Computationally restoring the potency of a clinical antibody against Omicron.

The COVID-19 pandemic underscored the promise of monoclonal antibody-based prophylactic and therapeutic drugs1-3 and revealed how quickly viral escape can curtail effective options4,5. When the SARS-CoV-2 Omicron variant emerged in 2021, many antibody drug products lost potency, including Evusheld and its constituent, cilgavimab4-6. Cilgavimab, like its progenitor COV2-2130, is a class 3 antibody that is compatible with other antibodies in combination4 and is challenging to replace with existing approaches. Rapidly modifying such high-value antibodies to restore efficacy against emerging variants is a compelling mitigation strategy. We sought to redesign and renew the efficacy of COV2-2130 against Omicron BA.1 and BA.1.1 strains while maintaining efficacy against the dominant Delta variant. Here we show that our computationally redesigned antibody, 2130-1-0114-112, achieves this objective, simultaneously increases neutralization potency against Delta and subsequent variants of concern, and provides protection in vivo against the strains tested: WA1/2020, BA.1.1 and BA.5. Deep mutational scanning of tens of thousands of pseudovirus variants reveals that 2130-1-0114-112 improves broad potency without increasing escape liabilities. Our results suggest that computational approaches can optimize an antibody to target multiple escape variants, while simultaneously enriching potency. Our computational approach does not require experimental iterations or pre-existing binding data, thus enabling rapid response strategies to address escape variants or lessen escape vulnerabilities.

Hexagonal Lattices of HIV Capsid Proteins Explored by Simulations Based on a Thermodynamically Consistent Model.

HIV capsid proteins (CAs) may self-assemble into a variety of shapes under in vivo and in vitro conditions. Here, we employed simulations based on a residue-level coarse-grained (CG) model with full conformational flexibility to investigate hexagonal lattices, which are the underlying structural pattern for CA aggregations. Facilitated by enhanced sampling simulations to rigorously calculate CA dimerization and polymerization affinities, we calibrated our model to reproduce the experimentally measured affinities. Using the calibrated model, we performed unbiased simulations on several large systems consisting of 1512 CA subunits, allowing reversible binding and unbinding of the CAs in a thermodynamically consistent manner. In one simulation, a preassembled hexagonal CA sheet developed spontaneous curvatures reminiscent of those observed in experiments, and the edges of the sheet exhibited local curvatures larger than those of the interior. In other simulations starting with randomly distributed CAs at different concentrations, existing CA assemblies grew by binding free capsomeres to the edges and by merging with other assemblies. At high CA concentrations, rapid establishment of predominant aggregates was followed by much slower adjustments toward more regular hexagonal lattices, with increasing numbers of intact CA hexamers and pentamers being formed. Our approach of adapting a general CG model to specific systems by using experimental binding data represents a practical and effective strategy for simulating and elucidating intricate protein aggregations.

Three-dimensional High-definition Exoscope in Minimally Invasive Transforaminal Lumbar Interbody Fusion: A Retrospective Cohort Study.

