Entropic Overcompensation of the N501Y Mutation on SARS-CoV-2 S Binding to ACE2.

Recent experimental work has shown that the N501Y mutation in the SARS-CoV-2 S glycoprotein's receptor binding domain (RBD) increases binding affinity to the angiotensin-converting enzyme 2 (ACE2), primarily by overcompensating for a less favorable enthalpy of binding by greatly reducing the entropic penalty for complex formation, but the basis for this entropic overcompensation is not clear [Prévost et al. J. Biol. Chem.2021, 297, 101151]. We use all-atom molecular dynamics simulations and free-energy calculations to qualitatively assess the impact of the N501Y mutation on the enthalpy and entropy of binding of RBD to ACE2. Our calculations correctly predict that N501Y causes a less favorable enthalpy of binding to ACE2 relative to the original strain. Furthermore, we show that this is overcompensated for by a more entropically favorable increase in large-scale quaternary flexibility and intraprotein root mean square fluctuations of residue positions upon binding in both RBD and ACE2. The enhanced quaternary flexibility stems from N501Y's ability to remodel the inter-residue interactions between the two proteins away from interactions central to the epitope and toward more peripheral interactions. These findings suggest that an important factor in determining protein-protein binding affinity is the degree to which fluctuations are distributed throughout the complex and that residue mutations that may seem to result in weaker interactions than their wild-type counterparts may yet result in increased binding affinity thanks to their ability to suppress unfavorable entropy changes upon binding.

Impact of temperature on the affinity of SARS-CoV-2 Spike glycoprotein for host ACE2.

The seasonal nature of outbreaks of respiratory viral infections with increased transmission during low temperatures has been well established. Accordingly, temperature has been suggested to play a role on the viability and transmissibility of SARS-CoV-2, the virus responsible for the COVID-19 pandemic. The receptor-binding domain (RBD) of the Spike glycoprotein is known to bind to its host receptor angiotensin-converting enzyme 2 (ACE2) to initiate viral fusion. Using biochemical, biophysical, and functional assays to dissect the effect of temperature on the receptor-Spike interaction, we observed a significant and stepwise increase in RBD-ACE2 affinity at low temperatures, resulting in slower dissociation kinetics. This translated into enhanced interaction of the full Spike glycoprotein with the ACE2 receptor and higher viral attachment at low temperatures. Interestingly, the RBD N501Y mutation, present in emerging variants of concern (VOCs) that are fueling the pandemic worldwide (including the B.1.1.7 (α) lineage), bypassed this requirement. This data suggests that the acquisition of N501Y reflects an adaptation to warmer climates, a hypothesis that remains to be tested.

Target identification for repurposed drugs active against SARS-CoV-2 via high-throughput inverse docking.

Screening already approved drugs for activity against a novel pathogen can be an important part of global rapid-response strategies in pandemics. Such high-throughput repurposing screens have already identified several existing drugs with potential to combat SARS-CoV-2. However, moving these hits forward for possible development into drugs specifically against this pathogen requires unambiguous identification of their corresponding targets, something the high-throughput screens are not typically designed to reveal. We present here a new computational inverse-docking protocol that uses all-atom protein structures and a combination of docking methods to rank-order targets for each of several existing drugs for which a plurality of recent high-throughput screens detected anti-SARS-CoV-2 activity. We demonstrate validation of this method with known drug-target pairs, including both non-antiviral and antiviral compounds. We subjected 152 distinct drugs potentially suitable for repurposing to the inverse docking procedure. The most common preferential targets were the human enzymes TMPRSS2 and PIKfyve, followed by the viral enzymes Helicase and PLpro. All compounds that selected TMPRSS2 are known serine protease inhibitors, and those that selected PIKfyve are known tyrosine kinase inhibitors. Detailed structural analysis of the docking poses revealed important insights into why these selections arose, and could potentially lead to more rational design of new drugs against these targets.

Roles of the Coupling Agent and Surfactant in Droplet Structure in Sizing Emulsions: A Molecular Dynamics Simulations Study.

