Chih-chin Huang

Structure of a V3-containing HIV-1 gp120 core.

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.

Access of Antibody Molecules to the Conserved Coreceptor Binding Site on Glycoprotein gp120 Is Sterically Restricted on Primary Human Immunodeficiency Virus Type 1

ABSTRACT Anti-human immunodeficiency virus type 1 (HIV-1) antibodies whose binding to gp120 is enhanced by CD4 binding (CD4i antibodies) are generally considered nonneutralizing for primary HIV-1 isolates. However, a novel CD4i-specific Fab fragment, X5, has recently been found to neutralize a wide range of primary isolates. To investigate the precise nature of the extraordinary neutralizing ability of Fab X5, we evaluated the abilities of different forms (immunoglobulin G [IgG], Fab, and single-chain Fv) of X5 and other CD4i monoclonal antibodies to neutralize a range of primary HIV-1 isolates. Our results show that, for a number of isolates, the size of the neutralizing agent is inversely correlated with its ability to neutralize. Thus, the poor ability of CD4i-specific antibodies to neutralize primary isolates is due, at least in part, to steric factors that limit antibody access to the gp120 epitopes. Studies of temperature-regulated neutralization or fusion-arrested intermediates suggest that the steric effects are important in limiting the binding of IgG to the viral envelope glycoproteins after HIV-1 has engaged CD4 on the target cell membrane. The results identify hurdles in using CD4i epitopes as targets for antibody-mediated neutralization in vaccine design but also indicate that the CD4i regions could be efficiently targeted by small molecule entry inhibitors.

Structure of HIV-1 gp120 with gp41-interactive region reveals layered envelope architecture and basis of conformational mobility.

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 β-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 β-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 β-sandwich clamp maintains gp120–gp41 interaction and regulates gp41 transitions.

Tyrosine Sulfation of Human Antibodies Contributes to Recognition of the CCR5 Binding Region of HIV-1 gp120

Sulfated tyrosines at the amino terminus of the principal HIV-1 coreceptor CCR5 play a critical role in its ability to bind the HIV-1 envelope glycoprotein gp120 and mediate HIV-1 infection. Here, we show that a number of human antibodies directed against gp120 are tyrosine sulfated at their antigen binding sites. Like that of CCR5, antibody association with gp120 is dependent on sulfate moieties, enhanced by CD4, and inhibited by sulfated CCR5-derived peptides. Most of these antibodies preferentially associate with gp120 molecules of CCR5-utilizing (R5) isolates and neutralize primary R5 isolates more efficiently than laboratory-adapted isolates. These studies identify a distinct subset of CD4-induced HIV-1 neutralizing antibodies that closely emulate CCR5 and demonstrate that tyrosine sulfation can contribute to the potency and diversity of the human humoral response.

Enhancing Protein Crystallization through Precipitant Synergy

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.

Structural Definition of an Antibody-Dependent Cellular Cytotoxicity Response Implicated in Reduced Risk for HIV-1 Infection

ABSTRACT 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.

Combinatorial Optimization of a CD4-Mimetic Miniprotein and Cocrystal Structures with HIV-1 gp120 Envelope Glycoprotein

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.

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.

Replication-Competent Variants of Human Immunodeficiency Virus Type 2 Lacking the V3 Loop Exhibit Resistance to Chemokine Receptor Antagonists

ABSTRACT 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.

P09-13. Structure of HIV-1 gp41 interactive region: layered architecture and basis of conformational mobility

Background Crystal structures of unliganded, CD4-bound, and antibody-bound gp120 core show large conformational rearrangements upon ligand binding. Moreover, cryo-EM tomograms of the HIV-1 viral spike (gp120/gp41) also show large ligand-induced shifts in gp120 orientation. Conformational change is one mechanism that HIV-1 has developed to evade the host immune response. We hypothesized that the key to understanding this mobility resided in the critical gp41-interactive region of gp120. However, previously determined core structures all contained deletions of the gp41-interactive region.