Mapping the Binding Interface between an HIV-1 Inhibiting Intrabody and the Viral Protein Rev

HIV-1 Rev is the key protein in the nucleocytoplasmic export and expression of the late viral mRNAs. An important aspect for its function is its ability to multimerize on these mRNAs. We have recently identified a llama single-domain antibody (Nb₁₉₀) as the first inhibitor targeting the Rev multimerization function in cells. This nanobody is a potent intracellular antibody that efficiently inhibits HIV-1 viral production. In order to gain insight into the Nb₁₉₀-Rev interaction interface, we performed mutational and docking studies to map the interface between the nanobody paratope and the Rev epitope. Alanine mutants of the hyper-variable domains of Nb₁₉₀ and the Rev multimerization domains were evaluated in different assays measuring Nb₁₉₀-Rev interaction or viral production. Seven residues within Nb₁₉₀ and five Rev residues are demonstrated to be crucial for epitope recognition. These experimental data were used to perform docking experiments and map the Nb₁₉₀-Rev structural interface. This Nb₁₉₀-Rev interaction model can guide further studies of the Nb₁₉₀ effect on HIV-1 Rev function and could serve as starting point for the rational development of smaller entities binding to the Nb₁₉₀ epitope, aimed at interfering with protein-protein interactions of the Rev N-terminal domain.     

HIV-1 structural gene expression requires the binding of multiple Rev monomers to the viral RRE: implications for HIV-1 latency

Expression of the structural proteins of HIV-1 requires the direct interaction of the viral Rev trans-activator with its cis-acting RNA target sequence, the Rev response element or RRE. Here, we demonstrate that this specific RNA-binding event is, as expected, mediated by the conserved arginine-rich motif of Rev. However, we also show that amino acid residues located proximal to this basic domain that are critical for in vivo Rev function are dispensable for sequence-specific binding to the RRE. Instead, these sequences are required for the multimerization of Rev on the viral RRE target sequence. The observation that Rev function requires the sequential binding of multiple Rev molecules to the RRE provides a biochemical explanation for the observed threshold effect for Rev function in vivo and suggests a molecular model for the high incidence of latent infection by HIV-1.     

Functional Analysis of the Human Immunodeficiency Virus Type 1 Rev Protein Oligomerization Interface

The expression of human immunodeficiency virus type 1 (HIV-1) structural proteins requires the action of the viral trans-regulatory protein Rev. Rev is a nuclear shuttle protein that directly binds to its cis-acting Rev response element (RRE) RNA target sequence. Subsequent oligomerization of Rev monomers on the RRE and interaction of Rev with a cellular cofactor(s) result in the cytoplasmic accumulation of RRE-containing viral mRNAs. Moreover, Rev by itself is exported from the nucleus to the cytoplasm. Although it has been demonstrated that Rev multimerization is critically required for Rev activity and hence for HIV-1 replication, the number of Rev monomers required to form a trans-activation-competent complex on the RRE is unknown. Here we report a systematic analysis of the putative multimerization domains within the Rev trans-activator protein. We identify the amino acid residues which are part of the proposed single hydrophobic surface patch in the Rev amino terminus that mediates intermolecular interactions. Furthermore, we show that the expression of a multimerization-deficient Rev mutant blocks HIV-1 replication in a trans-dominant (dominant-negative) fashion.     

HIV-1跨膜蛋白gp41与N-取代吡咯衍生物的结合模式研究

采用分子对接,分子动力学(MD)模拟和分子力学/泊松-波尔兹曼溶剂可有面积方法与分子力学/广义伯恩溶剂可及面积方法(MM-PBSA/MM-GBSA),预测两种N-取代吡咯衍生物与HIV-1 跨膜蛋白gp41疏水口袋的结合模式与作用机理．分子对接采用多种受体构象,并从结果中选取几种可能的结合模式进行MD 模拟,然后通过MM-PBSA计算结合能的方法识别最优的结合模式. MM-PBSA计算结果表明,范德华相互作用是结合的主要驱动力,而极性相互作用决定了配体在结合过程中的取向．进一步的结合能分解显示,配体的羧基与gp41残基Arg579的静电相互作用对结合有重要贡献．上述工作为进一步优化N-取代吡咯衍生物类的HIV-1融合抑制剂建立了良好的理论基础．     

Comparative molecular dynamics simulations of HIV-1 integrase and the T66I/M154I mutant: binding modes and drug resistance to a diketo acid inhibitor

