RESUMO
UNLABELLED: The RV144 vaccine trial implicated epitopes in the C1 region of gp120 (A32-like epitopes) as targets of potentially protective antibody-dependent cellular cytotoxicity (ADCC) responses. A32-like epitopes are highly immunogenic, as infected or vaccinated individuals frequently produce antibodies specific for these determinants. Antibody titers, as measured by enzyme-linked immunosorbent assay (ELISA) against these epitopes, however, do not consistently correlate with protection. Here, we report crystal structures of CD4-stabilized gp120 cores complexed with the Fab fragments of two nonneutralizing, A32-like monoclonal antibodies (MAbs), N5-i5 and 2.2c, that compete for antigen binding and have similar antigen-binding affinities yet exhibit a 75-fold difference in ADCC potency. We find that these MAbs recognize overlapping epitopes formed by mobile layers 1 and 2 of the gp120 inner domain, including the C1 and C2 regions, but bind gp120 at different angles via juxtaposed VH and VL contact surfaces. A comparison of structural and immunological data further showed that antibody orientation on bound antigen and the capacity to form multivalent antigen-antibody complexes on target cells were key determinants of ADCC potency, with the latter process having the greater impact. These studies provide atomic-level definition of A32-like epitopes implicated as targets of protective antibodies in RV144. Moreover, these studies establish that epitope structure and mode of antibody binding can dramatically affect the potency of Fc-mediated effector function against HIV-1. These results provide key insights for understanding, refining, and improving the outcome of HIV vaccine trials, in which relevant immune responses are facilitated by A32-like elicited responses. IMPORTANCE: HIV-1 Env is a primary target for antibodies elicited during infection. Although a small number of infected individuals elicit broadly neutralizing antibodies, the bulk of the humoral response consists of antibodies that do not neutralize or do so with limited breadth but may effect protection through Fc receptor-dependent processes, such as antibody-dependent cellular cytotoxicity (ADCC). Understanding these nonneutralizing responses is an important aspect of elucidating the complete spectrum of immune response against HIV-1 infection. With this report, we provide the first atomic-level definition of nonneutralizing CD4-induced epitopes in the N-terminal region of the HIV-1 gp120 (A32-like epitopes). Further, our studies point to the dominant role of precise epitope targeting and mode of antibody attachment in ADCC responses even when largely overlapping epitopes are involved. Such information provides key insights into the mechanisms of Fc-mediated function of antibodies to HIV-1 and will help us understand the outcome of vaccine trials based on humoral immunity.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Cristalografia por Raios X , Epitopos/química , Epitopos/imunologia , Anticorpos Anti-HIV/química , Proteína gp120 do Envelope de HIV/química , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Modelos Moleculares , Ligação Proteica , Conformação ProteicaRESUMO
G protein-coupled receptor (GPCR) kinases (GRKs) instigate the desensitization of activated GPCRs via phosphorylation that promotes interaction with arrestins, thereby preventing the interaction of GPCRs with heterotrimeric G proteins. A current proposed model of GRK1 activation involves the binding of activated rhodopsin (Rho*) to the N-terminal region of GRK1. Perhaps concomitantly, this N-terminal region also stabilizes a closed, active conformation of the kinase domain. To further probe this model, we mapped changes in the backbone flexibility of GRK1 as it binds to its two substrates, adenosine triphosphate (Mg(2+)·ATP) and Rho*. We found that the conformational flexibility of GRK1 was reduced in the presence of either Mg(2+)·ATP or Rho*, with Mg(2+)·ATP having the greatest effect. In a truncated form of GRK1 lacking the N-terminal region (ΔN-GRK1), peptides that directly interact with ATP were not as dramatically stabilized by adding Mg(2+)·ATP, and dynamics were greater in the interface between the large lobe of the kinase domain and the regulator of the G protein signaling homology domain. In the presence of Mg(2+)·ATP, the influence of Rho* versus Rho on GRK1 dynamics was negligible.
Assuntos
Receptor Quinase 1 Acoplada a Proteína G/química , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Animais , Bovinos , Células Cultivadas , Receptor Quinase 1 Acoplada a Proteína G/genética , Receptor Quinase 1 Acoplada a Proteína G/metabolismo , Conformação Molecular , Simulação de Dinâmica Molecular , Fosforilação , Estrutura Terciária de Proteína , Rodopsina/química , Segmento Externo da Célula Bastonete/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
G protein-coupled receptors (GPCRs) represent the largest class of integral membrane protein receptors in the human genome. Despite the great diversity of ligands that activate these GPCRs, they interact with a relatively small number of intracellular proteins to induce profound physiological change. Both heterotrimeric G proteins and GPCR kinases are well known for their ability to specifically recognize GPCRs in their active state. Recent structural studies now suggest that heterotrimeric G proteins and GPCR kinases identify activated receptors via a common molecular mechanism despite having completely different folds.