Sizing emulsions used as glass fiber surface treatments in composites manufacturing are aqueous suspensions of hydrophobic film formers, surface coupling agents, and surfactants. We employ all-atom molecular dynamics simulations to characterize droplet structures in several aqueous blends of the film-former diglycidyl ether of bisphenol A, coupling agent glycidoxypropyl trimethoxysilane, and a triblock copolymer surfactant (Pluronic L35 PEO/PPO copolymer). We show that the quasi-equilibrium states of emulsion droplets are invariant to different initial configurations. We examine the role of the surfactant in determining coupling agent partitioning between the droplet shell and corona and coupling agent cluster size distributions. This work takes a step toward systematic understanding of the sizing chemistry to optimize the interface between the glass and the resin in commercially relevant composites.

Atomistic simulation of volumetric properties of epoxy networks: effect of monomer length.

Properties of epoxy thermosets can be varied broadly to suit design requirements by altering the chemistry of the component agents. Atomistically-detailed molecular dynamics simulations are well-suited for molecular insight into the structure-property relationship for a rational tailoring of the chemistry. Since the macroscopic properties of interest for applications emerge hierarchically from molecular-scale chemical interactions, seamless integration of experiment, computation, and theory is of great interest. Recently, a Specific Volume-Cooling Rate analysis protocol was successfully developed to quantitatively compare the volumetric properties of an epoxy network model with experimental results in the literature, in spite of the nine orders of magnitude mismatch in the accessible time-scales. Here, we extend the application of the method for two epoxy networks in the same class of chemistry but whose monomers have a higher number of repeating units compared to the previous one for validating the generality of our approach. We observed that atomistic simulations are able to predict the experimental temperature trend of the specific volume within 0.4% for both these networks. Using the William-Landel-Ferry equation to account for rate differences, we also see good agreement between the computational and experimental values of the glass transition temperature.

Identification of a glycan cluster in gp120 essential for irreversible HIV-1 lytic inactivation by a lectin-based recombinantly engineered protein conjugate.

We previously discovered a class of recombinant lectin conjugates, denoted lectin DLIs ('dual-acting lytic inhibitors') that bind to the HIV-1 envelope (Env) protein trimer and cause both lytic inactivation of HIV-1 virions and cytotoxicity of Env-expressing cells. To facilitate mechanistic investigation of DLI function, we derived the simplified prototype microvirin (MVN)-DLI, containing an MVN domain that binds high-mannose glycans in Env, connected to a DKWASLWNW sequence (denoted 'Trp3') derived from the membrane-associated region of gp41. The relatively much stronger affinity of the lectin component than Trp3 argues that the lectin functions to capture Env to enable Trp3 engagement and consequent Env membrane disruption and virolysis. The relatively simplified engagement pattern of MVN with Env opened up the opportunity, pursued here, to use recombinant glycan knockout gp120 variants to identify the precise Env binding site for MVN that drives DLI engagement and lysis. Using mutagenesis combined with a series of biophysical and virological experiments, we identified a restricted set of residues, N262, N332 and N448, all localized in a cluster on the outer domain of gp120, as the essential epitope for MVN binding. By generating these mutations in the corresponding HIV-1 virus, we established that the engagement of this glycan cluster with the lectin domain of MVN*-DLI is the trigger for DLI-derived virus and cell inactivation. Beyond defining the initial encounter step for lytic inactivation, this study provides a guide to further elucidate DLI mechanism, including the stoichiometry of Env trimer required for function, and downstream DLI optimization.

Metastable HIV-1 Surface Protein Env Sensitizes Cell Membranes to Transformation and Poration by Dual-Acting Virucidal Entry Inhibitors.

