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...

HIV-1 coreceptor usage plays a critical role in virus tropism and pathogenesis. A switch from CCR5-to CXCR4-using viruses occurs during the course of HIV-1 infection and correlates with subsequent disease progression. A single mutation at position 322 within the V3 loop of the HIV-1 envelope glycoprotein gp120, from a negatively to a positively charged residue, was found to be sufficient to switch an R5 virus to an X4 virus. In this study, the NMR structure of the V3 region of an R5 strain, HIV-1JR-FL, in complex with an HIV-1-neutralizing antibody was determined. Positively charged and negatively charged residues at positions 304 and 322, respectively, oppose each other in the ␤-hairpin structure, enabling a favorable electrostatic interaction that stabilizes the postulated R5 conformation. Comparison of the R5 conformation with the postulated X4 conformation of the V3 region (positively charged residue at position 322) reveals that electrostatic repulsion between residues 304 and 322 in X4 strains triggers the observed one register shift in the N-terminal strand of the V3 region. We posit that electrostatic interactions at the base of the V3 ␤-hairpin can modulate the conformation and thereby influence the phenotype switch. In addition, we suggest that interstrand cation-interactions between positively charged and aromatic residues induce the switch to the X4 conformation as a result of the S306R mutation. The existence of three pairs of identical (or very similar) amino acids in the V3 C-terminal strand facilitates the switch between the R5 and X4 conformations.447-52D ͉ gp120 ͉ NMR T he third variable (V3) region of the HIV type 1 (HIV-1) envelope glycoprotein gp120 binds to chemokine receptors CCR5 and CXCR4, which are involved in HIV-1 infection. The amino acid sequence of V3 determines whether the virus binds to CCR5 (''R5 viruses'') and infects predominantly macrophages or to CXCR4 (''X4 viruses'') and infects mostly T cells (1). The presence of a basic residue at V3 positions 306 or 322 is associated with X4 and dual-tropic, X4R5 viruses, whereas the presence of a negatively charged residue and a neutral residue at positions 322 and 306, respectively, is correlated with R5 viruses (the ''11͞25 rule'') (2). Numerous investigations have confirmed that mutation of a negatively charged residue at position 322 to a positively charged one converts an R5 strain into an X4 strain (2-4).To gain insight into the structure of the V3 region and the mechanism for phenotype conversion, we used solution NMR spectroscopy to study the conformation of synthetic V3 peptides in complex with V3-specific anti-gp120 antibodies. An assumption underlying this approach is that the native conformation of V3 is induced in linear V3 peptides upon binding to V3-directed antibodies that were elicited against the entire gp120 protein. We studied two V3-specific antibodies. The first, murine mAb 0.5␤, is a potent strain-specific HIV-1-neutralizing antibody that was raised against a full-length gp120 protein of the X4 virus HIV-1 IIIB (...

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