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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Available from: Jeff Skinner, Oct 09, 2015
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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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    ABSTRACT: Background Interactions between the HIV-1 envelope glycoprotein (Env) and its primary receptor CD4 are influenced by the physiological setting in which these events take place. In this study, we explored the surface chemistry of HIV-1 Env constructs at a range of pH and salinities relevant to mucosal and systemic compartments through electrophoretic mobility (EM) measurements. Sexual transmission events provide a more acidic environment for HIV-1 compared to dissemination and spread of infection occurring in blood or lymph node. We hypothesize functional, trimeric Env behaves differently than monomeric forms. Results The dynamic electrophoretic fingerprint of trimeric gp140 revealed a change in EM from strongly negative to strongly positive as pH increased from that of the lower female genital tract (pHx) to that of the blood (pHy). Similar findings were observed using a trimeric influenza Haemagglutinin (HA) glycoprotein, indicating that this may be a general attribute of trimeric viral envelope glycoproteins. These findings were supported by computationally modeling the surface charge of various gp120 and HA crystal structures. To identify the behavior of the infectious agent and its target cells, EM measurements were made on purified whole HIV-1 virions and primary T-lymphocytes. Viral particles had a largely negative surface charge, and lacked the regions of positivity near neutral pH that were observed with trimeric Env. T cells changed their surface chemistry as a function of activation state, becoming more negative over a wider range of pH after activation. Soluble recombinant CD4 (sCD4) was found to be positively charged under a wide range of conditions. Binding studies between sCD4 and gp140 show that the affinity of CD4-gp140 interactions depends on pH. Conclusions Taken together, these findings allow a more complete model of the electrochemical forces involved in HIV-1 Env functionality. These results indicate that the influence of the localized environment on the interactions of HIV with target cells are more pronounced than previously appreciated. There is differential chemistry of trimeric, but not monomeric, Env under conditions which mimic the mucosa compared to those found systemically. This should be taken into consideration during design of immunogens which targets virus at mucosal portals of entry.
    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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    ABSTRACT: NMR spectroscopic characterization of the structure or the dynamics of proteins generally requires the production of samples isotopically enriched in 15N, 13C, or 2H. The bacterial expression systems currently in use to obtain isotopic enrichment, however, cannot produce a number of eukaryotic proteins, especially those that require post-translational modifications such as N-linked glycosylation for proper folding or activity. Here, we report the use of an adenovirus vector-based mammalian expression system to produce isotopically enriched 15N or 15N/13C samples of an outer domain variant of the HIV-1 gp120 envelope glycoprotein with 15 sites of N-linked glycosylation. Yields for the 15N- and 15N/13C-labeled gp120s after affinity chromatography were 45 and 44 mg/l, respectively, with an average of over 80% isotope incorporation. Recognition of the labeled gp120 by cognate antibodies that recognize complex epitopes showed affinities comparable to the unlabeled protein. NMR spectra, including 1H-15N and 1H-13C HSQCs, 15N-edited NOESY-HSQC, and 3D HNCO, were of high quality, with signal-to-noise consistent with an efficient level of isotope incorporation, and with chemical shift dispersion indicative of a well-folded protein. The exceptional protein yields, good isotope incorporation, and ability to obtain well-folded post-translationally modified proteins make this mammalian system attractive for the production of isotopically enriched eukaryotic proteins for NMR spectroscopy. Electronic supplementary material The online version of this article (doi:10.1007/s10858-011-9506-4) contains supplementary material, which is available to authorized users.
    Journal of Biomolecular NMR 06/2011; 50(3):197-207. DOI:10.1007/s10858-011-9506-4 · 3.14 Impact Factor
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