Dual-acting virucidal entry inhibitors (DAVEIs) have previously been shown to cause irreversible inactivation of HIV-1 Env-presenting pseudovirus by lytic membrane transformation. This study examined whether this transformation could be generalized to include membranes of Env-presenting cells. Flow cytometry was used to analyze HEK293T cells transiently transfected with increasing amounts of DNA encoding JRFL Env, loaded with calcein dye, and treated with serial dilutions of microvirin (Q831K/M83R)-DAVEI. Comparing calcein retention against intact Env expression (via Ab 35O22) on individual cells revealed effects proportional to Env expression. "Low-Env" cells experienced transient poration and calcein leakage, while "high-Env" cells were killed. The cell-killing effect was confirmed with an independent mitochondrial activity-based cell viability assay, showing dose-dependent cytotoxicity in response to DAVEI treatment. Transfection with increasing quantities of Env DNA showed further shifts toward "High-Env" expression and cytotoxicity, further reinforcing the Env dependence of the observed effect. Controls with unlinked DAVEI components showed no effect on calcein leakage or cell viability, confirming a requirement for covalently linked DAVEI compounds to achieve Env transformation. These data demonstrate that the metastability of Env is an intrinsic property of the transmembrane protein complex and can be perturbed to cause membrane disruption in both virus and cell contexts.

Mapping saddles and minima on free energy surfaces using multiple climbing strings.

Locating saddle points on free energy surfaces is key in characterizing multistate transition events in complicated molecular-scale systems. Because these saddle points represent transition states, determining minimum free energy pathways to these saddles and measuring their free energies relative to their connected minima are further necessary, for instance, to estimate transition rates. In this work, we propose a new multistring version of the climbing string method in collective variables to locate all saddles and corresponding pathways on free energy surfaces. The method uses dynamic strings to locate saddles and static strings to keep a history of prior strings converged to saddles. Interaction of the dynamic strings with the static strings is used to avoid the convergence to already-identified saddles. Additionally, because the strings approximate curves in collective-variable space, and we can measure free energy along each curve, identification of any saddle's two connected minima is guaranteed. We demonstrate this method to map the network of stationary points in the 2D and 4D free energy surfaces of alanine dipeptide and alanine tripeptide, respectively.

Restricted HIV-1 Env glycan engagement by lectin-reengineered DAVEI protein chimera is sufficient for lytic inactivation of the virus.

We previously reported a first-generation recombinant DAVEI construct, a dual action virus entry inhibitor composed of cyanovirin-N (CVN) fused to a membrane proximal external region or its derivative peptide Trp3. DAVEI exhibits potent and irreversible inactivation of HIV-1 (human immunodeficiency virus) viruses by dual engagement of gp120 and gp41. However, the promiscuity of CVN to associate with multiple glycosylation sites in gp120 and its multivalency limit current understanding of the molecular arrangement of the DAVEI molecules on trimeric spike. Here, we constructed and investigated the virolytic function of second-generation DAVEI molecules using a simpler lectin, microvirin (MVN). MVN is a monovalent lectin with a single glycan-binding site in gp120, is structurally similar to CVN and exhibits no toxicity or mitogenicity, both of which are liabilities with CVN. We found that, like CVN-DAVEI-L2-3Trp (peptide sequence DKWASLWNW), MVN-DAVEI2-3Trp exploits a similar mechanism of action for inducing HIV-1 lytic inactivation, but by more selective gp120 glycan engagement. By sequence redesign, we significantly increased the potency of MVN-DAVEI2-3Trp protein. Unlike CVN-DAVEI2-3Trp, re-engineered MVN-DAVEI2-3Trp(Q81K/M83R) virolytic activity and its interaction with gp120 were both competed by 2G12 antibody. That the lectin domain in DAVEIs can utilize MVN without loss of virolytic function argues that restricted HIV-1 Env (envelope glycoprotein) glycan engagement is sufficient for virolysis. It also shows that DAVEI lectin multivalent binding with gp120 is not required for virolysis. MVN-DAVEI2-3Trp(Q81K/M83R) provides an improved tool to elucidate productive molecular arrangements of Env-DAVEI enabling virolysis and also opens the way to form DAVEI fusions made up of gp120-binding small molecules linked to Trp3 peptide.

Multiscale implementation of infinite-swap replica exchange molecular dynamics.