Assuntos
Quinases de Receptores Acoplados a Proteína G , Proteínas Heterotriméricas de Ligação ao GTP , Dobramento de Proteína , Animais , Ativação Enzimática , Quinases de Receptores Acoplados a Proteína G/química , Quinases de Receptores Acoplados a Proteína G/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , HumanosRESUMO
G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors (GPCRs) to initiate receptor desensitization. In addition to the canonical phosphoacceptor site of the kinase domain, activated receptors bind to a distinct docking site that confers higher affinity and activates GRKs allosterically. Recent mutagenesis and structural studies support a model in which receptor docking activates a GRK by stabilizing the interaction of its â¼20-amino acid N-terminal region with the kinase domain. This interaction in turn stabilizes a closed, more active conformation of the enzyme. To investigate the importance of this interaction for the process of GRK activation, we first validated the functionality of the N-terminal region in rhodopsin kinase (GRK1) by site-directed mutagenesis and then introduced a disulfide bond to cross-link the N-terminal region of GRK1 with its specific binding site on the kinase domain. Characterization of the kinetic and biophysical properties of the cross-linked protein showed that disulfide bond formation greatly enhances the catalytic efficiency of the peptide phosphorylation, but receptor-dependent phosphorylation, Meta II stabilization, and inhibition of transducin activation were unaffected. These data indicate that the interaction of the N-terminal region with the kinase domain is important for GRK activation but does not dictate the affinity of GRKs for activated receptors.
Assuntos
Receptor Quinase 1 Acoplada a Proteína G/química , Receptor Quinase 1 Acoplada a Proteína G/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Estrutura Terciária de ProteínaRESUMO
G-protein-coupled receptor (GPCR) oligomerization has been observed in a wide variety of experimental contexts, but the functional significance of this phenomenon at different stages of the life cycle of class A GPCRs remains to be elucidated. Rhodopsin (Rh), a prototypical class A GPCR of visual transduction, is also capable of forming dimers and higher order oligomers. The recent demonstration that Rh monomer is sufficient to activate its cognate G protein, transducin, prompted us to test whether the same monomeric state is sufficient for rhodopsin phosphorylation and arrestin-1 binding. Here we show that monomeric active rhodopsin is phosphorylated by rhodopsin kinase (GRK1) as efficiently as rhodopsin in the native disc membrane. Monomeric phosphorylated light-activated Rh (P-Rh*) in nanodiscs binds arrestin-1 essentially as well as P-Rh* in native disc membranes. We also measured the affinity of arrestin-1 for P-Rh* in nanodiscs using a fluorescence-based assay and found that arrestin-1 interacts with monomeric P-Rh* with low nanomolar affinity and 1:1 stoichiometry, as previously determined in native disc membranes. Thus, similar to transducin activation, rhodopsin phosphorylation by GRK1 and high affinity arrestin-1 binding only requires a rhodopsin monomer.
Assuntos
Arrestinas/metabolismo , Receptor Quinase 1 Acoplada a Proteína G/metabolismo , Rodopsina/metabolismo , Transdução de Sinais/fisiologia , Visão Ocular/fisiologia , Sequência de Aminoácidos , Animais , Arrestinas/genética , Bovinos , Eletroquímica , Fluorescência , Leucina/metabolismo , Leucina/farmacologia , Lipídeos/química , Dados de Sequência Molecular , Mutação , Fosforilação/fisiologia , Ligação Proteica/fisiologia , Rodopsina/química , Rodopsina/genética , Trítio , beta-ArrestinasRESUMO
G protein-coupled receptor (GPCR) kinases (GRKs) selectively recognize and are allosterically regulated by activated GPCRs, but the molecular basis for this interaction is not understood. Herein, we report crystal structures of GRK6 in which regions known to be critical for receptor phosphorylation have coalesced to stabilize the kinase domain in a closed state and to form a likely receptor docking site. The crux of this docking site is an extended N-terminal helix that bridges the large and small lobes of the kinase domain and lies adjacent to a basic surface of the protein proposed to bind anionic phospholipids. Mutation of exposed, hydrophobic residues in the N-terminal helix selectively inhibits receptor, but not peptide phosphorylation, suggesting that these residues interact directly with GPCRs. Our structural and biochemical results thus provide an explanation for how receptor recognition, phospholipid binding, and kinase activation are intimately coupled in GRKs.