HIV-1 IN is an essential enzyme for viral replication and an interesting target for the design of new pharmaceuticals for use in multidrug therapy of AIDS. L-731,988 is one of the most active molecules of the class of beta-diketo acids. Individual and combined mutations of HIV-1 IN at residues T66, S153, and M154 confer important degrees of resistance to one or more inhibitors belonging to this class. In an effort to understand the molecular mechanism of the resistance of T66I/M154I IN to the inhibitor L-731,988 and its specific binding modes, we have carried out docking studies, explicit solvent MD simulations, and binding free energy calculations. The inhibitor was docked against different protein conformations chosen from prior MD trajectories, resulting in 2 major orientations within the active site. MD simulations have been carried out for the T66I/M154I DM IN, DM IN in complex with L-731,988 in 2 different orientations, and 1QS4 IN in complex with L-731,988. The results of these simulations show a similar dynamical behavior between T66I/M154I IN alone and in complex with L-731,988, while significant differences are observed in the mobility of the IN catalytic loop (residues 138-149). Water molecules bridging the inhibitor to residues from the active site have been identified, and residue Gln62 has been found to play an important role in the interactions between the inhibitor and the protein. This work provides information about the binding modes of L-731,988, as well as insight into the mechanism of inhibitor-resistance in HIV-1 integrase.     

Understanding microscopic binding of macrophage migration inhibitory factor with phenolic hydrazones by molecular docking, molecular dynamics simulations and free energy calculations

Macrophage migration inhibitory factor (MIF), an immunoregulatory protein, is a potential target for a number of inflammatory diseases. In the current work, the interactions between MIF and a series of phenolic hydrazones were studied by molecular docking, molecular dynamics (MD) simulations, binding free energy calculations, and binding energy decomposition analysis to determine the structural requirement for achieving favorable biological activity of phenolic hydrazones. First, molecular docking was used to predict the binding modes of inhibitors in the binding site of MIF. The good correlation between the predicted docking scores and the experimental activities shows that the binding conformations of the inhibitors in the active site of MIF are well predicted. Moreover, our results suggest that the flexibility of MIF is essential in ligand binding process. Then, MD simulations and MM/GBSA free energy calculations were employed to determine the dynamic binding process and compare the binding modes of the inhibitors with different activities. The predicted binding free energies given by MM/GBSA are not well correlated with the experimental activities for the two subsets of the inhibitors; however, for each subset, a good correlation between the predicted binding free energies and the experimental activities is achieved. The MM/GBSA free energy decomposition analysis highlights the importance of hydrophobic residues for the MIF binding of the studied inhibitors. Based on the essential factors for MIF-inhibitor interactions derived from the theoretical predictions, some derivatives were designed and the higher inhibitory activities of several candidates were confirmed by molecular docking studies. The structural insights obtained from our study are useful for designing potent inhibitors of MIF.     

Molecular insight into pseudolysin inhibition using the MM-PBSA and LIE methods

Pseudolysin, the extracellullar elastase of Pseudomonas aeruginosa (EC: 3.4.24.26) plays an important role in the pathogenesis of P. aeruginosa infections. In the present study, molecular dynamics simulations and theoretical affinity predictions were used to gain molecular insight into pseudolysin inhibition. Four low molecular weight inhibitors were docked at their putative binding sites and molecular dynamics (MD) simulations were performed for 5.0 ns, and the free energy of binding was calculated by the linear interaction energy method. The number and the contact surface area of stabilizing hydrophobic, aromatic, and hydrogen bonding interactions appears to reflect the affinity differences between the inhibitors. The proteinaceous inhibitor, Streptomyces metalloproteinase inhibitor (SMPI) was docked in three different binding positions and MD simulations were performed for 3.0 ns. The MD trajectories were used for molecular mechanics-Poisson-Boltzmann surface area analysis of the three binding positions. Computational alanine scanning of the average pseudolysin-SMPI complexes after MD revealed residues at the pseudolysin-SMPI interface giving the main contribution to the free energy of binding. The calculations indicated that SMPI interacts with pseudolysin via the rigid active site loop, but that also contact sites outside this loop contribute significantly to the free energy of association.     