Replica exchange molecular dynamics (REMD) is a popular method to accelerate conformational sampling of complex molecular systems. The idea is to run several replicas of the system in parallel at different temperatures that are swapped periodically. These swaps are typically attempted every few MD steps and accepted or rejected according to a Metropolis-Hastings criterion. This guarantees that the joint distribution of the composite system of replicas is the normalized sum of the symmetrized product of the canonical distributions of these replicas at the different temperatures. Here we propose a different implementation of REMD in which (i) the swaps obey a continuous-time Markov jump process implemented via Gillespie's stochastic simulation algorithm (SSA), which also samples exactly the aforementioned joint distribution and has the advantage of being rejection free, and (ii) this REMD-SSA is combined with the heterogeneous multiscale method to accelerate the rate of the swaps and reach the so-called infinite-swap limit that is known to optimize sampling efficiency. The method is easy to implement and can be trivially parallelized. Here we illustrate its accuracy and efficiency on the examples of alanine dipeptide in vacuum and C-terminal ß-hairpin of protein G in explicit solvent. In this latter example, our results indicate that the landscape of the protein is a triple funnel with two folded structures and one misfolded structure that are stabilized by H-bonds.

Roles of conserved tryptophans in trimerization of HIV-1 membrane-proximal external regions: Implications for virucidal design via alchemical free-energy molecular simulations.

The Dual-Action Virolytic Entry Inhibitors, or "DAVEI's," are a class of recombinant fusions of a lectin, a linker polypeptide, and a 15-residue fragment from the membrane-proximal external region (MPER) of HIV-1 gp41. DAVEI's trigger rupture of HIV-1 virions, and the interaction site between DAVEI MPER and HIV-1 lies in the gp41 component of the envelope glycoprotein Env. Here, we explore the hypothesis that DAVEI MPER engages Env gp41 in a mode structurally similar to a crystallographic MPER trimer. We used alchemical free-energy perturbation to assess the thermodynamic roles of each of the four conserved tryptophan residues on each protomer of MPER3 . We found that a W666A mutation had a large positive ΔΔG for all three protomers, while W672A had a large positive ΔΔG for only two of the three protomers, with the other tryptophans remaining unimportant contributors to MPER3 stability. The protomer for which W672 is not important is unique in the placement of its W666 sidechain between the other two protomers. We show that the unique orientation of this W666 sidechain azimuthally rotates its protomer away from the orientation it would have if the trimer were symmetric, resulting in the diminished interaction of this W672 with the rest of MPER3 . Our findings are consistent with our previous experimental study of W-to-A mutants of DAVEI. This suggests that DAVEI MPER may engage HIV-1 Env to form a mixed trimer state in which one DAVEI MPER forms a trimer by displacing a more weakly interacting protomer of the endogenous Env MPER trimer.

Lipid flip-flop vs. lateral diffusion in the relaxation of hemifusion diaphragms.

Molecular dynamics simulations of a solvent-free coarse-grained lipid model are used to characterize the mechanisms by which lipid-bilayer hemifusion diaphragm (HD) intermediates relax, across a range of global compositions of negative intrinsic curvature (NIC) lipids and neutral-curvature lipids. At low concentrations of NIC lipids, rapid fission produces a double bilayer end state through a lateral diffusion-based mechanism enabled by spontaneous rim-pore defects. At moderately higher NIC lipid concentrations, rim pores are absent and stable leaflet three-junctions persist, revealing an HD relaxation mechanism entirely reliant on lipid flip-flop, and end states that are either stable fusion pores or stable HD's. These fusogenic systems exhibit dynamics highly dependent on NIC lipid concentration via an underlying sensitivity of flip-flop rates for neutral lipids on NIC lipid concentration. This work illustrates that HD dynamics may be altered through regulation of lipid composition in the immediate three-junction region. This work further highlights the potential role of flippases in biological fusion and the importance of lipid composition on fusion dynamics.

Rate of hemifusion diaphragm dissipation and ability to form three-junction bound HD determined by lipid composition.