OBJECTIVES: The operative microscope (OM) has revolutionized the field of modern spine surgery, however, it remains limited by several drawbacks. Recently, the exoscope (EX) system has been designed to assistant spine surgery. It provides a three-dimensional (3D) high-definition (HD) operative experience and becomes an alternative to the OM. The aim of the study was to evaluate the clinical outcomes, advantages and limitations of EX-assisted minimally invasive transforaminal lumbar interbody fusion (EMIS-TLIF) and OM-assisted MIS-TLIF (OMIS-TLIF). METHODS: The clinical outcomes were assessed in 47 patients with lumbar degenerative diseases (LDD) who underwent MIS-TLIF assisted with the OM or EX between January 2019 and September 2020. A total of 22 were treated with EMIS-TLIF, and 25 received OMIS-TLIF. Perioperative parameters (including sex, age, number of fusion levels and body mass index), perioperative parameters (operation time, intraoperative blood loss, postoperative drainage, postoperative hospitalization stay, and duration of follow-up), visual analogue scale (VAS) of back pain, VAS of leg pain, Oswestry disability index (ODI) scores and clinical outcomes were assessed and compared. Image quality, handling of equipment, ergonomics, 3D glasses and educational usefulness were scored according to a questionnaire. RESULTS: Operation time in the OMIS-TLIF group (121.92 ± 16.92 min) was significantly increased compared with that in the EMIS-TLIF group (111.00 ± 19.87 min) (P < 0.05). The VAS of the back pain and ODI scores in the EMIS-TLIF group were significantly lower compared with the OMIS-TLIF group at 1 week postoperatively (P < 0.05). The good-excellent outcomes rate was 90.91% in the EMIS-TLIF group and 88.00% in the OMIS-TLIF group, and there was no significant difference. A total of 44 visits completed the questionnaire. The results of the questionnaire showed that the EX has exhibited advantages regarding handing of equipment, ergonomics and educational usefulness, and comparable image quality as compared with the OM, however, operating surgeons complained uncomfortable sensation when wearing 3D glasses. CONCLUSIONS: The EMIS-TLIF was a safe and effective procedure in the management of LDD as compared with the OMIS-LIF. Meanwhile, EMIS-TLIF might resulted in a short operation time.

Computationally restoring the potency of a clinical antibody against SARS-CoV-2 Omicron subvariants.

The COVID-19 pandemic underscored the promise of monoclonal antibody-based prophylactic and therapeutic drugs1-3, but also revealed how quickly viral escape can curtail effective options4,5. With the emergence of the SARS-CoV-2 Omicron variant in late 2021, many clinically used antibody drug products lost potency, including Evusheld™ and its constituent, cilgavimab4,6. Cilgavimab, like its progenitor COV2-2130, is a class 3 antibody that is compatible with other antibodies in combination4 and is challenging to replace with existing approaches. Rapidly modifying such high-value antibodies with a known clinical profile to restore efficacy against emerging variants is a compelling mitigation strategy. We sought to redesign COV2-2130 to rescue in vivo efficacy against Omicron BA.1 and BA.1.1 strains while maintaining efficacy against the contemporaneously dominant Delta variant. Here we show that our computationally redesigned antibody, 2130-1-0114-112, achieves this objective, simultaneously increases neutralization potency against Delta and many variants of concern that subsequently emerged, and provides protection in vivo against the strains tested, WA1/2020, BA.1.1, and BA.5. Deep mutational scanning of tens of thousands pseudovirus variants reveals 2130-1-0114-112 improves broad potency without incurring additional escape liabilities. Our results suggest that computational approaches can optimize an antibody to target multiple escape variants, while simultaneously enriching potency. Because our approach is computationally driven, not requiring experimental iterations or pre-existing binding data, it could enable rapid response strategies to address escape variants or pre-emptively mitigate escape vulnerabilities.

Large-scale application of free energy perturbation calculations for antibody design.

Alchemical free energy perturbation (FEP) is a rigorous and powerful technique to calculate the free energy difference between distinct chemical systems. Here we report our implementation of automated large-scale FEP calculations, using the Amber software package, to facilitate antibody design and evaluation. In combination with Hamiltonian replica exchange, our FEP simulations aim to predict the effect of mutations on both the binding affinity and the structural stability. Importantly, we incorporate multiple strategies to faithfully estimate the statistical uncertainties in the FEP results. As a case study, we apply our protocols to systematically evaluate variants of the m396 antibody for their conformational stability and their binding affinity to the spike proteins of SARS-CoV-1 and SARS-CoV-2. By properly adjusting relevant parameters, the particle collapse problems in the FEP simulations are avoided. Furthermore, large statistical errors in a small fraction of the FEP calculations are effectively reduced by extending the sampling, such that acceptable statistical uncertainties are achieved for the vast majority of the cases with a modest total computational cost. Finally, our predicted conformational stability for the m396 variants is qualitatively consistent with the experimentally measured melting temperatures. Our work thus demonstrates the applicability of FEP in computational antibody design.