Assuntos
Ativação Enzimática/fisiologia , Quinases de Receptores Acoplados a Proteína G/química , Quinases de Receptores Acoplados a Proteína G/metabolismo , Modelos Moleculares , Conformação Proteica , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Sítios de Ligação/genética , Cristalização , Análise Mutacional de DNA , Ativação Enzimática/genética , Cinética , Dados de Sequência Molecular , Fosforilação , Nucleosídeos de Pirimidina/química , Nucleosídeos de Pirimidina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/genéticaRESUMO
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.
Assuntos
Antígenos CD4/metabolismo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/química , Sequência de Aminoácidos , Cristalografia por Raios X , Medição da Troca de Deutério , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , Interações Hospedeiro-Patógeno , Humanos , Técnicas In Vitro , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
Human immunodeficiency virus (HIV-1) entry into cells is mediated by a trimeric complex consisting of noncovalently associated gp120 (exterior) and gp41 (transmembrane) envelope glycoproteins. The binding of gp120 to receptors on the target cell alters the gp120-gp41 relationship and activates the membrane-fusing capacity of gp41. Interaction of gp120 with the primary receptor, CD4, results in the exposure of the gp120 third variable (V3) loop, which contributes to binding the CCR5 or CXCR4 chemokine receptors. We show here that insertions in the V3 stem or polar substitutions in a conserved hydrophobic patch near the V3 tip result in decreased gp120-gp41 association (in the unliganded state) and decreased chemokine receptor binding (in the CD4-bound state). Subunit association and syncytium-forming ability of the envelope glycoproteins from primary HIV-1 isolates were disrupted more by V3 changes than those of laboratory-adapted HIV-1 envelope glycoproteins. Changes in the gp120 beta2, beta19, beta20, and beta21 strands, which evidence suggests are proximal to the V3 loop in unliganded gp120, also resulted in decreased gp120-gp41 association. Thus, a gp120 element composed of the V3 loop and adjacent beta strands contributes to quaternary interactions that stabilize the unliganded trimer. CD4 binding dismantles this element, altering the gp120-gp41 relationship and rendering the hydrophobic patch in the V3 tip available for chemokine receptor binding.
Assuntos
Antígenos CD4/metabolismo , Proteína gp120 do Envelope de HIV/química , Sequência de Aminoácidos , Epitopos , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp120 do Envelope de HIV/fisiologia , Proteína gp41 do Envelope de HIV/química , Dados de Sequência Molecular , Conformação Proteica , Subunidades Proteicas , Receptores CCR5/metabolismo , Relação Estrutura-AtividadeRESUMO
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
G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate activated GPCRs and initiate their desensitization. Many prior studies suggest that activated GPCRs dock to an allosteric site on the GRKs and thereby stimulate kinase activity. The extreme N-terminal region of GRKs is clearly involved in this process, but its role is not understood. Using our recent structure of bovine GRK1 as a guide, we generated mutants of solvent-exposed residues in the GRK1 kinase domain that are conserved among GRKs but not in the extended protein kinase A, G, and C family and evaluated their catalytic activity. Mutation of select residues in strands beta1 and beta3 of the kinase small lobe, alphaD of the kinase large lobe, and the protein kinase A, G, and C kinase C-tail greatly impaired receptor phosphorylation. The most dramatic effect was observed for mutation of an invariant arginine on the beta1-strand (approximately 1000-fold decrease in k(cat)/K(m)). These residues form a continuous surface that is uniquely available in GRKs for protein-protein interactions. Surprisingly, these mutants, as well as a 19-amino acid N-terminal truncation of GRK1, also show decreased catalytic efficiency for peptide substrates, although to a lesser extent than for receptor phosphorylation. Our data suggest that the N-terminal region and the newly identified surface interact and stabilize the closed, active conformation of the kinase domain. Receptor binding is proposed to promote this interaction, thereby enhancing GRK activity.
Assuntos
Quinase 2 de Receptor Acoplado a Proteína G/química , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Quinases de Receptores Acoplados a Proteína G/química , Quinases de Receptores Acoplados a Proteína G/metabolismo , Regulação Alostérica , Sítio Alostérico , Sequência de Aminoácidos , Animais , Bovinos , Ativação Enzimática , Quinase 2 de Receptor Acoplado a Proteína G/genética , Quinases de Receptores Acoplados a Proteína G/genética , Humanos , Técnicas In Vitro , Cinética , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Peptídeos/química , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Human immunodeficiency virus (HIV-1) interaction with the primary receptor, CD4, induces conformational changes in the viral envelope glycoproteins that allow binding to the CCR5 second receptor and virus entry into the host cell. The small molecule NBD-556 mimics CD4 by binding the gp120 exterior envelope glycoprotein, moderately inhibiting virus entry into CD4-expressing target cells and enhancing CCR5 binding and virus entry into CCR5-expressing cells lacking CD4. Studies of NBD-556 analogs and gp120 mutants suggest that (1) NBD-556 binds within the Phe 43 cavity, a highly conserved, functionally important pocket formed as gp120 assumes the CD4-bound conformation; (2) the NBD-556 phenyl ring projects into the Phe 43 cavity; (3) enhancement of CD4-independent infection by NBD-556 requires the induction of conformational changes in gp120; and (4) increased affinity of NBD-556 analogs for gp120 improves antiviral potency during infection of CD4-expressing cells.