Though the hemifusion diaphragm (HD) is widely accepted as an intermediate in bilayer membrane fusion, lipid contributions toward HD stability and dynamics are still not fully understood. In this paper, we study large, binary, protein-free HD systems at varying compositions of negative intrinsic curvature (NIC) lipids using molecular dynamics (MD) simulations of a solvent-free coarse-grained lipid model. Under MD, initially created HDs are found to relax to three major end states depending on the composition and lipid intrinsic curvature. Low compositions of NIC lipids or weak intrinsic curvature result in double-bilayer end states, and moderate compositions of moderate to strong NIC lipids result in metastable fusion pores. Importantly, high compositions of moderate NIC lipids result in a metastable HD that persists beyond µs time scales. NIC lipids stabilize the HD by filling the junction core around the HD. Sorting of NIC lipids toward the three-junction region occurs in fused-endpoint systems, but no significant sorting was seen in systems that end in a double bilayer indicating that high line tension at the triple junction drives HD dissipation faster than sorting can enrich that junction enough to lower that line tension. The appearance of three end states dependent on the NIC lipid composition highlights the necessity of NIC lipids for non-leaky fusion.

Lytic Inactivation of Human Immunodeficiency Virus by Dual Engagement of gp120 and gp41 Domains in the Virus Env Protein Trimer.

We recently reported the discovery of a recombinant chimera, denoted DAVEI (dual-acting virucidal entry inhibitor), which is able to selectively cause specific and potent lytic inactivation of both pseudotyped and fully infectious human immunodeficiency virus (HIV-1) virions. The chimera is composed of the lectin cyanovirin-N (CVN) fused to the 20-residue membrane-proximal external region (MPER) of HIV-1 gp41. Because the Env gp120-binding CVN domain on its own is not lytic, we sought here to determine how the MPER(DAVEI) domain is able to endow the chimera with virolytic activity. We used a protein engineering strategy to identify molecular determinants of MPER(DAVEI) that are important for function. Recombinant mutagenesis and truncation demonstrated that the MPER(DAVEI) domain could be significantly minimized without loss of function. The dependence of lysis on specific MPER sequences of DAVEI, determination of minimal linker length, and competition by a simplified MPER surrogate peptide suggested that the MPER domain of DAVEI interacts with the Env spike trimer, likely with the gp41 region. This conclusion was further supported by observations from binding of the biotinylated MPER surrogate peptide to Env protein expressed on cells, monoclonal antibody competition, a direct binding enzyme-linked immunosorbent assay on viruses with varying numbers of trimeric spikes on their surfaces, and comparison of maximal interdomain spacing in DAVEI to that in high-resolution structures of Env. The finding that MPER(DAVEI) in CVN-MPER linker sequences can be minimized without loss of virolytic function provides an improved experimental path for constructing size-minimized DAVEI chimeras and molecular tools for determining how simultaneous engagement of gp120 and gp41 by these chimeras can disrupt the metastable virus Env spike.

Computational Evaluation of HIV-1 gp120 Conformations of Soluble Trimeric gp140 Structures as Targets for de Novo Docking of First- and Second-Generation Small-Molecule CD4 Mimics.

Small-molecule CD4 mimics (SMCM's) bind to the gp120 subunit of the HIV-1 envelope glycoprotein (Env) and have been optimized to block cell infection in vitro. The lack of the V1/2 and V3 loops and the presence of the ß2/3 and ß20/21 strands (bridging sheet) in the available structures of the monomeric gp120 core may limit its applicability as a target for further synthetic optimization of SMCM potency and/or breadth. Here, we employ a combination of binding-site search, docking, estimation of protein-ligand interaction energy, all-atom molecular dynamics, and ELISA-based CD4-binding competition assays to create, characterize, and rationalize models of first- and second-generation of SMCM's bound to the distinct, trimeric BG505 SOSIP.664 structures 4NCO and 4TVP containing V1/2 and V3 loops with no bridging sheet. We demonstrate that the in silico neutralization of the highly conserved D368 is necessary to obtain the correct orientation of SMCM in their binding site when docking against the monomeric gp120 core. The computational results correlate with IC50's measured in CD4 binding competition ELISA and with KD's measured on gp120 core monomer. This supports the hypothesis that the 4NCO trimeric structure represents a viable target for further SMCM's optimization with advantages over both the 4TVP trimer and gp120 core monomer. Finally, the docking protocol has been optimized to screen compounds that can clearly interact with the highly conserved residue D368, increasing the likelihood of future optimizations to arrive at SMCM's with a broader spectrum of activity.