Toward Convergence in Free Energy Calculations for Protein Conformational Changes: A Case Study on the Thin Gate of Mhp1 Transporter.

It has been challenging to obtain reliable free energies for protein conformational changes from all-atom molecular dynamics simulations, despite the availability of many enhanced sampling techniques. To alleviate the difficulties associated with the enormous complexity of the conformational space, here we propose a few practical strategies for such calculations, including (1) a stringent method to examine convergence by comparing independent simulations starting from different initial coordinates, (2) adoption of multistep schemes in which the complete conformational change consists of multiple transition steps, each sampled using a distinct reaction coordinate, and (3) application of boundary restraints to simplify the conformational space. We demonstrate these strategies on the conformational changes between the outward-facing and outward-occluded states of the Mhp1 membrane transporter, obtaining the equilibrium thermodynamics of the relevant metastable states, the kinetic rates between these states, and the reactive trajectories that reveal the atomic details of spontaneous transitions. Our approaches thus promise convergent and reliable calculations to examine intuition-based hypotheses and to eventually elucidate the underlying molecular mechanisms of reversible conformational changes in complex protein systems.

Three-dimensional high-definition exoscope (Kestrel View II) in anterior cervical discectomy and fusion: a valid alternative to operative microscope-assisted surgery.

BACKGROUND: Operative microscope (OM) has greatly advanced modern spine surgery, but remains limited by several drawbacks. Therefore, a three-dimensional (3D) high-definition (HD) exoscope (EX) (Kestrel View II, Mataka Kohli, Japan) system has been developed and used as an alternative to the OM. The aim of this study was to assess and compare the perioperative data and clinical outcomes of anterior cervical discectomy and fusion (ACDF) procedure with either an EX or OM. METHODS: Forty-eight patients with cervical spondylotic myelopathy (CSM) underwent ACDF assisted by the EX or OM between January 2019 and December 2019. We collected and compared data on operative time, intraoperative bleeding, postoperative hospitalization stay, complications, and clinical outcomes between the two groups. The clinical outcomes were evaluated by using visual analogue scale (VAS) scores, Japanese Orthopedic Association (JOA) scores, the recovery rate of JOA scores, and Odom criteria. RESULTS: The operative time in the EX group was significantly shorter than that in the OM group (P < 0.05). The VAS and JOA scores were significantly improved in both groups after surgery (P < 0.05). In addition, the VAS scores in the EX group were significantly lower than those in the OM group at 1 week postoperatively (P < 0.05). The good-to-excellent outcome rates were 90.48 and 88.89% in the EX group and OM group, respectively, whereas the complication occurrence rates of the EX group and OM group were 4.76 and 11.11%, respectively. CONCLUSIONS: EX-assisted and OM-assisted ACDF resulted in similar clinical outcomes for CSM, while EX-assisted surgery may be related to a short operative time and fewer complications.

Discovery of Small-Molecule Inhibitors of SARS-CoV-2 Proteins Using a Computational and Experimental Pipeline.