Assuntos
Proteína gp120 do Envelope de HIV/química , HIV-1/fisiologia , Síndrome da Imunodeficiência Adquirida/virologia , Antígenos CD4/química , Antígenos CD4/fisiologia , Calorimetria , Sequência Conservada , HIV-1/química , HIV-1/patogenicidade , Humanos , Modelos Moleculares , Fenilalanina/química , Conformação Proteica , Receptores CXCR4/química , Proteínas Recombinantes/metabolismo , TermodinâmicaRESUMO
Miniproteins provide a bridge between proteins and small molecules. Here we adapt methods from combinatorial chemistry to optimize CD4M33, a synthetic miniprotein into which we had previously transplanted the HIV-1 gp120 binding surface of the CD4 receptor. Iterative deconvolution of generated libraries produced CD4M47, a derivative of CD4M33 that had been optimized at four positions. Surface plasmon resonance demonstrated fourfold to sixfold improvement in CD4M47 affinity for gp120 to a level about threefold tighter than that of CD4 itself. Assessment of the neutralization properties of CD4M47 against a diverse range of isolates spanning from HIV-1 to SIVcpz showed that CD4M47 retained the extraordinary breadth of the parent CD4M33, but yielded only limited improvements in neutralization potencies. Crystal structures of CD4M47 and a phenylalanine variant ([Phe23]M47) were determined at resolutions of 2.4 and 2.6 A, in ternary complexes with HIV-1 gp120 and the 17b antibody. Analysis of these structures revealed a correlation between mimetic affinity for gp120 and overall mimetic-gp120 interactive surface. A correlation was also observed between CD4- and mimetic-induced gp120 structural similarity and CD4- and mimetic-induced gp120 affinity for the CCR5 coreceptor. Despite mimetic substitutions, including a glycine-to-(d)-proline change, the gp120 conformation induced by CD4M47 was as close or closer to the conformation induced by CD4 as the one induced by the parent CD4M33. Our results demonstrate the ability of combinatorial chemistry to optimize a disulfide-containing miniprotein, and of structural biology to decipher the resultant interplay between binding affinity, neutralization breadth, molecular mimicry, and induced affinity for CCR5.
Assuntos
Antígenos CD4/química , Técnicas de Química Combinatória , Proteína gp120 do Envelope de HIV/química , HIV-1/química , Mimetismo Molecular , Conformação Proteica , Sequência de Aminoácidos , Animais , Antígenos CD4/genética , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Testes de Neutralização , Receptores CCR5/química , Receptores CCR5/genética , Ressonância de Plasmônio de SuperfícieRESUMO
The CCR5 co-receptor binds to the HIV-1 gp120 envelope glycoprotein and facilitates HIV-1 entry into cells. Its N terminus is tyrosine-sulfated, as are many antibodies that react with the co-receptor binding site on gp120. We applied nuclear magnetic resonance and crystallographic techniques to analyze the structure of the CCR5 N terminus and that of the tyrosine-sulfated antibody 412d in complex with gp120 and CD4. The conformations of tyrosine-sulfated regions of CCR5 (alpha-helix) and 412d (extended loop) are surprisingly different. Nonetheless, a critical sulfotyrosine on CCR5 and on 412d induces similar structural rearrangements in gp120. These results now provide a framework for understanding HIV-1 interactions with the CCR5 N terminus during viral entry and define a conserved site on gp120, whose recognition of sulfotyrosine engenders posttranslational mimicry by the immune system.