Effect of intrinsic curvature and edge tension on the stability of binary mixed-membrane three-junctions.

We use a combination of coarse-grained molecular dynamics simulations and theoretical modeling to examine three-junctions in mixed lipid bilayer membranes. These junctions are localized defect lines in which three bilayers merge in such a way that each bilayer shares one monolayer with one of the other two bilayers. The resulting local morphology is non-lamellar, resembling the threefold symmetric defect lines in inverse hexagonal phases, but it regularly occurs during membrane fission and fusion events. We realize a system of junctions by setting up a honeycomb lattice, which in its primitive cell contains two hexagons and four three-line junctions, permitting us to study their stability as well as their line tension. We specifically consider the effects of lipid composition and intrinsic curvature in binary mixtures, which contain a fraction of negatively curved lipids in a curvature-neutral background phase. Three-junction stability results from a competition between the junction and an open edge, which arises if one of the three bilayers detaches from the other two. We show that the stable phase is the one with the lower defect line tension. The strong and opposite monolayer curvatures present in junctions and edges enhance the mole fraction of negatively curved lipids in junctions and deplete it in edges. This lipid sorting affects the two line tensions and in turn the relative stability of the two phases. It also leads to a subtle entropic barrier for the transition between junction and edge that is absent in uniform membranes.

Characterization of the water defect at the HIV-1 gp41 membrane spanning domain in bilayers with and without cholesterol using molecular simulations.

The membrane spanning domain (MSD) of human immunodeficiency virus 1 (HIV-1) envelope glycoprotein gp41 is important for fusion and infection. We used molecular dynamics (MD) simulations (3.4 µs total) to relate membrane and peptide properties that lead to water solvation of the α-helical gp41 MSD's midspan arginine in pure dipalmitoylphosphatidylcholine (DPPC) and in 50/50 DPPC/cholesterol membranes. We find that the midspan arginine is solvated by water that penetrates the inner leaflet, leading to a so-called water defect. The water defect is surprisingly robust across initial conditions and membrane compositions, but the presence of cholesterol modulates its behavior in several key ways. In the cholesterol-containing membranes, fluctuations in membrane thickness and water penetration depth are localized near the midspan arginine, and the MSD helices display a tightly regulated tilt angle. In the cholesterol-free membranes, thickness fluctuations are not as strongly correlated to the peptide position and tilt angles vary significantly depending on protein position relative to boundaries between domains of differing thickness. Cholesterol in an HIV-1 viral membrane is required for infection. Therefore, this work suggests that the colocalized water defect and membrane thickness fluctuations in cholesterol-containing viral membranes play an important role in fusion by bringing the membrane closer to a stability limit that must be crossed for fusion to occur.

Full kinetics of CO entry, internal diffusion, and exit in myoglobin from transition-path theory simulations.

We use Markovian milestoning molecular dynamics (MD) simulations on a tessellation of the collective variable space for CO localization in myoglobin to estimate the kinetics of entry, exit, and internal site-hopping. The tessellation is determined by analysis of the free-energy surface in that space using transition-path theory (TPT), which provides criteria for defining optimal milestones, allowing short, independent, cell-constrained MD simulations to provide properly weighted kinetic data. We coarse grain the resulting kinetic model at two levels: first, using crystallographically relevant internal cavities and their predicted interconnections and solvent portals; and second, as a three-state side-path scheme inspired by similar models developed from geminate recombination experiments. We show semiquantitative agreement with experiment on entry and exit rates and in the identification of the so-called "histidine gate" at position 64 through which ≈90% of flux between solvent and the distal pocket passes. We also show with six-dimensional calculations that the minimum free-energy pathway of escape through the histidine gate is a "knock-on" mechanism in which motion of the ligand and the gate are sequential and interdependent. In total, these results suggest that such TPT simulations are indeed a promising approach to overcome the practical time-scale limitations of MD to allow reliable estimation of transition mechanisms and rates among metastable states.

Covalent conjugation of a peptide triazole to HIV-1 gp120 enables intramolecular binding site occupancy.