A rapid response is necessary to contain emergent biological outbreaks before they can become pandemics. The novel coronavirus (SARS-CoV-2) that causes COVID-19 was first reported in December of 2019 in Wuhan, China and reached most corners of the globe in less than two months. In just over a year since the initial infections, COVID-19 infected almost 100 million people worldwide. Although similar to SARS-CoV and MERS-CoV, SARS-CoV-2 has resisted treatments that are effective against other coronaviruses. Crystal structures of two SARS-CoV-2 proteins, spike protein and main protease, have been reported and can serve as targets for studies in neutralizing this threat. We have employed molecular docking, molecular dynamics simulations, and machine learning to identify from a library of 26 million molecules possible candidate compounds that may attenuate or neutralize the effects of this virus. The viability of selected candidate compounds against SARS-CoV-2 was determined experimentally by biolayer interferometry and FRET-based activity protein assays along with virus-based assays. In the pseudovirus assay, imatinib and lapatinib had IC50 values below 10 µM, while candesartan cilexetil had an IC50 value of approximately 67 µM against Mpro in a FRET-based activity assay. Comparatively, candesartan cilexetil had the highest selectivity index of all compounds tested as its half-maximal cytotoxicity concentration 50 (CC50) value was the only one greater than the limit of the assay (>100 µM).

Assessing the Perturbing Effects of Drugs on Lipid Bilayers Using Gramicidin Channel-Based In Silico and In Vitro Assays.

Partitioning of bioactive molecules, including drugs, into cell membranes may produce indiscriminate changes in membrane protein function. As a guide to safe drug development, it therefore becomes important to be able to predict the bilayer-perturbing potency of hydrophobic/amphiphilic drugs candidates. Toward this end, we exploited gramicidin channels as molecular force probes and developed in silico and in vitro assays to measure drugs' bilayer-modifying potency. We examined eight drug-like molecules that were found to enhance or suppress gramicidin channel function in a thick 1,2-dierucoyl-sn-glycero-3-phosphocholine (DC22:1PC) but not in thin 1,2-dioleoyl-sn-glycero-3-phosphocholine (DC18:1PC) lipid bilayer. The mechanism underlying this difference was attributable to the changes in gramicidin dimerization free energy by drug-induced perturbations of lipid bilayer physical properties and bilayer-gramicidin interactions. The combined in silico and in vitro approaches, which allow for predicting the perturbing effects of drug candidates on membrane protein function, have implications for preclinical drug safety assessment.

Binding Affinity Prediction by Pairwise Function Based on Neural Network.

We present a new approach to estimate the binding affinity from given three-dimensional poses of protein-ligand complexes. In this scheme, every protein-ligand atom pair makes an additive free-energy contribution. The sum of these pairwise contributions then gives the total binding free energy or the logarithm of the dissociation constant. The pairwise contribution is calculated by a function implemented via a neural network that takes the properties of the two atoms and their distance as input. The pairwise function is trained using a portion of the PDBbind 2018 data set. The model achieves good accuracy for affinity predictions when evaluated with PDBbind 2018 and with the CASF-2016 benchmark, comparing favorably to many scoring functions such as that of AutoDock Vina. The framework here may be extended to incorporate other factors to further improve its accuracy and power.

Calculating Single-Channel Permeability and Conductance from Transition Paths.

Permeability and conductance are the major transport properties of membrane channels, quantifying the rate of channel crossing by the solute. It is highly desirable to calculate these quantities in all-atom molecular dynamics simulations. When the solute crossing rate is low, however, direct methods would require prohibitively long simulations, and one thus typically adopts alternative strategies based on the free energy of single solute along the channel. Here we present a new method to calculate the crossing rate by initiating unbiased trajectories in which the solute is released at the free energy barrier. In this method, the total time the solute spends in the barrier region during a channel crossing (transition path) is used to determine the kinetic rate. Our method achieves a significantly higher statistical accuracy than the classical reactive flux method, especially for diffusive barrier crossing. Our test on ion permeation through a carbon nanotube verifies that the method correctly predicts the crossing rate and reproduces the spontaneous crossing events as in long equilibrium simulations. The rigorous and efficient method here will be valuable for quantitatively connecting simulations to experimental measurement of membrane channels.

Vitamin E Has Reduced Affinity for a Polyunsaturated Phospholipid: An Umbrella Sampling Molecular Dynamics Simulations Study.

