental observations of pathways, to test theoretical predictions, and discovery of new mechanisms. Combining LP‐EM with tomography, fluorescence, and mass spectroscopy allows for probing multi‐dimensional structural and dynamic information. Challenges remain in obtaining high‐quality data in large quantities, which can be improved by developing new liquid cell platforms and machine learning‐based data analysis.

Nanoprecipitates of γ-cyclodextrin/epigallocatechin-3-gallate inclusion complexes as efficient antioxidants for preservation of shrimp surimi products: synthesis, performance and mechanism.

Epigallocatechin-3-gallate (EGCG) is well known for excellent chain-breaking antioxidant capability. However, browning by oxidation and aggregation of EGCG is a non-negligible defect that hinders its applications as an antioxidant in various foodstuffs. Therefore, how to eliminate or mitigate browning efficiently, while retaining functionalities as food additive is a challenge in the food industry. Our results demonstrated that EGCG could be anchored within the internal cavity of γ-cyclodextrin (γ-CD) to form an inclusion structure, where hydrophobic interaction, hydrogen bonding, and π-stacking were identified to be the primary drivers. The interplay between two molecules and the steric hindrance from γ-CD could restrict the motion and aggregation of EGCG efficiently, thus alleviating the browning effect. In addition, the conformational adaption of EGCG within the inclusions would result in general decreases in hydrogen-bond dissociation enthalpies for the pyrogallol-type structure on the b ring, thus enhancing the antioxidant capability. In practical application, the nanoscale γ-CD/EGCG inclusion complexes were validated preliminarily as efficient additives in the preservation of shrimp surimi, presenting significant effects on prolonging the shelf-life of products. Here, nanoscale γ-CD/EGCG inclusion complexes as alternatives to EGCG were tailored as food antioxidants for the preservation of shrimp surimi products, exerting antioxidant effects while mitigating the browning effects of EGCG on products. Through self-assembly, EGCG would be anchored with the cavity of γ-CD, which could regulate the release modes and restrict the aggregation of EGCG. This facile strategy has great potential in food preservation. © 2023 Society of Chemical Industry.

Solvent Mediated Nanoscale Quasi-Periodic Chirality Reversal in Self-Assembled Molecular Networks Featuring Mirror Twin Boundaries.

Grain boundaries in polycrystals have a prominent impact on the properties of a material, therefore stimulating the research on grain boundary engineering. Structure determination of grain boundaries of molecule-based polycrystals with submolecular resolution remains elusive. Reducing the complexity to monolayers has the potential to simplify grain boundary engineering and may offer real-space imaging with submolecular resolution using scanning tunneling microscopy (STM). Herein, the authors report the observation of quasi-periodic nanoscale chirality switching in self-assembled molecular networks, in combination with twinning, as revealed by STM at the liquid/solid interface. The width of the chiral domain structure peaks at 12-19nm. Adjacent domains having opposite chirality are connected continuously through interdigitated alkoxy chains forming a 1D defect-free domain border, reflecting a mirror twin boundary. Solvent co-adsorption and the inherent conformational adaptability of the alkoxy chains turn out to be crucial factors in shaping grain boundaries. Moreover, the epitaxial interaction with the substrate plays a role in the nanoscale chirality reversal as well.

DFT-aided infrared and electronic circular dichroism spectroscopic study of cyclopeptide S-PK6 and the exploration of its antitumor potential by molecular docking

Cyclic peptides have been widely explored as new chemical modalities in drug design, yet the structural study of bioactive cyclic peptides is often difficult due to the irregular constrained structure. In this article, we reported the infrared (IR) and electronic circular dichroism (ECD) spectroscopic study of an isoindolinone-containing analog (S-PK6) of natural antitumor cyclopeptide Phakellistatin 6. The peak positions of various amino acid residues in the IR spectrum of S-PK6 were specified with the aid of DFT-calculations. To establish a feasible and accurate method for simulating the ECD of small cyclic peptides, we performed a comparison of experimental ECD of S-PK6 with the theoretical spectra calculated by different methods, which indicated that the simulation at the TDDFT/CAM-B3LTP/cc-pVDZ level could provide a decent match for the experimental ECD spectrum of cyclopeptide. Motivated by our previous finding that the isoindolinone-containing cyclopeptides could efficiently inhibit the proliferation of HepG2 cells by decreasing the murine double minute2 (MDM2) protein level, we have investigated the possible binding mode between S-PK6 and MDM2 by molecular docking. Finally, molecular docking of S-PK6 with different cancer-related proteins have been performed, which suggested that diverse intramolecular hydrogen bonding networks might be formed in S-PK6 upon binding to different target protein structures, revealing a target-induced conformational adaptation mechanism. This research provides critical insights into the structure of small cyclopeptides and hints towards the design of peptidyl drugs.

