Structural and dynamics investigations of HIV-1 capsid and its interactions with cyclophilin A: atomic-level insights from magic angle spinning NMR
Author(s) | Lu, Manman | |
Date Accessioned | 2022-09-20T13:30:30Z | |
Date Available | 2022-09-20T13:30:30Z | |
Publication Date | 2017 | |
SWORD Update | 2022-08-11T19:09:05Z | |
Abstract | In a mature HIV-1 virion, the viral capsid (CA) protein assembles into a conical capsid, enclosing the viral genome and viral proteins. The capsid is a multi-role key player in HIV-1 infection and a potential target for HIV-1 therapy. Despite extensive studies of CA protein assemblies by various methods, the detailed and precise molecular mechanisms of capsid’s roles in viral life cycle remain elusive. In this dissertation, we study the HIV-1 CA assemblies using solid-state magic angle spinning (MAS) NMR, which has emerged as a powerful method to investigate large protein assemblies. ☐ One major direction of this dissertation is to determine the three-dimensional structure of CA assemblies at atomic resolution. We have accomplished nearly complete resonance assignments for 13C and 15N resonances. Intramolecular and intermolecular distance restraints have been obtained using homo- and heteronuclear experiments, from which the 3D structure of the CA monomer was determined. The structure of the hexameric building block was determined using an integrated approach in which the NMR experimental restraints are refined into the cryoEM density map. ☐ We have explored the novel ultrafast MAS approach (frequencies above 100 kHz), which enables high-quality proton-detected NMR experiments for fully protonated protein assemblies. The dramatic increase in sensitivity and resolution, as well as the attainable 1H chemical shifts, permits one to streamline structure analysis and dynamics investigations, with greatly shortened experiment time and use of a small amount (0.1 – 0.3 mg) of sample. We have collected 3D 1H-detected datasets for proton resonance assignments, acquired 1H-1H distance restraints, and measured 1H-15N dipolar and 1H CSA tensors. ☐ Another main focus of this dissertation is to understand the interaction of HIV-1 capsid and a host cell factor cyclophilin A (CypA) better. CypA is packaged in the HIV-1 virions, and it directly binds with the HIV-1 capsid and modulates viral infectivity through an unknown mechanism. We have addressed the role of conformational dynamics on the nanosecond to millisecond timescales in the escape from CypA dependence, by MAS NMR and molecular dynamics. 1H-15N and 1H-13C dipolar order parameters (S) obtained from MAS NMR experiments on CA assemblies, CypA escape mutants A92E and G94D, and CA/CypA assemblies are in quantitative agreement with those calculated from MD trajectories. Our data demonstrated that CA assemblies are dynamic on multiple timescales, especially in the CypA binding loop. These motions are significantly reduced in CA/CypA assemblies. Remarkably, the CypA escape mutant assemblies exhibit dynamic behavior similar to that of the CA/CypA assemblies. Together, these findings suggest that a dynamic allostery mechanism governs the CA escape from CypA dependence. ☐ We characterized the binding interfaces between CA and CypA, by comparing MAS NMR spectra of CA/CypA assemblies with varying CA:CypA ratios. We have detected numerous chemical shift perturbations and peak intensity changes upon the formation of CA/CypA complex. Surprisingly, these spectral changes are associated not only with CA and CypA residues comprising the canonical binding sites, but also with additional residues distal to the canonical binding sites, which suggests a secondary binding site or allosteric effects, or both. Together with evidence from cryo-EM experiments and molecular dynamics (MD) simulations, we report a previously unidentified non-canonical interface for capsid and CypA interaction. | en_US |
Advisor | Polenova, Tatyana | |
Degree | Ph.D. | |
Department | University of Delaware, Department of Chemistry and Biochemistry | |
DOI | https://doi.org/10.58088/8q4e-6z18 | |
Unique Identifier | 1345245081 | |
URL | https://udspace.udel.edu/handle/19716/31389 | |
Language | en | |
Publisher | University of Delaware | en_US |
URI | https://login.udel.idm.oclc.org/login?url=https://www.proquest.com/dissertations-theses/structural-dynamics-investigations-hiv-1-capsid/docview/2708547580/se-2?accountid=10457 | |
Keywords | CypA dependence | en_US |
Keywords | Viral capsid protein | en_US |
Keywords | Solid-state magic angle spinning | en_US |
Title | Structural and dynamics investigations of HIV-1 capsid and its interactions with cyclophilin A: atomic-level insights from magic angle spinning NMR | en_US |
Type | Thesis | en_US |