Vitamin E is an essential micronutrient. The primary function of this lipid-soluble antioxidant is to protect membrane phospholipids from oxidation. Whether vitamin E preferentially interacts with polyunsaturated phospholipids to optimize protection of the lipid species most vulnerable to oxidative attack has been an unanswered question for a long time. In this work, we compared the binding of α-tocopherol (αtoc), the form of vitamin E retained by the human body, in bilayers composed of polyunsaturated 1-stearoyl-2-docosahexaenoylphosphatidylcholine (SDPC, 18:0-22:6PC) and, as a control, monounsaturated 1-stearoyl-2-oleoylphosphatidylcholine (SOPC, 18:0-18:1PC) by umbrella sampling molecular dynamics simulations. From the potential of mean force as a function depth within the bilayer, we find that the binding energy of αtoc is less in SDPC (Δ Gbind = 16.7 ± 0.3 kcal/mol) than that in SOPC (Δ Gbind = 18.3 ± 0.4 kcal/mol). The lower value in SDPC is ascribed to the high disorder of polyunsaturated fatty acids that produces a less tightly packed arrangement. Deformation of the bilayer is observed during desorption, indicating that phosphatidylcholine (PC)-PC and αtoc-PC interactions contribute to the binding energy. Our results do not support the proposal that vitamin E interacts more favorably with polyunsaturated phospholipids.

Calculating transition rates from durations of transition paths.

Calculating the kinetic rates for rare transitions is an important objective for molecular simulations. Here I prove equalities that relate the transition rates to the equilibrium free energy and the statistics of the transition paths. In particular, the durations of the transition paths within given intervals of the reaction coordinate provide the kinetic pre-factor in the rate formula. Based on the available free energy, the transition rates can further be rigorously calculated by initiating forward and backward simulations and evaluating the duration of each transition path. Validation on a model system confirms that the approach correctly predicts the transition rates from the simulations and demonstrates that whereas the relations here are general and valid for any chosen reaction coordinate, a good reaction coordinate will enable a more efficient sampling of the transition paths and thus a more reliable rate calculation.

Parameter Optimization for Interaction between C-Terminal Domains of HIV-1 Capsid Protein.

HIV-1 capsid proteins (CAs) assemble into a capsid that encloses the viral RNA. The binding between a pair of C-terminal domains (CTDs) constitutes a major interface in both the CA dimers and the large CA assemblies. Here, we attempt to use a general residue-level coarse-grained model to describe the interaction between two isolated CTDs in Monte Carlo simulations. With the standard parameters that depend only on the residue types, the model predicts a much weaker binding in comparison to the experiments. Detailed analysis reveals that some Lennard-Jones parameters are not compatible with the experimental CTD dimer structure, thus resulting in an unfavorable interaction energy. To improve the model for the CTD binding, we introduce ad hoc modifications to a small number of Lennard-Jones parameters for some specific pairs of residues at the binding interface. Through a series of extensive Monte Carlo simulations, we identify the optimal parameters for the CTD-CTD interactions. With the refined model parameters, both the binding affinity (with a dissociation constant of 13 ± 2 µM) and the binding mode are in good agreement with the experimental data. This study demonstrates that the general interaction model based on the Lennard-Jones potential, with some modest adjustment of the parameters for key residues, could correctly reproduce the reversible protein binding, thus potentially applicable for simulating the thermodynamics of the CA assemblies.

Thermodynamics of Protein Folding Studied by Umbrella Sampling along a Reaction Coordinate of Native Contacts.

