NMR mapping of the IFNAR1-EC binding site on IFNalpha2 reveals allosteric changes in the IFNAR2-EC binding site.

All type I interferons (IFNs) bind to a common cell-surface receptor consisting of two subunits. IFNs initiate intracellular signal transduction cascades by simultaneous interaction with the extracellular domains of its receptor subunits, IFNAR1 and IFNAR2. In this study, we mapped the surface of IFNalpha2 interacting with the extracellular domain of IFNAR1 (IFNAR1-EC) by following changes in or the disappearance of the (1)H-(15)N TROSY-HSQC cross peaks of IFNalpha2 caused by the binding of the extracellular domain of IFNAR1 (IFNAR1-EC) to the binary complex of IFNalpha2 with IFNAR2-EC. The NMR study of the 89 kDa complex was conducted at pH 8 and 308 K using an 800 MHz spectrometer. IFNAR1 binding affected a total of 47 of 165 IFNalpha2 residues contained in two large patches on the face of the protein opposing the binding site for IFNAR2 and in a third patch located on the face containing the IFNAR2 binding site. The first two patches form the IFNAR1 binding site, and one of these matches the IFNAR1 binding site previously identified by site-directed mutagenesis. The third patch partially matches the IFNalpha2 binding site for IFNAR2-EC, indicating allosteric communication between the binding sites for the two receptor subunits.

Intermolecular interactions in a 44 kDa interferon-receptor complex detected by asymmetric reverse-protonation and two-dimensional NOESY.

Type I interferons (IFNs) make up a family of homologous helical cytokines initiating strong antiviral and antiproliferative 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 IFNalpha2 using asymmetric reverse-protonation of the different complex components and two-dimensional homonuclear NOESY. This new approach revealed with an 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 mass (44 kDa). Sequential and side chain assignment of IFNAR2-EC and IFNalpha2 in their binary complex helped assign the intermolecular NOEs to the corresponding protons. A docking model of the IFNAR2-EC-IFNalpha2 complex was calculated on the basis of the intermolecular interactions found in this 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 IFNalpha2. Our docking model doubles the buried surface area of the previous model and significantly increases the number of intermolecular hydrogen bonds, salt bridges, and van der Waals interactions. Furthermore, our model reveals the participation of several new regions in the binding site such as the N-terminus and A helix of IFNalpha2 and the C domain of IFNAR2-EC. As a result of these additions, the orientation of IFNAR2-EC relative to IFNalpha2 has changed by 30 degrees 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 IFNalpha2 upon binding of IFNAR1-EC to the binary IFNalpha2-IFNAR2-EC complex.

The membrane proximal external region of the HIV-1 envelope glycoprotein gp41 contributes to the stabilization of the six-helix bundle formed with a matching N' peptide.

The HIV-1 envelope glycoprotein gp41 undergoes a sequence of extensive conformational changes while participating in the fusion of the virus with the host cell. Since the discovery of its postfusion conformation, the structure and function of the protease-resistant six-helix bundle (6-HB) have been the subject of extensive investigation. In this work, we describe additional determinants (S528-Q540 and W666-N677) in the fusion peptide proximal region (FP-PR) and the membrane proximal external region (MPER) that stabilize the six-helix bundle and are involved in the interaction of T-20 (FUZEON, an anti-HIV-1 fusion inhibitor drug) with the gp41 FP-PR. Circular dichroism and sedimentation equilibrium measurements indicate that the 1:1 mixture of N' and C' peptides comprising residues A541-T569 and I635-K665 from the gp41 first and second helical repeats, HR1 and HR2, respectively, fail to form a stable six-helix bundle. Triglutamic acid and triarginine tags were added to these N' and C' peptides, respectively, at the termini distant from the FP-PR and the MPER to alter their pI and increase their solubility at pH 3.5. The tagged HR1 and HR2 peptides were elongated by addition of residues S528-Q540 from the FP-PR and residues W666-N677 from the MPER, respectively. A 1:1 complex of the elongated peptides formed a stable six-helix bundle which melted at 60 degrees C. These results underscore the importance of a detailed high-resolution characterization of MPER interactions, the results of which may improve our understanding of the structure-function relationship of gp41 and its role in HIV-1 fusion.

The C4 region as a target for HIV entry inhibitors--NMR mapping of the interacting segments of T20 and gp120.

The peptide T20, which corresponds to a sequence in the C-terminal segment of the HIV-1 transmembrane glycoprotein gp41, is a strong entry inhibitor of HIV-1. It has been assumed that T20 inhibits HIV-1 infection by binding to the trimer formed by the N-terminal helical region (HR1) of gp41, preventing the formation of a six helix bundle by the N- and C-terminal helical regions of gp41. In addition to binding to gp41, T20 was found to bind to gp120 of X4 viruses and this binding was suggested to be responsible for an alternative mechanism of HIV-1 inhibition by this peptide. In the present study, T20 also was found to bind R5 gp120. Using NMR spectroscopy, the segments of T20 that interact with both gp120 and a gp120/CD4M33 complex were mapped. A peptide corresponding to the fourth constant region of gp120, sC4, was found to partially recapitulate gp120 binding to T20 and the segment of this peptide interacting with T20 was mapped. The present study concludes that an amphiphilic helix on the T20 C-terminus binds through mostly hydrophobic interactions to a nonpolar gp120 surface formed primarily by the C4 region. The ten- to thousand-fold difference between the EC50 of T20 against viral fusion and the affinity of T20 to gp120 implies that binding to gp120 is not a major factor in T20 inhibition of HIV-1 fusion. Nevertheless, this hydrophobic gp120 surface could be a target for anti-HIV therapeutics.