An Intrabody Based on a Llama Single-domain Antibody Targeting the N-terminal α-Helical Multimerization Domain of HIV-1 Rev Prevents Viral Production

The human immunodeficiency virus, type 1 (HIV-1)-encoded Rev protein is essential for the expression of late viral mRNAs. Rev forms a large organized multimeric protein-protein complex on the Rev response element of these viral mRNA species and transports them from the nucleus to the cytoplasm, exploiting the CRM1-mediated cellular machinery. Here we report the selection of a nanobody, derived from a llama heavy-chain only antibody, that efficiently blocks the assembly of Rev multimers. The nanobody inhibits HIV-1 replication in cells and specifically suppresses the Rev-dependent expression of partially spliced and unspliced HIV-1 RNA. In HIV-susceptible cells, this nanobody thus has potential as an effective anti-HIV agent using genetic immunization strategies. Its binding site was mapped to Rev residues Lys-20 and Tyr-23 located in the N-terminal α-helical multimerization domain. In the presence of this nanobody, we observed an accumulation of dimeric Rev species, supporting a head-to-head/tail-to-tail molecular model for Rev assembly. The results indicate that the oligomeric assembly of Rev follows an ordered stepwise process and identify a new epitope within Rev that could guide strategies for the development of novel HIV inhibitors.     

A comparative study of trypsin specificity based on QM/MM molecular dynamics simulation and QM/MM GBSA calculation

Hydrogen bonding and polar interactions play a key role in identification of protein-inhibitor binding specificity. Quantum mechanics/molecular mechanics molecular dynamics (QM/MM MD) simulations combined with DFT and semi-empirical Hamiltonian (AM1d, RM1, PM3, and PM6) methods were performed to study the hydrogen bonding and polar interactions of two inhibitors BEN and BEN1 with trypsin. The results show that the accuracy of treating the hydrogen bonding and polar interactions using QM/MM MD simulation of PM6 can reach the one obtained by the DFT QM/MM MD simulation. Quantum mechanics/molecular mechanics generalized Born surface area (QM/MM-GBSA) method was applied to calculate binding affinities of inhibitors to trypsin and the results suggest that the accuracy of binding affinity prediction can be significantly affected by the accurate treatment of the hydrogen bonding and polar interactions. In addition, the calculated results also reveal the binding specificity of trypsin: (1) the amidinium groups of two inhibitors generate favorable salt bridge interaction with Asp189 and form hydrogen bonding interactions with Ser190 and Gly214, (2) the phenyl of inhibitors can produce favorable van der Waals interactions with the residues His58, Cys191, Gln192, Trp211, Gly212, and Cys215. This systematic and comparative study can provide guidance for the choice of QM/MM MD methods and the designs of new potent inhibitors targeting trypsin.     

Probing the “fingers” domain binding pocket of Hepatitis C virus NS5B RdRp and D559G resistance mutation via molecular docking,molecular dynamics simulation and binding free energy calculations

The NS5B RdRp polymerase is a prominent enzyme for the replication of Hepatitis C virus (HCV). During the HCV replication, the template RNA binding takes place in the “fingers” sub-domain of NS5B. The “fingers” domain is a new emerging allosteric site for the HCV drug development. The inhibitors of the “fingers” sub-domain adopt a new antiviral mechanism called RNA intervention. The details of essential amino acid residues, binding mode of the ligand, and the active site intermolecular interactions of RNA intervention reflect that this mechanism is ambiguous in the experimental study. To elucidate these details, we performed molecular docking analysis of the fingers domain inhibitor quercetagetin (QGN) with NS5B polymerase. The detailed analysis of QGN-NS5B intermolecular interactions was carried out and found that QGN interacts with the binding pocket amino acid residues Ala97, Ala140, Ile160, Phe162, Gly283, Gly557, and Asp559; and also forms π?π stacking interaction with Phe162 and hydrogen bonding interaction with Gly283. These are found to be the essential interactions for the RNA intervention mechanism. Among the strong hydrogen bonding interactions, the QGN?Ala140 is a newly identified important hydrogen bonding interaction by the present work and this interaction was not resolved by the previously reported crystal structure. Since D559G mutation at the fingers domain was reported for reducing the inhibition percentage of QGN to sevenfold, we carried out molecular dynamics (MD) simulation for wild and D559G mutated complexes to study the stability of protein conformation and intermolecular interactions. At the end of 50?ns MD simulation, the π?π stacking interaction of Phe162 with QGN found in the wild-type complex is altered into T-shaped π stacking interaction, which reduces the inhibition strength. The origin of the D559G resistance mutation was studied using combined MD simulation, binding free energy calculations and principal component analysis. The results were compared with the wild-type complex. The mutation D559G reduces the binding affinity of the QGN molecule to the fingers domain. The free energy decomposition analysis of each residue of wild-type and mutated complexes revealed that the loss of non-polar energy contribution is the origin of the resistance.

