MicroED structures of HIV-1 Gag CTD-SP1 reveal binding interactions with the maturation inhibitor bevirimat

HIV-1 protease (PR) cleavage of the Gag polyprotein triggers the assembly of mature, infectious particles. Final cleavage of Gag occurs at the junction helix between the capsid protein CA and the SP1 spacer peptide. Here we used MicroED to delineate the binding interactions of the maturation inhibitor bevirimat (BVM) using very thin frozen-hydrated, 3D microcrystals of a CTD-SP1 Gag construct with and without bound BVM. The 2.9-Å MicroED structure revealed that a single BVM molecule stabilizes the six-helix bundle via both electrostatic interactions with the dimethylsuccinyl moiety and hydrophobic interactions with the pentacyclic triterpenoid ring. These results provide insight into the mechanism of action of BVM and related maturation inhibitors that will inform further drug discovery efforts. This study also demonstrates the capabilities of MicroED for structure-based drug design.

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MicroED Structures of HIV-1 Gag CTD-SP1 Reveal Binding Interactions with the Maturation Inhibitor Bevirimat

10.1101/241182 ◽

2017 ◽

Author(s):

Michael D. Purdy ◽

Dan Shi ◽

Jakub Chrustowicz ◽

Johan Hattne ◽

Tamir Gonen ◽

...

Keyword(s):

Drug Discovery ◽

Electrostatic Interactions ◽

Hydrophobic Interactions ◽

Three Dimensional ◽

Binding Interactions ◽

Structure Based Drug Design ◽

Gag Polyprotein ◽

Pentacyclic Triterpenoid ◽

Maturation Inhibitor ◽

Hiv 1

AbstractHIV-1 protease (PR) cleavage of the Gag polyprotein triggers the assembly of mature, infectious particles. Final cleavage of Gag occurs at the junction helix between the capsid protein CA and the SP1 spacer peptide. Here we used MicroED to delineate the binding interactions of the maturation inhibitor bevirimat (BVM) using very thin frozen-hydrated, three-dimensional microcrystals of a CTD-SP1 Gag construct with and without bound BVM. The 2.9-Å MicroED structure revealed that a single BVM molecule stabilizes the 6-helix bundle via both electrostatic interactions with the dimethysuccinyl moiety and hydrophobic interactions with the pentacyclic triterpenoid ring. These results provide insight into the mechanism of action of BVM and related maturation inhibitors that will inform further drug discovery efforts. This study also demonstrates the capabilities of MicroED for structure-based drug design.One-sentence summaryThe MicroED structure of HIV-1 Gag CTD-SP1 at 2.9 Å resolution reveals the molecular basis for binding of the maturation inhibitor bevirimat that will inform further drug discovery efforts.

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Insight into the HIV-1 Vif SOCS-box–ElonginBC interaction

Open Biology ◽

10.1098/rsob.130100 ◽

2013 ◽

Vol 3 (11) ◽

pp. 130100 ◽

Cited By ~ 7

Author(s):

Zhisheng Lu ◽

Julien R. C. Bergeron ◽

R. Andrew Atkinson ◽

Torsten Schaller ◽

Dennis A. Veselkov ◽

...

Keyword(s):

Solution Structure ◽

Hydrophobic Interactions ◽

Dynamic Information ◽

Ubiquitin Ligase Complex ◽

Biophysical Studies ◽

Viral Infectivity Factor ◽

Β Sheet ◽

Socs Box ◽

Insight Into ◽

Hiv 1

The HIV-1 viral infectivity factor (Vif) neutralizes cell-encoded antiviral APOBEC3 proteins by recruiting a cellular ElonginB (EloB)/ElonginC (EloC)/Cullin5-containing ubiquitin ligase complex, resulting in APOBEC3 ubiquitination and proteolysis. The suppressors-of-cytokine-signalling-like domain (SOCS-box) of HIV-1 Vif is essential for E3 ligase engagement, and contains a BC box as well as an unusual proline-rich motif. Here, we report the NMR solution structure of the Vif SOCS–ElonginBC (EloBC) complex. In contrast to SOCS-boxes described in other proteins, the HIV-1 Vif SOCS-box contains only one α-helical domain followed by a β-sheet fold. The SOCS-box of Vif binds primarily to EloC by hydrophobic interactions. The functionally essential proline-rich motif mediates a direct but weak interaction with residues 101–104 of EloB, inducing a conformational change from an unstructured state to a structured state. The structure of the complex and biophysical studies provide detailed insight into the function of Vif's proline-rich motif and reveal novel dynamic information on the Vif–EloBC interaction.