Comparative evaluation of trimeric envelope glycoproteins derived from subtype C and B HIV-1 R5 isolates.

We previously reported that an envelope (Env) glycoprotein immunogen (o-gp140DeltaV2SF162) containing a partial deletion in the second variable loop (V2) derived from the R5-tropic HIV-1 isolate SF162 partially protected vaccinated rhesus macaques against pathogenic SHIV(SF162P4) virus. Extending our studies to subtype C isolate TV1, we have purified o-gp140DeltaV2TV1 (subtype C DeltaV2 trimer) to homogeneity, performed glycosylation analysis, and determined its ability to bind CD4, as well as a panel of well-characterized neutralizing monoclonal antibodies (mAb). In general, critical epitopes are preserved on the subtype C DeltaV2 trimer; however, we did not observe significant binding for the b12 mAb. The molecular mass of subtype C DeltaV2 trimer was found to be 450 kDa, and the hydrodynamic radius was found to be 10.87 nm. Our data suggest that subtype C DeltaV2 trimer binds to CD4 with an affinity comparable to o-gp140DeltaV2SF162 (subtype B DeltaV2 trimer). Using isothermal titration calorimetric (ITC) analysis, we demonstrated that all three CD4 binding sites (CD4-BS) in both subtype C and B trimers are exposed and accessible. However, compared to subtype B trimer, the three CD4-BS in subtype C trimer have different affinities for CD4, suggesting a cooperativity of CD4 binding in subtype C trimer but not in subtype B trimer. Negative staining electron microscopy of the subtype C DeltaV2 trimer has demonstrated that it is in fact a trimer. These results highlight the importance of studying subtype C Env, and also of developing appropriate subtype C-specific reagents that may be used for better immunological characterization of subtype C Env for developing an AIDS vaccine.

The 2F5 epitope is helical in the HIV-1 entry inhibitor T-20.

The HIV-1 envelope glycoprotein gp41 is responsible for viral fusion with the host cell. The fusion process, as well as the full structure of gp41, is not completely understood. One of the strongest inhibitors of HIV-1 fusion is a 36-residue peptide named T-20, gp41(638-673) (Fuzeon, also called Enfuvirtide or DP-178; residues are numbered according to the HXB2 gp160 variant) now used as an anti HIV-1 drug. This peptide also contains the immunogenic sequences that represent the full or partial recognition epitope for the broadly neutralizing human monoclonal antibodies 2F5 and 4E10, respectively. Due to its hydrophobicity, T-20 tends to aggregate at high concentrations in water, and therefore the structure of this molecule in aqueous solution has not been previously determined. We expressed a uniformly 13C/15N-labeled 42-residue peptide NN-T-20-NITN (gp41(636-677)) and used heteronuclear 2D and 3D NMR methods to determine its structure. Due to the additional gp41-native hydrophilic residues, NN-T-20-NITN dissolved in water, enabling for the first time determination of its secondary structure at near physiological conditions. Our results show that the NN-T-20-NITN peptide is composed of a mostly unstructured N-terminal region and a helical region beginning at the center of T-20 and extending toward the C-terminus. The helical region is found under various conditions and has been observed also in a 13-residue peptide gp41(659-671). We suggest that this helical conformation is maintained in most of the different tertiary structures of the gp41 envelope protein that form during the process of viral fusion. Accordingly, an important element of the immunogenicity of gp41 and the inhibitory properties of Fuzeon may be the propensity of specific sequences in these polypeptides to assume helical structures.

A monomeric 3(10)-helix is formed in water by a 13-residue peptide representing the neutralizing determinant of HIV-1 on gp41.

The peptide gp41(659-671) (ELLELDKWASLWN) comprises the entire epitope for one of the three known antibodies capable of neutralizing a broad spectrum of primary HIV-1 isolates and is the only such epitope that is sequential. Here we present the NMR structure of gp41(659-671) in water. This peptide forms a monomeric 3(10)-helix stabilized by i,i+3 side chain-side chain interactions favored by its primary sequence. In this conformation the peptide presents an exposed surface, which is mostly hydrophobic and consists of conserved HIV-1 residues. The presence of the 3(10)-helix is confirmed by its characteristic CD pattern. Studies of the 3(10)-helix have been hampered by the absence of a model peptide adopting this conformation. gp41(659-671) can serve as such a model to investigate the spectral characteristics of the 3(10)-helix, the factors that influence its stability, and the propensity of different amino acids to form a 3(10)-helix. The observation that the 3(10)-helical conformation is highly populated in the peptide gp41(659-671) indicates that the corresponding segment in the cognate protein is an autonomous folding unit. As such, it is very likely that the helical conformation is maintained in gp41 throughout the different tertiary structures of the envelope protein that form during the process of viral fusion. However, the exposure of the gp41(659-671) segment may vary, leading to changes in the reactivity of anti-gp41 antibodies in the different stages of viral fusion. Since gp41(659-671) is an autonomous folding unit, peptide immunogens consisting of the complete gp41(659-671) sequence are likely to induce antibodies highly cross-reactive with HIV-1.