Communicated by Ramaswamy H. Sarma     

Identification of potent inhibitors against snake venom metalloproteinase (SVMP) using molecular docking and molecular dynamics studies

Snake venom metalloproteinase (SVMP) (Echis coloratus (Carpet viper) is a multifunctional enzyme that is involved in producing several symptoms that follow a snakebite, such as severe local hemorrhage, nervous system effects and tissue necrosis. Because the three-dimensional (3D) structure of SVMP is not known, models were constructed, and the best model was selected based on its stereo-chemical quality. The stability of the modeled protein was analyzed through molecular dynamics (MD) simulation studies. Structure-based virtual screening was performed, and 15 potential molecules with the highest binding energies were selected. Further analysis was carried out with induced fit docking, Prime/MM–GBSA (ΔG_Bind calculations), quantum-polarized ligand docking, and density functional theory calculations. Further, the stability of the lead molecules in the SVMP-active site was examined using MD simulation. The results showed that the selected lead molecules were highly stable in the active site of SVMP. Hence, these molecules could potentially be selective inhibitors of SVMP. These lead molecules can be experimentally validated, and their backbone structural scaffold could serve as building blocks in designing drug-like molecules for snake antivenom.     

Intermolecular insights into allosteric inhibition of histone lysine-specific demethylase 1

BackgroundHistone lysine-specific demethylase 1 (LSD1) has become a potential anticancer target for the novel drug discovery. Recent reports have shown that SP2509 and its derivatives strongly inhibit LSD1 as allosteric inhibitors. However, the binding mechanism of these allosteric inhibitors in the allosteric site of LSD1 is not known yet.MethodsThe stability and binding mechanism of allosteric inhibitors in the binding site of LSD1 were evaluated by molecular docking, ligand-based pharmacophore, molecular dynamics (MD) simulations, molecular mechanics generalized born surface area (MM/GBSA) analysis, quantum mechanics/molecular mechanics (QM/MM) calculation and Hirshfeld surface analysis.ResultsThe conformational geometry and the intermolecular interactions of allosteric inhibitors showed high binding affinity towards allosteric site of LSD1 with the neighboring amino acids (Gly358, Cys360, Leu362, Asp375 and Glu379). Meanwhile, MD simulations and MM/GBSA analysis were performed on selected allosteric inhibitors in complex with LSD1 protein, which confirmed the high stability and binding affinity of these inhibitors in the allosteric site of LSD1.ConclusionThe simulation results revealed the crucial factors accounting for allosteric inhibitors of LSD1, including different protein–ligand interactions, the positions and conformations of key residues, and the ligands flexibilities. Meanwhile, a halogen bond interaction between chlorine atom of ligand and key residues Trp531 and His532 was recurrent in our analysis confirming its importance.General significanceOverall, our research analyzed in depth the binding modes of allosteric inhibitors with LSD1 and could provide useful information for the design of novel allosteric inhibitors.     

Computational modeling suggests dimerization of equine infectious anemia virus Rev is required for RNA binding

Background

The lentiviral Rev protein mediates nuclear export of intron-containing viral RNAs that encode structural proteins or serve as the viral genome. Following translation, HIV-1 Rev localizes to the nucleus and binds its cognate sequence, termed the Rev-responsive element (RRE), in incompletely spliced viral RNA. Rev subsequently multimerizes along the viral RNA and associates with the cellular Crm1 export machinery to translocate the RNA-protein complex to the cytoplasm. Equine infectious anemia virus (EIAV) Rev is functionally homologous to HIV-1 Rev, but shares very little sequence similarity and differs in domain organization. EIAV Rev also contains a bipartite RNA binding domain comprising two short arginine-rich motifs (designated ARM-1 and ARM-2) spaced 79 residues apart in the amino acid sequence. To gain insight into the topology of the bipartite RNA binding domain, a computational approach was used to model the tertiary structure of EIAV Rev.

