RESUMO
The viral spike of HIV-1 is composed of three gp120 envelope glycoproteins attached noncovalently to three gp41 transmembrane molecules. Viral entry is initiated by binding to the CD4 receptor on the cell surface, which induces large conformational changes in gp120. These changes not only provide a model for receptor-triggered entry, but affect spike sensitivity to drug- and antibody-mediated neutralization. Although some of the details of the CD4-induced conformational change have been visualized by crystal structures and cryoelectron tomograms, the critical gp41-interactive region of gp120 was missing from previous atomic-level characterizations. Here we determine the crystal structure of an HIV-1 gp120 core with intact gp41-interactive region in its CD4-bound state, compare this structure to unliganded and antibody-bound forms to identify structurally invariant and plastic components, and use ligand-oriented cryoelectron tomograms to define component mobility in the viral spike context. Newly defined gp120 elements proximal to the gp41 interface complete a 7-stranded beta-sandwich, which appeared invariant in conformation. Loop excursions emanating from the sandwich form three topologically separate--and structurally plastic--layers, topped off by the highly glycosylated gp120 outer domain. Crystal structures, cryoelectron tomograms, and interlayer chemistry were consistent with a mechanism in which the layers act as a shape-changing spacer, facilitating movement between outer domain and gp41-associated beta-sandwich and providing for conformational diversity used in immune evasion. A "layered" gp120 architecture thus allows movement among alternative glycoprotein conformations required for virus entry and immune evasion, whereas a beta-sandwich clamp maintains gp120-gp41 interaction and regulates gp41 transitions.
Assuntos
Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Proteína gp120 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/química , HIV-1/fisiologia , Fusão de Membrana , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
The binding surface on CD4 for the HIV-1 gp120 envelope glycoprotein has been transplanted previously onto a scorpion-toxin scaffold. Here, we use X-ray crystallography to characterize atomic-level details of gp120 with this transplant, CD4M33. Despite known envelope flexibility, the conformation of gp120 induced by CD4M33 was so similar to that induced by CD4 that localized measures were required to distinguish ligand-induced differences from lattice variation. To investigate relationships between structure, function, and mimicry, an F23 analog of CD4M33 was devised. Structural and thermodynamic analyses showed F23 to be a better molecular mimic of CD4 than CD4M33. F23 also showed increased neutralization breadth, against diverse isolates of HIV-1, HIV-2, and SIVcpz. Our results lend insight into the stability of the CD4 bound conformation of gp120, define measures that quantify molecular mimicry as a function of evolutionary distance, and suggest how such evaluations might be useful in developing mimetic antagonists with increased neutralization breadth.
Assuntos
Fármacos Anti-HIV/química , Antígenos CD4/química , Proteína gp120 do Envelope de HIV/química , HIV-1/efeitos dos fármacos , Mimetismo Molecular , Animais , Fármacos Anti-HIV/farmacologia , Antígenos CD4/farmacologia , Cristalografia por Raios X , Humanos , Ligantes , Conformação Proteica , Venenos de Escorpião/química , TermodinâmicaRESUMO
Suitable conditions for protein crystallization are commonly identified by screening combinations of independent factors that affect crystal formation. Because precipitating agents are prime determinants of crystallization, we investigated whether a systematic exploration of combinations of mechanistically distinct precipitants would enhance crystallization. A crystallization screen containing 64 precipitant mixtures was devised. Tests with ten HIV envelope-related proteins demonstrated that use of precipitant mixtures significantly enhanced both the probability of crystallization as well as the quality of optimized crystals. Tests with hen egg white lysozyme generated a novel C2 crystal from a salt/organic solvent mixture; structure solution at 2 A resolution revealed a lattice held together by both hydrophobic and electrostatic dyad interactions. The results indicate that mechanistically distinct precipitants can synergize, with precipitant combinations adding unique dimensions to protein crystallization.
Assuntos
Proteínas/química , Animais , Precipitação Química , Galinhas , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Muramidase/química , Conformação ProteicaRESUMO
The third variable region (V3) of the HIV-1 gp120 envelope glycoprotein is immunodominant and contains features essential for coreceptor binding. We determined the structure of V3 in the context of an HIV-1 gp120 core complexed to the CD4 receptor and to the X5 antibody at 3.5 angstrom resolution. Binding of gp120 to cell-surface CD4 would position V3 so that its coreceptor-binding tip protrudes 30 angstroms from the core toward the target cell membrane. The extended nature and antibody accessibility of V3 explain its immunodominance. Together, the results provide a structural rationale for the role of V3 in HIV entry and neutralization.
Assuntos
Antígenos CD4/metabolismo , Proteína gp120 do Envelope de HIV/química , HIV-1/química , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Sítios de Ligação , Antígenos CD4/química , Cristalização , Cristalografia por Raios X , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/imunologia , HIV-1/metabolismo , Humanos , Ligação de Hidrogênio , Epitopos Imunodominantes , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Receptores CCR5/química , Receptores CCR5/metabolismo , Receptores CXCR4/química , Receptores CXCR4/metabolismoRESUMO
The conserved surface of the HIV-1 gp120 envelope glycoprotein that binds to the HIV-1 coreceptor is protected from humoral recognition by multiple layers of camouflage. Here we present sequence and genomic analyses for 12 antibodies that pierce these defenses and determine the crystal structures of 5. The data reveal mechanisms and atomic-level details for three unusual immune features: posttranslational mimicry of coreceptor by tyrosine sulfation of antibody, an alternative molecular mechanism controlling such sulfation, and highly selective V(H)-gene usage. When confronted by extraordinary viral defenses, the immune system unveils novel adaptive capabilities, with tyrosine sulfation enhancing the vocabulary of antigen recognition.
Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , Genes de Imunoglobulinas , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Tirosina/análogos & derivados , Tirosina/metabolismo , Sequência de Aminoácidos , Formação de Anticorpos , Antígenos CD4/imunologia , Cristalografia por Raios X , Infecções por HIV/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/química , Região de Junção de Imunoglobulinas/química , Região de Junção de Imunoglobulinas/genética , Região Variável de Imunoglobulina/química , Dados de Sequência Molecular , Sulfatos/metabolismoRESUMO
The ability of human immunodeficiency virus (HIV-1) to persist and cause AIDS is dependent on its avoidance of antibody-mediated neutralization. The virus elicits abundant, envelope-directed antibodies that have little neutralization capacity. This lack of neutralization is paradoxical, given the functional conservation and exposure of receptor-binding sites on the gp120 envelope glycoprotein, which are larger than the typical antibody footprint and should therefore be accessible for antibody binding. Because gp120-receptor interactions involve conformational reorganization, we measured the entropies of binding for 20 gp120-reactive antibodies. Here we show that recognition by receptor-binding-site antibodies induces conformational change. Correlation with neutralization potency and analysis of receptor-antibody thermodynamic cycles suggested a receptor-binding-site 'conformational masking' mechanism of neutralization escape. To understand how such an escape mechanism would be compatible with virus-receptor interactions, we tested a soluble dodecameric receptor molecule and found that it neutralized primary HIV-1 isolates with great potency, showing that simultaneous binding of viral envelope glycoproteins by multiple receptors creates sufficient avidity to compensate for such masking. Because this solution is available for cell-surface receptors but not for most antibodies, conformational masking enables HIV-1 to maintain receptor binding and simultaneously to resist neutralization.