The V3 loop of the HIV-1 envelope glycoprotein gp120 is involved in binding to the CCR5 and CXCR4 coreceptors. The structure of an HIV-1(MN) V3 peptide bound to the Fv of the broadly neutralizing human monoclonal antibody 447-52D was solved by NMR and found to be a beta hairpin. This structure of V3(MN) was found to have conformation and sequence similarities to beta hairpins in CD8 and CCR5 ligands MIP-1alpha, MIP-1beta, and RANTES and differed from the beta hairpin of a V3(IIIB) peptide bound to the strain-specific murine anti-gp120(IIIB) antibody 0.5beta. In contrast to the structure of the bound V3(MN) peptide, the V3(IIIB) peptide resembles a beta hairpin in SDF-1, a CXCR4 ligand. These data suggest that the 447-52D-bound V3(MN) and the 0.5beta-bound V3(IIIB) structures represent alternative V3 conformations responsible for selective interactions with CCR5 and CXCR4, respectively.

Type I interferons (IFNs) are a family of homologous helical cytokines that exhibit pleiotropic effects on a wide variety of cell types, including antiviral activity and antibacterial, antiprozoal, immunomodulatory, and cell growth regulatory functions. Consequently, IFNs are the human proteins most widely used in the treatment of several kinds of cancer, hepatitis C, and multiple sclerosis. All type I IFNs bind to a cell surface receptor consisting of two subunits, IFNAR1 and IFNAR2, associating upon binding of interferon. The structure of the extracellular domain of IFNAR2 (R2-EC) was solved recently. Here we study the complex and the binding interface of IFNa2 with R2-EC using multidimensional NMR techniques. NMR shows that IFNa2 does not undergo significant structural changes upon binding to its receptor, suggesting a lock-and-key mechanism for binding. Cross saturation experiments were used to determine the receptor binding site upon IFNa2. The NMR data and previously published mutagenesis data were used to derive a docking model of the complex with an RMSD of 1 Å , and its well-defined orientation between IFNa2 and R2-EC and the structural quality greatly improve upon previously suggested models. The relative ligand-receptor orientation is believed to be important for interferon signaling and possibly one of the parameters that distinguish the different IFN I subtypes. This structural information provides important insight into interferon signaling processes and may allow improvement in the development of therapeutically used IFNs and IFN-like molecules.Keywords: interferons; protein-protein docking; protein-protein interactions; multidimensional NMR; cross saturation Type I Interferons (IFNs) are a family of homologous helical cytokines initiating strong antiviral and antiproliferative activity. Since IFNs are at the forefront of defense against viral infection and promote a variety of biological effects, they are essential for the survival of higher vertebrates (Stark et al. 1998;Biron 2001). Not surprisingly, IFNs are the human proteins most widely used as therapeutics for the treatment of several kinds of cancer and viral diseases (e.g., Perry and Jarvis 2001; Kirkwood 2002). Human type I interferons include 13 IFNa isotypes (and allelic forms) and single forms of IFNb, IFNe, IFNk, and IFNv (Pestka et al. 2004). Sequence homology between all IFNa isotypes is high, with ;80% identity, and the identity of the IFNa isotypes to v, b, e, and k subtypes is 50%, 31%, 28%, and 27%, respectively. IFNg is the only known type II interferon (Pestka et al. 1987), and it shares only 10% identity with IFNa. The threedimensional structures of several type I IFNs have been solved, and a high resolution NMR structure of human IFNa2a (Klaus et al. 1997) and the X-ray structures of IFNa2b (Karpusas et al. 1997) and IFNb (Radhakrishnan et al. 1996) are available.Reprint requests to: Jacob Anglister, Department of Structural Biology, Weizmann Institute of Science, 76100 Rehovot, Israel; e-mail: jacob.anglister@wei...