The HIV-1 gp120 glycoprotein is the main viral surface protein responsible for initiation of the entry process and, as such, can be targeted for the development of entry inhibitors. We previously identified a class of broadly active peptide triazole (PT) dual antagonists that inhibit gp120 interactions at both its target receptor and coreceptor binding sites, induce shedding of gp120 from virus particles prior to host-cell encounter, and consequently can prevent viral entry and infection. However, our understanding of the conformational alterations in gp120 by which PT elicits its dual receptor antagonism and virus inactivation functions is limited. Here, we used a recently developed computational model of the PT-gp120 complex as a blueprint to design a covalently conjugated PT-gp120 recombinant protein. Initially, a single-cysteine gp120 mutant, E275CYU-2, was expressed and characterized. This variant retains excellent binding affinity for peptide triazoles, for sCD4 and other CD4 binding site (CD4bs) ligands, and for a CD4-induced (CD4i) ligand that binds the coreceptor recognition site. In parallel, we synthesized a PEGylated and biotinylated peptide triazole variant that retained gp120 binding activity. An N-terminally maleimido variant of this PEGylated PT, denoted AE21, was conjugated to E275C gp120 to produce the AE21-E275C covalent conjugate. Surface plasmon resonance interaction analysis revealed that the PT-gp120 conjugate exhibited suppressed binding of sCD4 and 17b to gp120, signatures of a PT-bound state of envelope protein. Similar to the noncovalent PT-gp120 complex, the covalent conjugate was able to bind the conformationally dependent mAb 2G12. The results argue that the PT-gp120 conjugate is structurally organized, with an intramolecular interaction between the PT and gp120 domains, and that this structured state embodies a conformationally entrapped gp120 with an altered bridging sheet but intact 2G12 epitope. The similarities of the PT-gp120 conjugate to the noncovalent PT-gp120 complex support the orientation of binding of PT to gp120 predicted in the molecular dynamics simulation model of the PT-gp120 noncovalent complex. The conformationally stabilized covalent conjugate can be used to expand the structural definition of the PT-induced "off" state of gp120, for example, by high-resolution structural analysis. Such structures could provide a guide for improving the subsequent structure-based design of inhibitors with the peptide triazole mode of action.

A model of peptide triazole entry inhibitor binding to HIV-1 gp120 and the mechanism of bridging sheet disruption.

Peptide triazole (PT) entry inhibitors prevent HIV-1 infection by blocking the binding of viral gp120 to both the HIV-1 receptor and the coreceptor on target cells. Here, we used all-atom explicit solvent molecular dynamics (MD) to propose a model for the encounter complex of the peptide triazoles with gp120. Saturation transfer difference nuclear magnetic resonance (STD NMR) and single-site mutagenesis experiments were performed to test the simulation results. We found that docking of the peptide to a conserved patch of residues lining the "F43 pocket" of gp120 in a bridging sheet naïve gp120 conformation of the glycoprotein led to a stable complex. This pose prevents formation of the bridging sheet minidomain, which is required for receptor-coreceptor binding, providing a mechanistic basis for dual-site antagonism of this class of inhibitors. Burial of the peptide triazole at the gp120 inner domain-outer domain interface significantly contributed to complex stability and rationalizes the significant contribution of hydrophobic triazole groups to peptide potency. Both the simulation model and STD NMR experiments suggest that the I-X-W [where X is (2S,4S)-4-(4-phenyl-1H-1,2,3-triazol-1-yl)pyrrolidine] tripartite hydrophobic motif in the peptide is the major contributor of contacts at the gp120-PT interface. Because the model predicts that the peptide Trp side chain hydrogen bonding with gp120 S375 contributes to the stability of the PT-gp120 complex, we tested this prediction through analysis of peptide binding to gp120 mutant S375A. The results showed that a peptide triazole KR21 inhibits S375A with 20-fold less potency than WT, consistent with predictions of the model. Overall, the PT-gp120 model provides a starting point for both the rational design of higher-affinity peptide triazoles and the development of structure-minimized entry inhibitors that can trap gp120 into an inactive conformation and prevent infection.