title: Local conformational stability of HIV-1 gp120 in unliganded and CD4-bound states as defined by amide hydrogen/deuterium exchange.
abstract: 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.
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-3027, USA.
authors.forename: Leopold
authors.initials: L
authors.lastname: Kong
authors.forename: Chih-Chin
authors.initials: CC
authors.lastname: Huang
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Coales
authors.forename: Kathleen S
authors.initials: KS
authors.lastname: Molnar
authors.forename: Jeff
authors.initials: J
authors.lastname: Skinner
authors.forename: Yoshitomo
authors.initials: Y
authors.lastname: Hamuro
authors.forename: Peter D
authors.initials: PD
authors.lastname: Kwong
mesh_terms: D000595:Amino Acid Sequence; D015704:CD4 Antigens / Q000378:metabolism*; D018360:Crystallography, X-Ray; D041961:Deuterium Exchange Measurement; D015699:HIV Envelope Protein gp120 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics; D054884:Host-Pathogen Interactions; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D055550:Protein Stability; D017433:Protein Structure, Secondary; D013816:Thermodynamics
chemical_list: D015704:CD4 Antigens; D015699:HIV Envelope Protein gp120; D008024:Ligands; D010446:Peptide Fragments; C514793:gp120 protein, Human immunodeficiency virus 1