Spontaneous transitions between the native and non-native protein conformations are normally rare events that hardly take place in typical unbiased molecular dynamics simulations. It was recently demonstrated that such transitions can be well described by a reaction coordinate, Q, that represents the collective fraction of the native contacts between the protein atoms. Here we attempt to use this reaction coordinate to enhance the conformational sampling. We perform umbrella sampling simulations with biasing potentials on Q for two model proteins, Trp-Cage and BBA, using the CHARMM force field. Hamiltonian replica exchange is implemented in these simulations to further facilitate the sampling. The simulations appear to have reached satisfactory convergence, resulting in unbiased free energies as a function of Q. In addition to the native structure, multiple folded conformations are identified in the reconstructed equilibrium ensemble. Some conformations without any native contacts nonetheless have rather compact geometries and are stabilized by hydrogen bonds not present in the native structure. Whereas the enhanced sampling along Q reasonably reproduces the equilibrium conformational space, we also find that the folding of an α-helix in Trp-Cage is a slow degree of freedom orthogonal to Q and therefore cannot be accelerated by biasing the reaction coordinate. Overall, we conclude that whereas Q is an excellent parameter to analyze the simulations, it is not necessarily a perfect reaction coordinate for enhanced sampling, and better incorporation of other slow degrees of freedom may further improve this reaction coordinate.

Structural Model of the Tubular Assembly of the Rous Sarcoma Virus Capsid Protein.

The orthoretroviral capsid protein (CA) assembles into polymorphic capsids, whose architecture, assembly, and stability are still being investigated. The N-terminal and C-terminal domains of CA (NTD and CTD, respectively) engage in both homotypic and heterotypic interactions to create the capsid. Hexameric turrets formed by the NTD decorate the majority of the capsid surface. We report nearly complete solid-state NMR (ssNMR) resonance assignments of Rous sarcoma virus (RSV) CA, assembled into hexamer tubes that mimic the authentic capsid. The ssNMR assignments show that, upon assembly, large conformational changes occur in loops connecting helices, as well as the short 310 helix initiating the CTD. The interdomain linker becomes statically disordered. Combining constraints from ssNMR and cryo-electron microscopy (cryo-EM), we establish an atomic resolution model of the RSV CA tubular assembly using molecular dynamics flexible fitting (MDFF) simulations. On the basis of comparison of this MDFF model with an earlier-derived crystallographic model for the planar assembly, the induction of curvature into the RSV CA hexamer lattice arises predominantly from reconfiguration of the NTD-CTD and CTD trimer interfaces. The CTD dimer and CTD trimer interfaces are also intrinsically variable. Hence, deformation of the CA hexamer lattice results from the variable displacement of the CTDs that surround each hexameric turret. Pervasive H-bonding is found at all interdomain interfaces, which may contribute to their malleability. Finally, we find helices at the interfaces of HIV and RSV CA assemblies have very different contact angles, which may reflect differences in the capsid assembly pathway for these viruses.

Finite Temperature String Method with Umbrella Sampling: Application on a Side Chain Flipping in Mhp1 Transporter.

Protein conformational change is of central importance in molecular biology. Here we demonstrate a computational approach to characterize the transition between two metastable conformations in all-atom simulations. Our approach is based on the finite temperature string method, and the implementation is essentially a generalization of umbrella sampling simulations with Hamiltonian replica exchange. We represent the transition pathway by a curve in the conformational space, with the curve parameter taken as the reaction coordinate. Our approach can efficiently refine a transition pathway and compute a one-dimensional free energy as a function of the reaction coordinate. A diffusion model can then be used to calculate the forward and backward transition rates, the major kinetic quantities for the transition. We applied the approach on a local transition in the ligand-free Mhp1 transporter, between its outward-facing conformation and an intermediate conformation with the side chain of Phe305 flipped to the outside of the protein. Our simulations predict that the flipped-out position of this side chain has a free energy 6.5 kcal/mol higher than the original position in the crystal structure, and that the forward and backward transition rates are in the millisecond and submicrosecond time scales, respectively.

Curcumin Suppresses Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells through the Inhibition of Akt/mTOR Pathway.