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Distinct Contributions of Different Domains within the HIV-1 Gag Polyprotein to Specific and Nonspecific Interactions with RNA

Viruses ◽

10.3390/v12040394 ◽

2020 ◽

Vol 12 (4) ◽

pp. 394 ◽

Cited By ~ 3

Author(s):

Tomas Kroupa ◽

Siddhartha A. K. Datta ◽

Alan Rein

Keyword(s):

Salt Concentration ◽

Electrostatic Interactions ◽

Rna Binding ◽

High Specificity ◽

Viral Rna ◽

Gag Protein ◽

Gag Polyprotein ◽

The Individual ◽

Hiv 1

Viral genomic RNA is packaged into virions with high specificity and selectivity. However, in vitro the Gag specificity towards viral RNA is obscured when measured in buffers containing physiological salt. Interestingly, when the binding is challenged by increased salt concentration, the addition of competing RNAs, or introducing mutations to Gag protein, the specificity towards viral RNA becomes detectable. The objective of this work was to examine the contributions of the individual HIV-1 Gag polyprotein domains to nonspecific and specific RNA binding and stability of the initial protein-RNA complexes. Using a panel of Gag proteins with mutations disabling different Gag-Gag or Gag-RNA interfaces, we investigated the distinct contributions of individual domains which distinguish the binding to viral and nonviral RNA by measuring the binding of the proteins to RNAs. We measured the binding affinity in near-physiological salt concentration, and then challenged the binding by increasing the ionic strength to suppress the electrostatic interactions and reveal the contribution of specific Gag–RNA and Gag–Gag interactions. Surprisingly, we observed that Gag dimerization and the highly basic region in the matrix domain contribute significantly to the specificity of viral RNA binding.

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Effects of an HIV-1 maturation inhibitor on the structure and dynamics of CA-SP1 junction helices in virus-like particles

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1917755117 ◽

2020 ◽

Vol 117 (19) ◽

pp. 10286-10293 ◽

Cited By ~ 3

Author(s):

Sebanti Gupta ◽

John M. Louis ◽

Robert Tycko

Keyword(s):

Chemical Shifts ◽

Isotopic Labeling ◽

Relaxation Rates ◽

Virus Like Particles ◽

Gag Polyprotein ◽

Structure And Dynamics ◽

Maturation Inhibitor ◽

Rna Maturation ◽

Segmental Isotopic Labeling ◽

Hiv 1

HIV-1 maturation involves conversion of the immature Gag polyprotein lattice, which lines the inner surface of the viral membrane, to the mature capsid protein (CA) lattice, which encloses the viral RNA. Maturation inhibitors such as bevirimat (BVM) bind within six-helix bundles, formed by a segment that spans the junction between the CA and spacer peptide 1 (SP1) subunits of Gag, and interfere with cleavage between CA and SP1 catalyzed by the HIV-1 protease (PR). We report solid-state NMR (ssNMR) measurements on spherical virus-like particles (VLPs), facilitated by segmental isotopic labeling, that provide information about effects of BVM on the structure and dynamics of CA–SP1 junction helices in the immature lattice. Although BVM strongly blocks PR-catalyzed CA–SP1 cleavage in VLPs and blocks conversion of VLPs to tubular CA assemblies, 15N and 13C ssNMR chemical shifts of segmentally labeled VLPs with and without BVM are very similar, indicating that interaction with BVM does not alter the six-helix bundle structure appreciably. Only the 15N chemical shift of A280 (the first residue of SP1) changes significantly, consistent with BVM binding to an internal ring of hydrophobic side chains of L279 residues. Measurements of transverse 15N spin relaxation rates reveal a reduction in the amplitudes and/or timescales of backbone N–H bond motions, corresponding to a rigidification of the six-helix bundles. Overall, our data show that inhibition of HIV-1 maturation by BVM involves changes in structure and dynamics that are surprisingly subtle, but still sufficient to produce a large effect on CA–SP1 cleavage.

