Variable regions 1 and 2 (V1/V2) of human immunodeficiency virus-1 (HIV-1) gp120 envelope glycoprotein are critical for viral evasion of antibody neutralization, and are themselves protected by extraordinary sequence diversity and N-linked glycosylation. Human antibodies such as PG9 nonetheless engage V1/V2 and neutralize 80% of HIV-1 isolates. Here we report the structure of V1/V2 in complex with PG9. V1/V2 forms a four-stranded β-sheet domain, in which sequence diversity and glycosylation are largely segregated to strand-connecting loops. PG9 recognition involves electrostatic, sequence-independent and glycan interactions: the latter account for over half the interactive surface but are of sufficiently weak affinity to avoid autoreactivity. The structures of V1/V2-directed antibodies CH04 and PGT145 indicate that they share a common mode of glycan penetration by extended anionic loops. In addition to structurally defining V1/V2, the results thus identify a paradigm of antibody recognition for highly glycosylated antigens, which—with PG9—involves a site of vulnerability comprising just two glycans and a strand.

The HIV-1-envelope (Env) trimer is covered by a glycan shield of ~90 N-linked oligosaccharides, which comprises roughly half its mass and is a key component of HIV evasion from humoral immunity. To understand how antibodies can overcome the barriers imposed by the glycan shield, we crystallized fully glycosylated Env trimers from clades A, B and G, visualizing the shield at 3.4-3.7 Å resolution. These structures reveal the HIV-1-glycan shield to comprise a network of interlocking oligosaccharides, substantially ordered by glycan crowding, which encase the protein component of Env and enable HIV-1 to avoid most antibody-mediated neutralization. The revealed features delineate a taxonomy of N-linked glycan-glycan interactions. Crowded and dispersed glycans are differently ordered, conserved, processed and recognized by antibody. The structures, along with glycan-array binding and molecular dynamics, reveal a diversity in oligosaccharide affinity and a requirement for accommodating glycans amongst known broadly neutralizing antibodies that target the glycan-shielded trimer.

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.

The solution structure of a complex between a truncated form of HMG-I(Y), consisting of the second and third DNA binding domains (residues 51-90), and a DNA dodecamer containing the PRDII site of the interferon-beta promoter has been solved by multidimensional nuclear magnetic resonance spectroscopy. The stoichiometry of the complex is one molecule of HMG-I(Y) to two molecules of DNA. The structure reveals a new architectural minor groove binding motif which stabilizes B-DNA, thereby facilitating the binding of other transcription factors in the opposing major groove. The interactions involve a central Arg-Gly-Arg motif together with two other modules that participate in extensive hydrophobic and polar contracts. The absence of one of these modules in the third DNA binding domain accounts for its-100 fold reduced affinity relative to the second one.

The HIV-1 envelope (Env) spike (gp120 3 /gp41 3 ) undergoes considerable structural rearrangements to mediate virus entry into cells and to evade the host immune response. Engagement of CD4, the primary human receptor, fixes a particular conformation and primes Env for entry. The CD4-bound state, however, is prone to spontaneous inactivation and susceptible to antibody neutralization. How does unliganded HIV-1 maintain CD4-binding capacity and regulate transitions to the CD4-bound state? To define this mechanistically, we determined crystal structures of unliganded core gp120 from HIV-1 clades B, C, and E. Notably, all of these unliganded HIV-1 structures resembled the CD4-bound state. Conformational fixation with ligand selection and thermodynamic analysis of full-length and core gp120 interactions revealed that the tendency of HIV-1 gp120 to adopt the CD4-bound conformation was restrained by the V1/V2- and V3-variable loops. In parallel, we determined the structure of core gp120 in complex with the small molecule, NBD-556, which specifically recognizes the CD4-bound conformation of gp120. Neutralization by NBD-556 indicated that Env spikes on primary isolates rarely assume the CD4-bound conformation spontaneously, although they could do so when quaternary restraints were loosened. Together, the results suggest that the CD4-bound conformation represents a “ground state” for the gp120 core, with variable loop and quaternary interactions restraining unliganded gp120 from “snapping” into this conformation. A mechanism of control involving deformations in unliganded structure from a functionally critical state (e.g., the CD4-bound state) provides advantages in terms of HIV-1 Env structural diversity and resistance to antibodies and inhibitors, while maintaining elements essential for entry.

HIV-1 uses a diverse N-linked-glycan shield to evade recognition by antibody. Select human antibodies, such as the clonally related PG9 and PG16, recognize glycopeptide epitopes in the HIV-1 V1–V2 region and penetrate this shield, but their ability to accommodate diverse glycans is unclear. Here we report the structure of antibody PG16 bound to a scaffolded V1–V2, showing an epitope comprising both high mannose–type and complex-type N-linked glycans. We combined structure, NMR and mutagenesis analyses to characterize glycan recognition by PG9 and PG16. Three PG16-specific residues, arginine, serine and histidine (RSH), were critical for binding sialic acid on complex-type glycans, and introduction of these residues into PG9 produced a chimeric antibody with enhanced HIV-1 neutralization. Although HIV-1–glycan diversity facilitates evasion, antibody somatic diversity can overcome this and can provide clues to guide the design of modified antibodies with enhanced neutralization.

The solution structure of cyanovirin-N, a potent 11,000 Mr HIV-inactivating protein that binds with high affinity and specificity to the HIV surface envelope protein gp120, has been solved by nuclear magnetic resonance spectroscopy, including extensive use of dipolar couplings which provide a priori long range structural information. Cyanovirin-N is an elongated, largely beta-sheet protein that displays internal two-fold pseudosymmetry. The two sequence repeats (residues 1-50 and 51-101) share 32% sequence identity and superimpose with a backbone atomic root-mean-square difference of 1.3 A. The two repeats, however, do not form separate domains since the overall fold is dependent on numerous contacts between them. Rather, two symmetrically related domains are formed by strand exchange between the two repeats. Analysis of surface hydrophobic clusters suggests the location of potential binding sites for protein-protein interactions.

To date, high-resolution structures have been solved for five different architectural proteins complexed to their DNA target sites. These include TATA-box-binding protein, integration host factor (IHF), high mobility group I(Y)[HMG I(Y)], and the HMG-box-containing proteins SRY and LEF-1. Each of these proteins interacts with DNA exclusively through minor groove contacts and alters DNA conformation. This paper reviews the structural features of these complexes and the roles they play in facilitating assembly of higher-order protein-DNA complexes and discusses elements that contribute to sequence-specific recognition and conformational changes.