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.

Type I Interferons (IFNs) are a family of homologous helical cytokines initiating strong anti-viral and anti-proliferative activity. All type I IFNs bind to a common cell surface receptor consisting of two subunits, IFNAR1 and IFNAR2, associating upon binding of interferon. We studied intermolecular interactions between IFNAR2-EC and IFNα2 using asymmetric reverse-protonation of the different complex components and 2D homonuclear NOESY. This new approach revealed with excellent signal-to-noise ratio 24 new intermolecular NOEs between the two molecules despite the low concentration of the complex (0.25 mM) and its high molecular weight (44 kDA). Sequential and side-chain assignment of IFNAR2-EC and IFNα2 in their binary complex helped assign the inter-molecular NOEs to the corresponding protons. A docking model of the IFNAR2-EC/IFNα2 complex was calculated based on the inter-molecular interactions found in the present study as well as four double mutant cycle constraints, previously observed NOEs between a single pair of residues and the NMR mapping of the binding sites on IFNAR2-EC and IFNα2. Our docking model doubles the buried surface area of the previous model and significantly increases the number of inter-molecular hydrogen bonds, salt bridges and Van der-Waals interactions. Furthermore, the current model reveals participation of several new regions in the binding site such as the N-terminus and A-helix of IFNα2 and the C-domain of IFNAR2-EC. As a result of these additions, the orientation of IFNAR2-EC relative to IFNα2 has changed by 30° in comparison with a previously calculated model that was based on NMR mapping of the binding sites and double mutant cycle constraints. In addition, the new model strongly supports the recently proposed allosteric changes in IFNα2 upon IFNAR1-EC binding to the binary IFNα2/ IFNAR2-EC complex.Type I IFNs are a major component of the innate immune system protecting against viral infection. They provide an early line of defense, hours to days ahead of the adaptive immune † This study was supported by the Israel Science Foundation, NIH Grant GM53329 and the Kimmelman Center. J.A. is the Dr. Joseph and Ruth Owades Professor of Chemistry. * To whom correspondence should be addressed. Jacob. . NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 June 29. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript response, and are essential for the survival of higher vertebrates (1-3). In addition to a strong anti-viral activity, type I IFNs are also attributed anti-proliferative and immunomodulatory properties (4,5).All human type I IFNs elicit their activity through the same cell surface receptor consisting of two trans-membranal protein subunits, IFNAR1 and IFNAR2 (6,7). IFNAR2 is the major ligand binding component and has nM affinity to IFNs without the presence of IFNAR1. The affinity of the IFNAR1 subunit to IFNs is much lower and the dissociation constant is in the μM range (8). The IFN signaling process begins ...

Chemokines constitute a large family of small proteins that regulate leukocyte trafficking to the site of inflammation by binding to specific cell-surface receptors belonging to the GPCR superfamily. The interactions between N-terminal (Nt-) peptides of these GPCRs and chemokines have been studied extensively using NMR spectroscopy. However, due to lower affinities of peptides representing the three extracellular loops (ECLs) of chemokine receptors to their respective chemokine ligands, information concerning these interactions is scarce. To overcome the low affinity of ECL peptides to chemokines, we linked two or three CCR5 extracellular domains by either biosynthesis in Escherichia coli or by chemical synthesis. Using such chimeras, CCR5 binding to RANTES was followed using 1H-15N-HSQC spectra to monitor titration of the chemokine with peptides corresponding to the extracellular surface of the receptor. Nt-CCR5 and ECL2 were found to be the major contributors to CCR5 binding to RANTES, creating a nearly closed ring around this protein by interacting with opposing faces of the chemokine. A RANTES positively charged surface involved in Nt-CCR5 binding resembles the positively charged surface in HIV-1 gp120 formed by the C4 and the base of the V3. The opposing surface on RANTES, composed primarily of β2-β3 hairpin residues, binds ECL2 and was found to be analogous to a surface in the crown of the gp120 V3. The chemical and biosynthetic approaches for linking GPCR surface regions discussed herein should be widely applicable to investigation of interactions of extracellular segments of chemokine receptors with their respective ligands.

The envelope spike of HIV-1, which consists of three external gp120 and three transmembrane gp41 glycoproteins, recognizes its target cells by successively binding to its primary CD4 receptor and a coreceptor molecule. Until recently, atomic-resolution structures were available primarily for monomeric HIV-1 gp120, in which the V1, V2 and V3 variable loops were omitted (gp120 core ), in complex with soluble CD4 (sCD4). Differences between the structure of HIV gp120 core in complex with sCD4 and the structure of unliganded simian immunodeficiency virus gp120 core led to the hypothesis that gp120 undergoes a major conformational change upon sCD4 binding. To investigate the conformational flexibility of gp120, we generated two forms of mutated gp120 amenable for NMR studies: one with V1, V2 and V3 omitted ( mut gp120 core ) and the other containing the V3 region N]Ile-labeled unliganded mut gp120 core showed many fewer crosspeaks than the expected number, and also many fewer crosspeaks in comparison with the labeled mut gp120 core bound to the CD4-mimic peptide, CD4M33. This finding suggests that in the unliganded form, mut gp120 core shows considerable flexibility and motions on the millisecond time scale. In contrast, most of the expected crosspeaks were observed for the unliganded mut gp120 core (+V3), and only a few changes in chemical shift were observed upon CD4M33 binding. These results indicate that mut gp120 core (+V3) does not show any significant conformational flexibility in its unliganded form and does not undergo any significant conformational change upon CD4M33 binding, underlining the importance of V3 in stabilizing the gp120 core conformation.

Voltage-gated sodium channels (Navs) are large transmembrane proteins that initiate action potential in electrically excitable cells. This central role in the nervous system has made them a primary target for a large number of neurotoxins. Scorpion alpha-neurotoxins bind to Navs with high affinity and slow their inactivation, causing a prolonged action potential. Despite the similarity in their mode of action and three-dimensional structure, alpha-toxins exhibit great variations in selectivity toward insect and mammalian Navs, suggesting differences in the binding surfaces of the toxins and the channels. The scorpion alpha-toxin binding site, termed neurotoxin receptor site 3, has been shown to involve the extracellular S3-S4 loop in domain 4 of the alpha-subunit of voltage-gated sodium channels (D4/S3-S4). In this study, the binding site for peptides corresponding to the D4/S3-S4 loop of the para insect Nav was mapped on the highly insecticidal alpha-neurotoxin, LqhalphaIT, from the scorpion Leiurus quinquestriatus hebraeus, by following changes in the toxin amide 1H and 15N chemical shifts upon binding. This analysis suggests that the five-residue turn (residues LqK8-LqC12) of LqhalphaIT and those residues in its vicinity interact with the D4/S3-S4 loop of Nav. Residues LqR18, LqW38, and LqA39 could also form a patch contributing to the interaction with D4/S3-S4. Moreover, a new bioactive residue, LqV13, was identified as being important for Nav binding and specifically for the interaction with the D4/S3-S4 loop. The contribution of LqV13 to NaV binding was further verified by mutagenesis. Future studies involving other extracellular regions of Navs are required for further characterization of the structure of the LqhalphaIT-Navs binding site.

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Heparin interacts with the nonapeptide oxytocin, the binding region preferentially involves the 6-O- and N-sulfates of glucosamine.

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