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ADS-J1 Disaggregates Semen-derived Amyloid Fibrils

10.1101/292342 ◽

2018 ◽

Author(s):

J. Li ◽

Z. Yang ◽

H. Liu ◽

Y. Lan ◽

T. Zhang ◽

...

Keyword(s):

Viral Infection ◽

Conformational Changes ◽

Electrostatic Interactions ◽

Amyloid Fibrils ◽

Hydrophobic Interactions ◽

Sexual Transmission ◽

Md Simulations ◽

Potential Mode ◽

Biochemical Measurements ◽

Hiv 1

ABSTRACTSemen-derived amyloid fibrils, composing SEVI (semen-derived enhancer of viral infection) fibrils and SEM1 fibrils, could remarkably enhance HIV-1 sexual transmission and thus, are potential targets for the development of an effective microbicide. Previously, we found that ADS-J1, apart from being an HIV-1 entry inhibitor, could also potently inhibit seminal amyloid fibrillization and block fibril-mediated enhancement of viral infection. However, the remodeling effects of ADS-J1 on mature seminal fibrils were unexplored. Herein, we investigated the capacity of ADS-J1 to disassemble seminal fibrils and the potential mode of action by applying several biophysical and biochemical measurements, combined with molecular dynamic (MD) simulations. We found that ADS-J1 effectively remodeled SEVI, SEM186-107 fibrils and endogenous seminal fibrils. Unlike epi-gallocatechin gallate (EGCG), a universal amyloid fibril breaker, ADS-J1 disaggregated SEVI fibrils into monomeric peptides, which was independent of oxidation reaction. MD simulations revealed that ADS-J1 displayed strong binding potency to the full-length PAP248-286 via electrostatic interactions, hydrophobic interactions and hydrogen bonds. ADS-J1 might initially bind to the fibrillar surface and then occupy the amyloid core, which eventually lead to fibril disassembly. Furthermore, the binding of ADS-J1 with PAP248-286 might induce conformational changes of PAP248-286. Disassembled PAP248-286 might not be favor to re-aggregate into fibrils. ADS-J1 also exerts abilities to remodel a panel of amyloid fibrils, including Aβ1-42, hIAPP1-37 and EP2 fibrils. ADS-J1 displays promising potential to be a combination microbicide and an effective lead-product to treat amyloidogenic diseases.

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HIV-1 Packaging Visualised by In-Gel SHAPE

Viruses ◽

10.3390/v13122389 ◽

2021 ◽

Vol 13 (12) ◽

pp. 2389

Author(s):

Aaron R. D’Souza ◽

Dhivya Jayaraman ◽

Ziqi Long ◽

Jingwei Zeng ◽

Liam J. Prestwood ◽

...

Keyword(s):

Rna Structure ◽

Structural Changes ◽

Structural Protein ◽

Packaging Signal ◽

Packaging Process ◽

Gag Polyprotein ◽

Rna Conformation ◽

Shape Selective ◽

Insight Into ◽

Hiv 1

HIV-1 packages two copies of its gRNA into virions via an interaction with the viral structural protein Gag. Both copies and their native RNA structure are essential for virion infectivity. The precise stepwise nature of the packaging process has not been resolved. This is largely due to a prior lack of structural techniques that follow RNA structural changes within an RNA–protein complex. Here, we apply the in-gel SHAPE (selective 2’OH acylation analysed by primer extension) technique to study the initiation of HIV-1 packaging, examining the interaction between the packaging signal RNA and the Gag polyprotein, and compare it with that of the NC domain of Gag alone. Our results imply interactions between Gag and monomeric packaging signal RNA in switching the RNA conformation into a dimerisation-competent structure, and show that the Gag–dimer complex then continues to stabilise. These data provide a novel insight into how HIV-1 regulates the translation and packaging of its genome.