Results

The tertiary structure of EIAV Rev was modeled using several protein structure prediction and model quality assessment servers. Two types of structures were predicted: an elongated structure with an extended central alpha helix, and a globular structure with a central bundle of helices. Assessment of models on the basis of biophysical properties indicated they were of average quality. In almost all models, ARM-1 and ARM-2 were spatially separated by >15 Å, suggesting that they do not form a single RNA binding interface on the monomer. A highly conserved canonical coiled-coil motif was identified in the central region of EIAV Rev, suggesting that an RNA binding interface could be formed through dimerization of Rev and juxtaposition of ARM-1 and ARM-2. In support of this, purified Rev protein migrated as a dimer in Blue native gels, and mutation of a residue predicted to form a key coiled-coil contact disrupted dimerization and abrogated RNA binding. In contrast, mutation of residues outside the predicted coiled-coil interface had no effect on dimerization or RNA binding.

Conclusions

Our results suggest that EIAV Rev binding to the RRE requires dimerization via a coiled-coil motif to juxtapose two RNA binding motifs, ARM-1 and ARM-2.

     

Sensitive in vitro analysis of HIV-1 Rev multimerization

Oligomerization of the Rev protein of human immuno-deficiency virus type 1 on its cognate response element is essential for export of the late viral mRNAs from the nucleus. Two regions of the protein, flanking the RNA binding site, have been defined as oligomerization sites after mutants (M4 and M7) had been reported to bind specifically to the response element but not to oligomerize in vivo or in vitro. These mutants are often used as paradigms for studies of Rev multimerization. We have re-examined the in vitro binding of these mutants to model Rev response elements, using improved gel mobility assays. We find that both mutants will form oligomers on the Rev response element, but have somewhat lower affinities for RNA than the wild-type protein. M7 has lower specific affinity, but shows little deficiency in oligomerization once binding starts. In contrast, M4 is multimerization deficient, as previously reported. Therefore, whilethe sites are correctly defined, it is inappropriate to employ the original M7 deletion mutant to study Rev oligomerization.     

In silico analyses of substrate interactions with human serum paraoxonase 1

Human paraoxonase (HuPON1) is a serum enzyme that exhibits a broad spectrum of hydrolytic activities, including the hydrolysis of various organophosphates, esters, and recently identified lactone substrates. Despite intensive site-directed mutagenesis and other biological studies, the structural basis for the specificity of substrate interactions of HuPON1 remains elusive. In this study, we apply homology modeling, docking, and molecular dynamic (MD) simulations to probe the binding interactions of HuPON1 with representative substrates. The results suggest that the active site of HuPON1 is characterized by two distinct binding regions: the hydrophobic binding site for arylesters/lactones, and the paraoxon binding site for phosphotriesters. The unique binding modes proposed for each type of substrate reveal a number of key residues governing substrate specificity. The polymorphic residue R/Q192 interacts with the leaving group of paraoxon, suggesting it plays an important role in the proper positioning of this substrate in the active site. MD simulations of the optimal binding complexes show that residue Y71 undergoes an "open-closed" conformational change upon ligand binding, and forms strong interactions with substrates. Further binding free energy calculations and residual decomposition give a more refined molecular view of the energetics and origin of HuPON1/substrate interactions. These studies provide a theoretical model of substrate binding and specificity associated with wild type and mutant forms of HuPON1, which can be applied in the rational design of HuPON1 variants as bioscavengers with enhanced catalytic activity.     

A Critical Role of the C-terminal Segment for Allosteric Inhibitor-induced Aberrant Multimerization of HIV-1 Integrase

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are a promising class of antiretroviral agents for clinical development. Although ALLINIs promote aberrant IN multimerization and inhibit IN interaction with its cellular cofactor LEDGF/p75 with comparable potencies in vitro, their primary mechanism of action in infected cells is through inducing aberrant multimerization of IN. Crystal structures have shown that ALLINIs bind at the IN catalytic core domain dimer interface and bridge two interacting subunits. However, how these interactions promote higher-order protein multimerization is not clear. Here, we used mass spectrometry-based protein footprinting to monitor surface topology changes in full-length WT and the drug-resistant A128T mutant INs in the presence of ALLINI-2. These experiments have identified protein-protein interactions that extend beyond the direct inhibitor binding site and which lead to aberrant multimerization of WT but not A128T IN. Specifically, we demonstrate that C-terminal residues Lys-264 and Lys-266 play an important role in the inhibitor induced aberrant multimerization of the WT protein. Our findings provide structural clues for exploiting IN multimerization as a new, attractive therapeutic target and are expected to facilitate development of improved inhibitors.