Curcumin has exhibited a protective effect against development of renal fibrosis in animal models, however, its underlying molecular mechanisms are largely unclear. Therefore, we investigated the anti-fibrosis effects of curcumin in transforming growth factor-ß1 (TGF-ß1)-induced epithelial-to-mesenchymal transition (EMT), and the mechanism by which it mediates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Human kidney tubular epithelial cells (HKCs) were treated with TGF-ß1 or curcumin alone, or TGF-ß1 in combination with curcumin. The effect of curcumin on cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Expression of E-cadherin, cytokeratin, vimentin, alpha smooth muscle actin (α-SMA), fibroblast-specific protein 1 (FSP1) and key proteins of Akt/mammalian target of rapamycin (mTOR) pathway were analyzed by immunocytochemistry, real-time PCR and Western blot. Low dose curcumin (3.125 and 25 µmol/L) effectively promoted HKC proliferation. When HKCs were co-incubated with TGF-ß1 and curcumin for 72 h, curcumin maintained the epithelial morphology in a dose-dependent manner, decreased expression of vimentin, α-SMA and FSP1 normally induced by TGF-ß1, and increased expression of E-cadherin, cytokeratin. Importantly, we found that curcumin reduced Akt, mTOR and P70S6K phosphorylation, effectively suppressing the activity of the Akt/mTOR pathway in HKCs. Curcumin also promoted HKC proliferation, and antagonized TGF-ß1-driven EMT through the inhibition of Akt/mTOR pathway activity, which may suggest an alternative therapy for renal fibrosis.

Construction of a novel coarse grain model for simulations of HIV capsid assembly to capture the backbone structure and inter-domain motions in solution.

We show the construction of a novel coarse grain model for simulations of HIV capsid assembly based on four structural models of HIV capsid proteins: isolated hexamer 3H47.pdb, tubular assembly 3J34.pdb, isolated pentamer 3P05.pdb and C-terminus dimer 2KOD.pdb. The data demonstrates the derivation of inter-domain motions from all atom Molecular Dynamics simulations and comparison with the motions derived from the analysis of solution NMR results defined in 2M8L.pdb. Snapshots from a representative Monte Carlo simulation with 128 dimeric subunit proteins based on 3J34.pdb are shown in addition to the quantitative analysis of its assembly pathway. Movies of the assembly process are compiled with snapshots of representative simulations of four structural models. The methods and data in this article were utilized in Qiao et al. (in press) [1] to probe the mechanism of polymorphism and curvature control of HIV capsid assembly.

Mechanism of polymorphism and curvature of HIV capsid assemblies probed by 3D simulations with a novel coarse grain model.

BACKGROUND: During the maturation process, HIV capsid proteins self-assemble into polymorphic capsids. The strong polymorphism precludes high resolution structural characterization under in vivo conditions. In spite of the determination of structural models for various in vitro assemblies of HIV capsid proteins, the assembly mechanism is still not well-understood. METHODS: We report 3D simulations of HIV capsid proteins by a novel coarse grain model that captures the backbone of the rigid segments in the protein accurately. The effects of protein dynamics on assembly are emulated by a static ensemble of subunits in conformations derived from molecular dynamics simulation. RESULTS: We show that HIV capsid proteins robustly assemble into hexameric lattices in a range of conditions where trimers of dimeric subunits are the dominant oligomeric intermediates. Variations of hexameric lattice curvatures are observed in simulations with subunits of variable inter-domain orientations mimicking the conformation distribution in solution. Simulations with subunits based on pentameric structural models lead to assembly of sharp curved structures resembling the tips of authentic HIV capsids, along a distinct pathway populated by tetramers and pentamers with the characteristic quasi-equivalency of viral capsids. CONCLUSIONS: Our results suggest that the polymorphism assembly is triggered by the inter-domain dynamics of HIV capsid proteins in solution. The assembly of highly curved structures arises from proteins in conformation with a highly specific inter-domain orientation. SIGNIFICANCE: Our work proposes a mechanism of HIV capsid assembly based on available structural data, which can be readily verified. Our model can be applied to other large biomolecular assemblies.