Cross-Species Permissivity: Structure of a Goat Adeno-Associated Virus and Its Complex with the Human Receptor AAVR.

Adeno-associated virus (AAV) is a small ssDNA satellite virus of high interest (in recombinant form) as a safe and effective gene therapy vector. AAV's human cell entry receptor (AAVR) contains polycystic kidney disease (PKD) domains bound by AAV. Seeking understanding of the spectrum of interactions, goat AAVGo.1 is investigated, because its host is the species most distant from human with reciprocal cross-species cell susceptibility. The structure of AAVGo.1, solved by cryo-EM to 2.9 Å resolution, is most similar to AAV5. Through ELISA (enzyme-linked immunosorbent assay) studies, it is shown that AAVGo.1 binds to human AAVR more strongly than do AAV2 or AAV5, and that it joins AAV5 in a class that binds exclusively to PKD domain 1 (PKD1), in contrast to other AAVs that interact primarily with PKD2. The AAVGo.1 cryo-EM structure of a complex with a PKD12 fragment of AAVR at 2.4 Å resolution shows PKD1 bound with minimal change in virus structure. There are only minor conformational adaptations in AAVR, but there is a near-rigid rotation of PKD1 with maximal displacement of the receptor domain by ~1 Å compared to PKD1 bound to AAV5. AAVGo.1 joins AAV5 as the second member of an emerging class of AAVs whose mode of receptor-binding is completely different from other AAVs, typified by AAV2. IMPORTANCE Adeno-associated virus (AAV) is a small ssDNA satellite parvovirus. As a recombinant vector with a protein shell encapsidating a transgene, recombinant AAV (rAAV) is a leading delivery vehicle for gene therapy, with two FDA-approved treatments and 150 clinical trials for 30 diseases. The human entry receptor AAVR has five PKD domains. To date, all serotypes, except AAV5, have interacted primarily with the second PKD domain, PKD2. Goat is the AAV host most distant from human with cross-species cell infectivity. AAVGo.1 is similar in structure to AAV5, the two forming a class with a distinct mode of receptor-binding. Within the two classes, binding interactions are mostly conserved, giving an indication of the latitude available in modulating delivery vectors.

Cryo-EM structure of disease-related prion fibrils provides insights into seeding barriers.

One of the least understood aspects of prion diseases is the structure of infectious prion protein aggregates. Here we report a high-resolution cryo-EM structure of amyloid fibrils formed by human prion protein with the Y145Stop mutation that is associated with a familial prion disease. This structural insight allows us not only to explain previous biochemical findings, but also provides direct support for the conformational adaptability model of prion transmissibility barriers.

Structure activity relationship of 3‐methylcytidine‐5’‐α,β‐methylenediphosphates as CD73 inhibitors

Adenosine in the tumor microenvironment acts on A2A and A2B adenosine receptors (ARs) on immune cells (T cells, dendritic cells, NK cells, macrophages and neutrophils) to prevent their activation. Therefore, a means of lowering extracellular adenosine would be beneficial in cancer immunotherapy either as a co‐therapy or monotherapy. Inhibitors of CD73 (ecto‐5’‐nucleotidase, the main extracellular adenosine‐forming enzyme) are already in clinical trials for cancer immunotherapy. Many purine (ADP) analogues have been shown to inhibit this enzyme, but we have explored pyrimidine (UDP/CDP) analogues. We recently reported N4‐substituted 3‐methylcytidine‐5’‐α,β‐methylenediphosphates as potent CD73 inhibitors. We now expand the structure activity relationship (SAR) of pyrimidine nucleotides as human CD73 inhibitors, focusing mainly on cytidine functionalization at the N4 position. 4‐Chloro (MRS4598 16, Ki 0.673 nM) and 4‐iodo (MRS4620 18, Ki 0.436 nM) substitution of the N4‐benzyloxy group increased inhibitory potency Ki by ~20‐fold. Primary alkylamine derivatives coupled through a p‐amido group and varying methylene chain length (24and 25) were functionalized congeners, for subsequent conjugation to carrier or reporter moieties. hCD73 X‐ray structures with two of the newly synthesized inhibitors indicated a ribose ring conformational adaptation, and the benzyloxyimino group (E configuration) binds to the same region (between C‐terminal and N‐terminal domains) as N4‐benzyl groups in potent adenine‐derived inhibitors. Molecular dynamics simulation identified stabilizing interactions and predicted conformational diversity around the N4‐benzyloxy moiety. Thus, by structure‐guided substitution of the N4‐benzyloxy moiety, we have greatly enhanced their inhibitory potency and added functionality enabling molecular probes. Their potential as anticancer drugs was confirmed by blocking CD73 activity in human head and neck squamous cell carcinoma (HNSCC) and palatine tonsils in situ.