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Determinants for Sensitivity of Human Immunodeficiency Virus Coreceptor CXCR4 to the Bicyclam AMD3100

Journal of Virology ◽

10.1128/jvi.72.8.6381-6388.1998 ◽

1998 ◽

Vol 72 (8) ◽

pp. 6381-6388 ◽

Cited By ~ 108

Author(s):

Béatrice Labrosse ◽

Anne Brelot ◽

Nikolaus Heveker ◽

Nathalie Sol ◽

Dominique Schols ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Electrostatic Interactions ◽

Hydrophobic Interactions ◽

Direct Interaction ◽

Single Amino Acid ◽

Amino Terminal ◽

Immunodeficiency Virus ◽

Hiv Entry ◽

Membrane Spanning ◽

Hiv 1

ABSTRACT The bicyclam AMD3100 is a potent and selective inhibitor of the replication of human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2). It was recently demonstrated that the compound inhibited HIV entry through CXCR4 but not through CCR5. Selectivity of AMD3100 for CXCR4 was further indicated by its lack of effect on HIV-1 and HIV-2 infection mediated by the CCR5, CCR3, Bonzo, BOB, and US28, coreceptors. AMD3100 completely blocked HIV-1 infection mediated by a mutant CXCR4 bearing a deletion of most of the amino-terminal extracellular domain. In contrast, relative resistance to AMD3100 was conferred by different single amino acid substitutions in the second extracellular loop (ECL2) or in the adjacent membrane-spanning domain, TM4. Only substitutions of a neutral residue for aspartic acid and of a nonaromatic residue for phenylalanine (Phe) were associated with drug resistance. This suggests a direct interaction of AMD3100 with these amino acids rather than indirect effects of their mutation on the CXCR4 structure. The interaction of aspartic acids of ECL2 and TM4 with AMD3100 is consistent with the positive charge of bicyclams, which might block HIV-1 entry by preventing electrostatic interactions between CXCR4 and the HIV-1 envelope protein gp120. Other features of AMD3100 must account for its high antiviral activity, in particular the presence of an aromatic linker between the cyclam units. This aromatic group might engage in hydrophobic interactions with the Phe-X-Phe motifs of ECL2 or TM4. These results confirm the importance of ECL2 for the HIV coreceptor activity of CXCR4.

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Emergence of compensatory mutations reveal the importance of electrostatic interactions between HIV-1 integrase and genomic RNA

10.1101/2021.12.16.472884 ◽

2021 ◽

Author(s):

Christian Shema Mugisha ◽

Tung Dinh ◽

Kasyap Tenneti ◽

Jenna Eve Eschbach ◽

Keanu Davis ◽

...

Keyword(s):

Electrostatic Interactions ◽

Rna Binding ◽

Class Ii ◽

Integrase Inhibitors ◽

Ribonucleoprotein Complexes ◽

Compensatory Mutations ◽

Insight Into ◽

Hiv 1 ◽

Molecular Nature

Independent of its catalytic activity, HIV-1 integrase (IN) enzyme regulates proper particle maturation by binding to and packaging the viral RNA genome (gRNA) inside the mature capsid lattice. Allosteric integrase inhibitors (ALLINIs) and class II IN substitutions inhibit the binding of IN to the gRNA and cause the formation of non-infectious virions characterized by mislocalization of the viral ribonucleoprotein complexes between the translucent conical capsid lattice and the viral lipid envelope. To gain insight into the molecular nature of IN-gRNA interactions, we have isolated compensatory substitutions in the background of a class II IN (R269A/K273A) variant that directly inhibits IN binding to the gRNA. We found that additional D256N and D270N substitutions in the C-terminal domain (CTD) of IN restored its ability to bind gRNA and led to the formation of infectious particles with correctly matured morphology. Furthermore, reinstating the overall positive electrostatic potential of the CTD through individual D256R or D256K substitutions was sufficient to restore IN-RNA binding and infectivity for the R269A/K273A as well as the R262A/R263A class II IN mutants. The compensatory mutations did not impact functional IN oligomerization, suggesting that they directly contributed to IN binding to the gRNA. Interestingly, HIV-1 IN R269A/K273A, but not IN R262A/R263A, bearing compensatory mutations was more sensitive to ALLINIs providing key genetic evidence that specific IN residues required for RNA binding also influence ALLINI activity. Structural modeling provided further insight into the molecular nature of IN-gRNA interactions and ALLINI mechanism of action. Taken together, our findings highlight an essential role of IN-gRNA interactions for proper virion maturation and reveal the importance of electrostatic interactions between the IN CTD and the gRNA.

