Robyn L. Stanfield

An αβ T cell receptor structure at 2.5 Å and its orientation in the TCR-MHC complex

The central event in the cellular immune response to invading microorganisms is the specific recognition of foreign peptides bound to major histocompatibility complex (MHC) molecules by the αβ T cell receptor (TCR). The x-ray structure of the complete extracellular fragment of a glycosylated αβ TCR was determined at 2.5 angstroms, and its orientation bound to a class I MHC-peptide (pMHC) complex was elucidated from crystals of the TCR-pMHC complex. The TCR resembles an antibody in the variable Vα and Vβ domains but deviates in the constant Cα domain and in the interdomain pairing of Cα with Cβ. Four of seven possible asparagine-linked glycosylation sites have ordered carbohydrate moieties, one of which lies in the Cα-Cβ interface. The TCR combining site is relatively flat except for a deep hydrophobic cavity between the hypervariable CDR3s (complementarity-determining regions) of the α and β chains. The 2C TCR covers the class I MHC H-2Kb binding groove so that the Vα CDRs 1 and 2 are positioned over the amino-terminal region of the bound dEV8 peptide, the Vβ chain CDRs 1 and 2 are over the carboxyl-terminal region of the peptide, and the Vα and Vβ CDR3s straddle the peptide between the helices around the central position of the peptide.

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.

The Broadly Neutralizing Anti-Human Immunodeficiency Virus Type 1 Antibody 2G12 Recognizes a Cluster of α1→2 Mannose Residues on the Outer Face of gp120

ABSTRACT 2G12 is a broadly neutralizing human monoclonal antibody against human immunodeficiency virus type-1 (HIV-1) that has previously been shown to bind to a carbohydrate-dependent epitope on gp120. Here, site-directed mutagenesis and carbohydrate analysis were used to define further the 2G12 epitope. Extensive alanine scanning mutagenesis showed that elimination of the N-linked carbohydrate attachment sequences associated with residues N295, N332, N339, N386, and N392 by N→A substitution produced significant decreases in 2G12 binding affinity to gp120JR-CSF. Further mutagenesis suggested that the glycans at N339 and N386 were not critical for 2G12 binding to gp120JR-CSF. Comparison of the sequences of isolates neutralized by 2G12 was also consistent with a lesser role for glycans attached at these positions. The mutagenesis studies provided no convincing evidence for the involvement of gp120 amino acid side chains in 2G12 binding. Antibody binding was inhibited when gp120 was treated with Aspergillus saitoi mannosidase, Jack Bean mannosidase, or endoglycosidase H, indicating that Manα1→2Man-linked sugars of oligomannose glycans on gp120 are required for 2G12 binding. Consistent with this finding, the binding of 2G12 to gp120 could be inhibited by monomeric mannose but not by galactose, glucose, or N-acetylglucosamine. The inability of 2G12 to bind to gp120 produced in the presence of the glucose analogue N-butyl-deoxynojirimycin similarly implicated Manα1→2Man-linked sugars in 2G12 binding. Competition experiments between 2G12 and the lectin cyanovirin for binding to gp120 showed that 2G12 only interacts with a subset of available Manα1→2Man-linked sugars. Consideration of all the data, together with inspection of a molecular model of gp120, suggests that the most likely epitope for 2G12 is formed from mannose residues contributed by the glycans attached to N295 and N332, with the other glycans playing an indirect role in maintaining epitope conformation.

Crystal Structure of a Soluble Cleaved HIV-1 Envelope Trimer

Knowing the Enemy Infection of host cells by HIV-1 is mediated by an envelope glycoprotein (Env) trimeric spike on the surface of the virus. Proteins comprising the Env trimer must be cleaved for infectivity, and thus viral fusion involves three Env conformations. The flexibility of the Env trimer has made it a challenge to determine a high-resolution structure, although such a structure is key both for understanding trimer function and for guiding vaccine design. Lyumkis et al. (p. 1484) and Julien et al. (p. 1477) studied soluble cleaved trimers stabilized by specific mutations but that have kept a near-native antigenicity profile. Lyumkis et al. present a high-resolution structure of the trimer in complex with a broadly neutralizing antibody, and Julien et al. present a crystal structure of the trimer in complex with another broadly neutralizing antibody. Key structural features dictate how the HIV envelope protein functions and interacts with the human immune system. HIV-1 entry into CD4+ target cells is mediated by cleaved envelope glycoprotein (Env) trimers that have been challenging to characterize structurally. Here, we describe the crystal structure at 4.7 angstroms of a soluble, cleaved Env trimer that is stabilized and antigenically near-native (termed the BG505 SOSIP.664 gp140 trimer) in complex with a potent broadly neutralizing antibody, PGT122. The structure shows a prefusion state of gp41, the interaction between the component gp120 and gp41 subunits, and how a close association between the gp120 V1/V2/V3 loops stabilizes the trimer apex around the threefold axis. The complete epitope of PGT122 on the trimer involves gp120 V1, V3, and several surrounding glycans. This trimer structure advances our understanding of how Env functions and is presented to the immune system, and provides a blueprint for structure-based vaccine design.

