Local Conformational Stability of HIV-1 gp120 in Unliganded and CD4-Bound States as Defined by Amide Hydrogen/Deuterium Exchange

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-3027, USA.
Journal of Virology (Impact Factor: 4.44). 10/2010; 84(19):10311-21. DOI: 10.1128/JVI.00688-10
Source: PubMed


The binding reaction of the HIV-1 gp120 envelope glycoprotein to the CD4 receptor involves exceptional changes in enthalpy and entropy. Crystal structures of gp120 in unliganded and various ligand-bound states, meanwhile, reveal an inner domain able to fold into diverse conformations, a structurally invariant outer domain, and, in the CD4-bound state, a bridging sheet minidomain. These studies, however, provide only hints as to the flexibility of each state. Here we use amide hydrogen/deuterium exchange coupled to mass spectrometry to provide quantifications of local conformational stability for HIV-1 gp120 in unliganded and CD4-bound states. On average, unliganded core gp120 displayed >10,000-fold slower exchange of backbone-amide hydrogens than a theoretically unstructured protein of the same composition, with binding by CD4 reducing the rate of gp120 amide exchange a further 10-fold. For the structurally constant CD4, alterations in exchange correlated well with alterations in binding surface (P value = 0.0004). For the structurally variable gp120, however, reductions in flexibility extended outside the binding surface, and regions of expected high structural diversity (inner domain/bridging sheet) displayed roughly 20-fold more rapid exchange in the unliganded state than regions of low diversity (outer domain). Thus, despite an extraordinary reduction in entropy, neither unliganded gp120 nor free CD4 was substantially unstructured, suggesting that most of the diverse conformations that make up the gp120 unliganded state are reasonably ordered. The results provide a framework for understanding how local conformational stability influences entropic change, conformational diversity, and structural rearrangements in the gp120-CD4 binding reaction.

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    • "The quaternary structure of Env trimers has primarily been inferred from analyses of monomers and remains controversial [8,21] although it is accepted that a number of folding events occur during a successful infection event. During the entry process trimeric Env undergoes a large structural transition from a closed, unliganded state to an open state when in complex with CD4. "
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    Retrovirology 03/2013; 10(1):33. DOI:10.1186/1742-4690-10-33 · 4.19 Impact Factor
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    • "PPI dynamics could also be studied by HDX and MS. For instance, Kong et al. (2010) comparatively studied the local conformational rearrangements within HIV-1 gp120 in the presence or absence of CD4. Monroe et al. (2010) also applied HDX combined with LTQ-FT MS to study the immature, mature, and mutant Gag polyprotein to further unravel the capsid assembly. "
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    ABSTRACT: Viruses could manipulate cellular machinery to ensure their continuous survival and thus become parasites of living organisms. Delineation of sophisticated host responses upon virus infection is a challenging task. It lies in identifying the repertoire of host factors actively involved in the viral infectious cycle and characterizing host responses qualitatively and quantitatively during viral pathogenesis. Mass spectrometry based proteomics could be used to efficiently study pathogen-host interactions and virus-hijacked cellular signaling pathways. Moreover, direct host and viral responses upon infection could be further investigated by activity-based functional validation studies. These approaches involve drug inhibition of secretory pathway, immunofluorescence staining, dominant negative mutant of protein target, real-time PCR, small interfering siRNA-mediated knockdown, and molecular cloning studies. In this way, functional validation could gain novel insights into the high-content proteomic dataset in an unbiased and comprehensive way.
    Frontiers in Microbiology 11/2012; 3:393. DOI:10.3389/fmicb.2012.00393 · 3.99 Impact Factor
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    • "Unfortunately, such information is not so easily attained. Despite a growing body of biophysical information for the HIV-1 gp120 glycoprotein—from crystal structures of gp120 in complex with various ligands (Kwong et al. 1998; Huang et al. 2005; Zhou et al. 2007, 2010; Chen et al. 2009; Pancera et al. 2010), isothermal titration calorimetry providing measurements of entropic and enthalpic changes (Myszka et al. 2000), and hydrogen deuterium exchange providing a coarse description of conformational stability (Kong et al. 2010)—an atomic-level description of HIV-1 gp120 flexibility has been missing, and NMR spectroscopy is one of the only ways to obtain such information. One barrier to obtaining NMR spectroscopic information is the production of isotopically labeled HIV-1 gp120. "
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