Interaction of CCR5 with the HIV-1 gp120-CD4 complex involves its amino-terminal domain (Nt-CCR5) and requires sulfation of 2-4 tyrosine residues in Nt-CCR5. The conformation of a 27-residue Nt-CCR5 peptide, sulfated at Y10 and Y14, was studied in both its free form and in a ternary complex with deglycosylated-gp120 and a CD4-mimic peptide. NMR experiments revealed a helical conformation at the center of Nt-CCR5(1-27) which is induced upon gp120 binding, as well as a helical propensity for the free peptide. A well-defined structure for the bound peptide was determined for residues 7-23, increasing by two-fold the length of Nt-CCR5's known structure. Two-dimensional saturation transfer experiments and measurement of relaxation-times highlighted Nt-CCR5 residues Y3, V5, P8-T16, E18, I23 and possibly D2 as the main binding determinant. A calculated docking model for Nt-CCR5(1-27) suggests that residues 2-22 of Nt-CCR5 interact with the bases of V3 and C4 while the C-terminal segment of Nt-CCR5(1-27) points towards the target cell membrane reflecting an Nt-CCR5 orientation that differs by 180° from a previous model. A gp120 site that could accommodate CCR5Y3 in a sulfated form has been identified. The present model attributes a structural basis for binding interactions to all gp120 residues previously implicated in Nt-CCR5 binding. Moreover, the strong interaction of sulfated CCR5Tyr14 with gp120Arg440 revealed by the model and the previously found correlation between E322 and R440 mutations shed light on the role of these residues in HIV-1 phenotype conversion furthering our understanding of CCR5 recognition by HIV-1.

Background: The protein 0.5β is a potent strain-specific human immunodeficiency virus type 1 (HIV-1) neutralizing antibody raised against the entire envelope glycoprotein (gp120) of the HIV-1 IIIB strain. The epitope recognized by 0.5β is located within the third hypervariable region (V3) of gp120. Recently, several HIV-1 V3 residues involved in co-receptor utilization and selection were identified.Results: Virtually complete sidechain assignment of the variable fragment (Fv) of 0.5β in complex with the V3 IIIB peptide P1053 (RKSIRIQRGPGRAFVTIG, in single-letter amino acid code) was accomplished and the combining site structure of 0.5β Fv complexed with P1053 was solved using multidimensional nuclear magnetic resonance (NMR). Five of the six complementarity determining regions (CDRs) of the antibody adopt standard canonical conformations, whereas CDR3 of the heavy chain assumes an unexpected fold. The epitope recognized by 0.5β encompasses 14 of the 18 P1053 residues. The bound peptide assumes a β-hairpin conformation with a QRGPGR loop located at the very center of the binding pocket. The Fv and peptide surface areas buried upon binding are 601 Å and 743 Å 2 , respectively, in the 0.5β Fv-P1053 mean structure. The surface of P1053 interacting with the antibody is more extensive and the V3 peptide orientation in the binding site is significantly different compared with those derived from the crystal structures of a V3 peptide of the HIV-1 MN strain (V3 MN ) complexed to three different anti-peptide antibodies. Conclusions:The surface of P1053 that is in contact with the anti-protein antibody 0.5β is likely to correspond to a solvent-exposed region in the native gp120 molecule. Some residues of this region of gp120 are involved in co-receptor binding, and in discrimination between different chemokine receptors utilized by the protein. Several highly variable residues in the V3 loop limit the specificity of the 0.5β antibody, helping the virus to escape from the immune system. The highly conserved GPG sequence might have a role in maintaining the β-hairpin conformation of the V3 loop despite insertions, deletions and mutations in the flanking regions.

The human interferon receptor (IFNAR) mediates the antiviral and antiproliferative activities of type I interferons (IFNs). This receptor is comprised of subunits IFNAR1 and IFNAR2, the latter exhibiting nanomolar affinity for IFNs. Here the extracellular domain of IFNAR2 (IFNAR2-EC), a soluble 25 kDa IFN-binding polypeptide, and its complex with IFN-alpha 2 were studied using multidimensional NMR. IFNAR2-EC is comprised of two fibronectin-III (FN-III) domains connected by a helical hinge region. The deduced global fold was utilized to improve the alignment of IFNAR2-EC against structurally related receptors and to model its structure. A striking feature of IFNAR2-EC is the limited and localized deviations in chemical shifts exhibited upon ligand binding, observed for only 15% of its backbone (1)H and (15)N nuclei. Analysis of these deviations maps the IFN-alpha 2 binding site upon IFNAR2-EC to a contiguous surface on the N-terminal domain, including the S3-S4 loop (residues 44-53), the S5-S6 loop and S6 beta-strand (residues 74-82), and the S7 beta-strand and the hinge region (residues 95-105). The C-terminal domain contributes only marginally to ligand binding, and no change in the hypothesized interdomain interface is observed. The proposed binding domain encompasses all residues implicated by mutagenesis studies in IFN binding, and suggests adjacent residues cooperate in forming the binding surface. D(2)O-exchange experiments indicate that binding of IFN-alpha2 induces tightening of the N-terminal domain of IFNAR2-EC. This increase in receptor rigidity may play an important role in initiating the intracellular stage of the IFN signaling cascade.