A potent and broad neutralizing antibody recognizes and penetrates the HIV glycan shield.

An HIV antibody achieves potency and breadth by binding simultaneously to two conserved glycans on the viral envelope protein. The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of antigen-binding fragments (Fabs) PGT 127 and 128 with Man9 at 1.65 and 1.29 angstrom resolution, respectively, and glycan binding data delineate a specific high mannose-binding site. Fab PGT 128 complexed with a fully glycosylated gp120 outer domain at 3.25 angstroms reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short β-strand segment of the gp120 V3 loop, accounting for its high binding affinity and broad specificify. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 immunoglobulin Gs may be mediated by cross-linking Env trimers on the viral surface.

Antibody-antigen interactions: new structures and new conformational changes

During the past year, many new antibody structures have been determined, increasing our understanding of these immunologically important molecules. Of special interest are new catalytic antibodies, antibody-peptide and antibody-virus complexes, NMR structures, and structures illustrating conformational changes and antibody cross-reactivity.

Structures of the CCR5 N Terminus and of a Tyrosine-Sulfated Antibody with HIV-1 gp120 and CD4

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 (α-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.

Antibody-antigen interactions

Abstract Different antibodies adopt substantially different shapes in order to recognize their respective antigens. Local and more global structural rearrangements, in addition to sequence and size diversity of complementarity determining loops, result in antigen-binding sites that can vary considerably in size, shape and charge distribution, even though the antibody framework structure remains invariant. The structures of several free and bound Fabs show that induced-fit mechanisms best describe the antibody-antigen recognition process. This affects the prediction, engineering and design of antibody combining sites.

Broadly Neutralizing Antibodies Present New Prospects to Counter Highly Antigenically Diverse Viruses

Certain human pathogens avoid elimination by our immune system by rapidly mutating the surface protein sites targeted by antibody responses, and consequently they tend to be problematic for vaccine development. The behavior described is prominent for a subset of viruses—the highly antigenically diverse viruses—which include HIV, influenza, and hepatitis C viruses. However, these viruses do harbor highly conserved exposed sites, usually associated with function, which can be targeted by broadly neutralizing antibodies. Until recently, not many such antibodies were known, but advances in the field have enabled increasing numbers to be identified. Molecular characterizations of the antibodies and, most importantly, of the sites of vulnerability that they recognize give hope for the discovery of new vaccines and drugs.

Hepatitis C virus E2 envelope glycoprotein core structure

Deciphering Hepatitis C Hepatitis C virus is a major cause of liver disease and cancer. Two envelope glycoproteins, E1 and E2, form a heterodimer that facilitates infection. The envelope proteins have been difficult to crystallize, hindering vaccine development. Kong et al. (p. 1090) designed an E2 core glycoprotein construct and solved the crystal structure of the glycosylated protein in complex with a broadly neutralizing antibody. The host cell receptor binding site was identified by electron microscopy and mutagenesis. The findings should help in future drug and vaccine design. The structure of a key viral surface protein provides insight for drug and vaccine development. Hepatitis C virus (HCV), a Hepacivirus, is a major cause of viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV envelope glycoproteins E1 and E2 mediate fusion and entry into host cells and are the primary targets of the humoral immune response. The crystal structure of the E2 core bound to broadly neutralizing antibody AR3C at 2.65 angstroms reveals a compact architecture composed of a central immunoglobulin-fold β sandwich flanked by two additional protein layers. The CD81 receptor binding site was identified by electron microscopy and site-directed mutagenesis and overlaps with the AR3C epitope. The x-ray and electron microscopy E2 structures differ markedly from predictions of an extended, three-domain, class II fusion protein fold and therefore provide valuable information for HCV drug and vaccine design.