Assuntos
Antígenos CD4/química , Anticorpos Anti-HIV/química , Proteína gp120 do Envelope de HIV/química , Receptores CCR5/química , Sequência de Aminoácidos , Antígenos CD4/imunologia , Cristalografia por Raios X , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Humanos , Modelos Moleculares , Mimetismo Molecular , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Receptores CCR5/metabolismo , Sulfatos/metabolismo , Tirosina/metabolismo , Internalização do VírusRESUMO
Entry of human immunodeficiency virus type 1 (HIV-1) and HIV-2 requires interactions between the envelope glycoprotein (Env) on the virus and CD4 and a chemokine receptor, either CCR5 or CXCR4, on the cell surface. The V3 loop of the HIV gp120 glycoprotein plays a critical role in this process, determining tropism for CCR5- or CXCR4-expressing cells, but details of how V3 interacts with these receptors have not been defined. Using an iterative process of deletion mutagenesis and in vitro adaptation of infectious viruses, variants of HIV-2 were derived that could replicate without V3, either with or without a deletion of the V1/V2 variable loops. The generation of these functional but markedly minimized Envs required adaptive changes on the gp120 core and gp41 transmembrane glycoprotein. V3-deleted Envs exhibited tropism for both CCR5- and CXCR4-expressing cells, suggesting that domains on the gp120 core were mediating interactions with determinants shared by both coreceptors. Remarkably, HIV-2 Envs with V3 deletions became resistant to small-molecule inhibitors of CCR5 and CXCR4, suggesting that these drugs inhibit wild-type viruses by disrupting a specific V3 interaction with the coreceptor. This study represents a proof of concept that HIV Envs lacking V3 alone or in combination with V1/V2 that retain functional domains required for viral entry can be derived. Such minimized Envs may be useful in understanding Env function, screening for new inhibitors of gp120 core interactions with chemokine receptors, and designing novel immunogens for vaccines.
Assuntos
Farmacorresistência Viral , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/metabolismo , HIV-2/metabolismo , Internalização do Vírus , Replicação Viral , Fármacos Anti-HIV/farmacologia , Benzilaminas , Antagonistas dos Receptores CCR5 , Antígenos CD4/genética , Antígenos CD4/metabolismo , Linhagem Celular , Ciclamos , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Proteína gp120 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/genética , HIV-2/genética , Compostos Heterocíclicos/farmacologia , Humanos , Oligopeptídeos/farmacologia , Estrutura Secundária de Proteína/genética , Receptores CCR5/genética , Receptores CCR5/metabolismo , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Deleção de Sequência/genética , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
CE offers the advantages of high speed, great efficiency, as well as the requirement of minimum amounts of sample and buffer for the analysis of proteins. In this review, we summarize the CE-based techniques coupled with absorption, LIF, and MS detection systems for the analysis of proteins mostly within the past 5 years. The basic principle of each technique and its advantages and disadvantages for protein analysis are discussed in brief. Advanced CE techniques, including on-column concentration techniques and high-efficiency multidimensional separation techniques, for high-throughput protein profiling of complex biological samples and/or of single cells are emphasized. Although the developed techniques provide improved peak capacity, they have not become practical tools for proteomics, mainly because of poor reproducibility, low-sample lading capacity, and low throughput due to ineffective interfaces between two separation dimensions and that between separation and MS systems. In order to identify the complexities and dynamics of the proteomes expressed by cells, tissues, or organisms, techniques providing improved analytical sensitivity, throughput, and dynamic ranges are still demanded.
Assuntos
Eletroforese Capilar/métodos , Proteínas/análise , Proteínas/isolamento & purificação , Humanos , Espectrometria de MassasRESUMO
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 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
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
Human immunodeficiency virus (HIV-1) enters target cells by binding its gp120 exterior envelope glycoprotein to CD4 and one of the chemokine receptors, CCR5 or CXCR4. CD4-induced (CD4i) antibodies bind gp120 more efficiently after CD4 binding and block the interaction with the chemokine receptor. Examples of CD4i antibodies are limited, and the prototypes of the CD4i antibodies exhibit only weak neutralizing activity against primary, clinical HIV-1 isolates. Here we report the identification of a novel antibody, E51, that exhibits CD4-induced binding to gp120 and neutralizes primary HIV-1 more efficiently than the prototypic CD4i antibodies. The E51 antibody blocks the interaction of gp120-CD4 complexes with CCR5 and binds to a highly conserved, basic gp120 element composed of the beta 19-strand and surrounding structures. Thus, on primary HIV-1 isolates, this gp120 region, which has been previously implicated in chemokine receptor binding, is accessible to a subset of CD4i antibodies.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos CD4/imunologia , Mapeamento de Epitopos , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Antígenos CD4/metabolismo , Linhagem Celular , Epitopos/imunologia , Proteína gp120 do Envelope de HIV/genética , Humanos , Modelos Moleculares , Testes de Neutralização , Receptores CCR5/química , Receptores CCR5/metabolismo , Receptores de HIV/química , Receptores de HIV/metabolismoRESUMO
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.