Alternative Conformations of HIV-1 V3 Loops Mimic β Hairpins in Chemokines, Suggesting a Mechanism for Coreceptor Selectivity

Michal Sharon, Naama Kessler, Rina Levy, Susan Zolla-Pazner, Matthias Görlach, Jacob Anglister
2003 Structure  
formed an R5 virus into an X4 virus. A double mutation, S313R and D329Q or D333N, caused the same phenotype conversion [4]. (Residues are numbered throughout according to the BH10 isolate sequence [5]). Thus, small changes in the V3 sequence are sufficient to switch Spectroscopy the binding of gp120 to either the CCR5 or CXCR4 chemokine receptor, thus abolishing fusion of the virus with Institute for Molecular Biotechnology (IMB) Beutenbergstrasse 11 its target cell [1, 2]. The structure of the
more » ... gp120 core of both an X4 labora-D-07745 Jena Germany tory-adapted virus and that of an R5 primary isolate in complex with a CD4 fragment and the Fab of a gp120specific antibody have been solved [7, 8]. However, crystals could be obtained only for gp120 lacking the Summary first three variable loops, including V3, and the structures of V4 and V5 have not been defined. Despite the The V3 loop of the HIV-1 envelope glycoprotein gp120 dramatic antigenic differences between the laboratoryis involved in binding to the CCR5 and CXCR4 corecepadapted X4 and the primary R5 isolates, the structures tors. The structure of an HIV-1 MN V3 peptide bound to of their gp120 cores are very similar [7]. These data, the Fv of the broadly neutralizing human monoclonal together with chimeric substitution and sequence analysis, antibody 447-52D was solved by NMR and found to indicate that coreceptor choice and neutralization resisbe a ␤ hairpin. This structure of V3 MN was found to tance involve the major variable loops, V1/V2 and V3. have conformation and sequence similarities to ␤ hair-As an alternative to studying the V3 conformation in pins in CD8 and CCR5 ligands MIP-1␣, MIP-1␤, and the intact gp120 molecule, complexes of V3 peptides RANTES and differed from the ␤ hairpin of a V3 IIIB pepwith antibodies elicited against gp120 or HIV-1 can be tide bound to the strain-specific murine anti-gp120 IIIB used. Wilson and coworkers determined the crystal strucantibody 0.5␤. In contrast to the structure of the bound tures of V3 MN peptides bound to three murine mono-V3 MN peptide, the V3 IIIB peptide resembles a ␤ hairpin clonal antibodies generated against a cyclic 40-residue in SDF-1, a CXCR4 ligand. These data suggest that V3 peptide comprising the entire V3 loop [9-11]. An the 447-52D-bound V3 MN and the 0.5␤-bound V3 IIIB extended conformation and multiple turn conformations structures represent alternative V3 conformations rewere observed, respectively, for the N-and C-terminal sponsible for selective interactions with CCR5 and segments of the V3 loop flanking the central GPGR se-CXCR4, respectively. quence. The GPGR segment itself was found to adopt dual conformations. However, the short epitopes recog-Introduction nized by these anti-peptide antibodies did not permit the determination of the global conformation of the V3 MN The binding of the human immunodeficiency virus loop. A strain-specific HIV-1-neutralizing murine monotype-1 (HIV-1) to its target cells is mediated primarily by clonal antibody, 0.5␤, raised against gp120 IIIB , recogthe envelope glycoprotein (gp120) of the virus. Binding nizes a significantly longer epitope in a V3 IIIB peptide. of gp120 to CD4, a molecule found on the surface of The peptide bound to this antibody formed a ␤ hairpin both T cells and macrophages, triggers conformational not observed in the X-ray studies [12]. The HIV-1 IIIB strain changes in gp120 that expose a binding site to either contains a QR insertion near the tip of the V3 loop pres-CCR5 or CXCR4 chemokine receptors. Only after bindent in less than 10% of HIV-1 isolates. This insertion is ing to the chemokine receptors can the virus penetrate not found in the MN strain, which is a representative of into the target cell. The third hypervariable region of the subtype B viruses common in Europe and North gp120 (V3 loop, residues 303-340) is directly involved America [13]. in the binding to the chemokine receptors [1, 2]. The V3 The human monoclonal antibody 447-52D is one of sequence determines whether the virus binds to CCR5 the most broadly neutralizing and most potent anti-V3 (designated R5 virus) and, therefore, infects macroantibodies that have been studied to date. It binds to phages or whether it binds to CXCR4 (designated the intact virions from clades A, B, D, F, G, and H [14] and X4 virus) and infects T cells ([3] and the references neutralizes primary isolates from several clades, includtherein). A single mutation in the V3 loop, D329R, trans- Key words: gp120 V3; NMR structure; HIV-1; coreceptor; neutralizing antibodies; chemokines *Correspondence: jacob.anglister@weizmann.ac.il filtration with vivaspin (Vivascience) with a 10 kDa cut-off. All samples contained 10 mM sodium acetate buffer and 0.05% NaN 3 (pH 5). We thank Professor Fred Naider for illuminating discussions and for careful editing of the manuscript, Mr. Yehezkiel Haik for peptide purification, Dr. Anat Zvi for cloning and sequencing 447-52D, Mrs. NMR Spectroscopy Min-Ji for expressing 447 Fv in E. Coli and developing the purification NMR spectra were acquired at 35ЊC on Bruker DMX 500 and DRX protocol, Mr. Ran Lati for Fv production, Dr. Vitali Tugarinov, Mr. 800 spectrometers with unlabeled 308-332 gp120 MN or peptide uniformly Jordan Chill, and Dr. Tali Scherf for help in the NMR experiments, labeled with 15 N or with 13 C and 15 N in complex with unlabeled 447 and Dr. Mirek Gorny for the immunological characterization of 447-Fv. ROESY and HOHAHA spectra with long mixing times (90 ms) 52D and helpful discussions. This study was supported by the Minwere used for epitope mapping. The mixing time was adjusted to erva Foundation, Germany (J.A.), and by NIH grants GM 53329 (J.A.), discriminate between crosspeaks of peptide protons that are immo-AI 36085 (S.Z.P.), and HL 59725 (S.Z.P.). J.A. is the Dr. Joseph and bilized in the complex because of interactions with the antibody Ruth Owades Professor of Chemistry. and that have a short T 1 relaxation time and those of protons that do not interact with the Fv and, therefore, retain considerable mobility and have a long T 1 . Two-dimensional spectra of the unlabeled Received: August 16, 2002 complex were measured at 30ЊC, 20ЊC, and 10ЊC and at pH 7, 5, Revised: December 26, 2002 and 4.25. The combination of the HOHAHA and ROESY spectra
doi:10.1016/s0969-2126(03)00011-x pmid:12575942 fatcat:45euhk6tqvggxovepzph2pvxge