Landscape-Based Protein Stability Analysis and Network Modeling of Multiple Conformational States of the SARS-CoV-2 Spike D614G Mutant: Conformational Plasticity and Frustration-Induced Allostery as Energetic Drivers of Highly Transmissible Spike Variants.

The structural and functional studies of the SARS-CoV-2 spike protein variants revealed an important role of the D614G mutation that is shared across many variants of concern (VOCs), suggesting the effect of this mutation on the enhanced virus infectivity and transmissibility. The recent structural and biophysical studies provided important evidence about multiple conformational substates of the D614G spike protein. The development of a plausible mechanistic model that can explain the experimental observations from a more unified thermodynamic perspective is an important objective of the current work. In this study, we employed efficient and accurate coarse-grained simulations of multiple structural substates of the D614G spike trimers together with the ensemble-based mutational frustration analysis to characterize the dynamics signatures of the conformational landscapes. By combining the local frustration profiling of the conformational states with residue-based mutational scanning of protein stability and network analysis of allosteric interactions and communications, we determine the patterns of mutational sensitivity in the functional regions and sites of variants. We found that the D614G mutation may induce a considerable conformational adaptability of the open states in the SARS-CoV-2 spike protein without compromising the folding stability and integrity of the spike protein. The results suggest that the D614G mutant may employ a hinge-shift mechanism in which the dynamic couplings between the site of mutation and the interprotomer hinge modulate the interdomain interactions, global mobility change, and the increased stability of the open form. This study proposes that mutation-induced modulation of the conformational flexibility and energetic frustration at the interprotomer interfaces may serve as an efficient mechanism for allosteric regulation of the SARS-CoV-2 spike proteins.

Variability in the Spatial Structure of the Central Loop in Cobra Cytotoxins Revealed by X-ray Analysis and Molecular Modeling.

Cobra cytotoxins (CTs) belong to the three-fingered protein family and possess membrane activity. Here, we studied cytotoxin 13 from Naja naja cobra venom (CT13Nn). For the first time, a spatial model of CT13Nn with both “water” and “membrane” conformations of the central loop (loop-2) were determined by X-ray crystallography. The “water” conformation of the loop was frequently observed. It was similar to the structure of loop-2 of numerous CTs, determined by either NMR spectroscopy in aqueous solution, or the X-ray method. The “membrane” conformation is rare one and, to date has only been observed by NMR for a single cytotoxin 1 from N. oxiana (CT1No) in detergent micelle. Both CT13Nn and CT1No are S-type CTs. Membrane-binding of these CTs probably involves an additional step—the conformational transformation of the loop-2. To confirm this suggestion, we conducted molecular dynamics simulations of both CT1No and CT13Nn in the Highly Mimetic Membrane Model of palmitoiloleoylphosphatidylglycerol, starting with their “water” NMR models. We found that the both toxins transform their “water” conformation of loop-2 into the “membrane” one during the insertion process. This supports the hypothesis that the S-type CTs, unlike their P-type counterparts, require conformational adaptation of loop-2 during interaction with lipid membranes.

D3PM: a comprehensive database for protein motions ranging from residue to domain

BackgroundKnowledge of protein motions is significant to understand its functions. While currently available databases for protein motions are mostly focused on overall domain motions, little attention is paid on local residue motions. Albeit with relatively small scale, the local residue motions, especially those residues in binding pockets, may play crucial roles in protein functioning and ligands binding.ResultsA comprehensive protein motion database, namely D3PM, was constructed in this study to facilitate the analysis of protein motions. The protein motions in the D3PM range from overall structural changes of macromolecule to local flip motions of binding pocket residues. Currently, the D3PM has collected 7679 proteins with overall motions and 3513 proteins with pocket residue motions. The motion patterns are classified into 4 types of overall structural changes and 5 types of pocket residue motions. Impressively, we found that less than 15% of protein pairs have obvious overall conformational adaptations induced by ligand binding, while more than 50% of protein pairs have significant structural changes in ligand binding sites, indicating that ligand-induced conformational changes are drastic and mainly confined around ligand binding sites. Based on the residue preference in binding pocket, we classified amino acids into “pocketphilic” and “pocketphobic” residues, which should be helpful for pocket prediction and drug design.ConclusionD3PM is a comprehensive database about protein motions ranging from residue to domain, which should be useful for exploring diverse protein motions and for understanding protein function and drug design. The D3PM is available on www.d3pharma.com/D3PM/index.php.