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ADS-J1 disaggregates semen-derived amyloid fibrils

Biochemical Journal ◽

10.1042/bcj20180886 ◽

2019 ◽

Vol 476 (6) ◽

pp. 1021-1035

Author(s):

Jinqing Li ◽

Zichao Yang ◽

Han Liu ◽

Mengjie Qiu ◽

Tingting Zhang ◽

...

Keyword(s):

Viral Infection ◽

Conformational Changes ◽

Electrostatic Interactions ◽

Amyloid Fibrils ◽

Hydrophobic Interactions ◽

Sexual Transmission ◽

Epigallocatechin Gallate ◽

Md Simulations ◽

Potential Mode ◽

Hiv 1

Abstract Semen-derived amyloid fibrils, comprising SEVI (semen-derived enhancer of viral infection) fibrils and SEM1 fibrils, could remarkably enhance HIV-1 sexual transmission and thus are potential targets for the development of an effective microbicide. Previously, we found that ADS-J1, apart from being an HIV-1 entry inhibitor, could also potently inhibit seminal amyloid fibrillization and block fibril-mediated enhancement of viral infection. However, the remodeling effects of ADS-J1 on mature seminal fibrils were unexplored. Herein, we investigated the capacity of ADS-J1 to disassemble seminal fibrils and the potential mode of action by applying several biophysical and biochemical measurements, combined with molecular dynamic (MD) simulations. We found that ADS-J1 effectively remodeled SEVI, SEM186–107 fibrils and endogenous seminal fibrils. Unlike epigallocatechin gallate (EGCG), a universal amyloid fibril breaker, ADS-J1 disaggregated SEVI fibrils into monomeric peptides, which was independent of oxidation reaction. MD simulations revealed that ADS-J1 displayed strong binding potency to the full-length PAP248–286 via electrostatic interactions, hydrophobic interactions and hydrogen bonds. ADS-J1 might initially bind to the fibrillar surface and then occupy the amyloid core, which eventually lead to fibril disassembly. Furthermore, the binding of ADS-J1 with PAP248–286 might induce conformational changes of PAP248–286. Disassembled PAP248–286 might not be favorable to re-aggregate into fibrils. ADS-J1 also exerts abilities to remodel a panel of amyloid fibrils, including Aβ1–42, hIAPP1–37 and EP2 fibrils. ADS-J1 displays promising potential to be a combination microbicide and an effective lead-product to treat amyloidogenic diseases.

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Use of Molecular Dynamics Simulations in Structure-Based Drug Discovery

Current Pharmaceutical Design ◽

10.2174/1381612825666190903153043 ◽

2019 ◽

Vol 25 (31) ◽

pp. 3339-3349 ◽

Cited By ~ 6

Author(s):

Indrani Bera ◽

Pavan V. Payghan

Keyword(s):

Molecular Dynamics ◽

Drug Discovery ◽

Molecular Dynamics Simulations ◽

Drug Target ◽

Structural Information ◽

Drug Binding ◽

Structure Based Drug Design ◽

Drug Designing ◽

Target Binding ◽

Dynamics Simulations

Background: Traditional drug discovery is a lengthy process which involves a huge amount of resources. Modern-day drug discovers various multidisciplinary approaches amongst which, computational ligand and structure-based drug designing methods contribute significantly. Structure-based drug designing techniques require the knowledge of structural information of drug target and drug-target complexes. Proper understanding of drug-target binding requires the flexibility of both ligand and receptor to be incorporated. Molecular docking refers to the static picture of the drug-target complex(es). Molecular dynamics, on the other hand, introduces flexibility to understand the drug binding process. Objective: The aim of the present study is to provide a systematic review on the usage of molecular dynamics simulations to aid the process of structure-based drug design. Method: This review discussed findings from various research articles and review papers on the use of molecular dynamics in drug discovery. All efforts highlight the practical grounds for which molecular dynamics simulations are used in drug designing program. In summary, various aspects of the use of molecular dynamics simulations that underline the basis of studying drug-target complexes were thoroughly explained. Results: This review is the result of reviewing more than a hundred papers. It summarizes various problems that use molecular dynamics simulations. Conclusion: The findings of this review highlight how molecular dynamics simulations have been successfully implemented to study the structure-function details of specific drug-target complexes. It also identifies the key areas such as stability of drug-target complexes, ligand binding kinetics and identification of allosteric sites which have been elucidated using molecular dynamics simulations.

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