A Monomeric 3 10 -Helix Is Formed in Water by a 13-Residue Peptide Representing the Neutralizing Determinant of HIV-1 on gp41 †,‡ Zohar Biron, § Sanjay Khare, | Abraham O. Samson, § Yehezkiel Hayek, § Fred Naider, | and Jacob Anglister* ,§ Department of Structural Biology, The Weizmann Institute of Science, RehoVot 76100, Israel, and Department of Chemistry, College of Staten Island and Graduate Center of the City UniVersity of New York, 2800 Victory BouleVard, Staten Island, New York 10314 ReceiVed June 7, 2002; ReVised Manuscript ReceiVed August 4, 2002 ABSTRACT: 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 a3 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. The protein gp160 of the human immunodeficiency virus type 1 (HIV-1) 1 is the precursor of the surface glycoproteins gp120 and gp41. The latter is a transmembrane protein that mediates the fusion of the virus with host cells. The three- dimensional structure of the HIV-1 gp41 core, made of two segments containing heptad leucine/isoleucine repeats, N36 (gp41 546-581 ) and C34 (gp41 628-661 ), was solved by X-ray crystallography (1-3), and the structure of the SIV gp41 core was solved by multidimensional NMR (4). The gp41 core is a six-helix bundle that exhibits structural similarity to other fusion proteins such as the hemagglutinin fusion protein of the influenza virus (5) and is assumed to be the fusogenic or postfusogenic form of this protein (6). Antibod- ies against this structure do not seem to neutralize the virus possibly because once the six-helical coiled-coil structure is formed in the fusion process, gp41 is not accessible to HIV-1 neutralizing antibodies (7). The C-terminal region (residues 660-683) of the gp41 ectodomain preceding the membrane-spanning domain is rich in tryptophan residues that are conserved in lentiviruses (8). Deletion of residues 660-670 (LLELDKWASLW) results in partial dissociation of the oligomeric structure of gp41 (9), abolishes fusion, and decreases gp160 precursor cleav- age. Mutation of the first three tryptophan residues (W666, W670, and W672) suffices to eliminate viral fusion (8). The C-terminal tryptophan-rich region overlaps the peptide T-20 (gp41 638-673 ), which is a strong inhibitor of HIV-1 fusion with an EC 50 of 0.5 ng/mL (10). This peptide is presently in a phase III clinical trial (11). In contrast to all currently available anti-HIV drugs that inhibit the virus in already infected cells, T-20 is expected to represent a new class of anti HIV-1 drugs that inhibit the fusion of the virus with its target cells, thus preventing infection of new cells. A peptide corresponding to the C-terminal residues 661- 673 of T-20 inhibits HIV-1 infection with an EC 50 of 20 µg mL -1 . † This study was supported by National Institutes of Health Grants R01 GM53329 (to J.A.) and GM22086 (to F.N.). J.A. is the Joseph and Ruth Owades Professor in Chemistry. F.N. is the Leonard and Esther Kurtz Term Professor at the College of Staten Island. ‡ The NMR data and the atomic coordinates of the 25 lowest energy structures (1LCX) and the average structure (1LB0) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http:// www.rcsb.org/). * Corresponding author. Phone: 011-972-8-934-3394. Fax: 011- 972-8-934-4136. E-mail: Jacob.Anglister@weizmann.ac.il. § The Weizmann Institute of Science. | College of Staten Island and the Graduate Center of CUNY. 1 Abbreviations: Ac, acetyl; Ac-gp41659-671-NH2, gp41659-671 acety- lated at the N-terminus and amidated at the C-terminus; CD, circular dichroism; DQF-COSY, double-quantum-filtered correlation spec- troscopy; EC 50, concentration that causes 50% inhibition of the virus; gp, glycoprotein; gp41659-671, a gp41 peptide comprising residues 659-671; HIV-1, human immunodeficiency virus type 1; NOESY, nuclear Overhauser effect spectroscopy; SIV, simian immunodeficiency virus. 12687 Biochemistry 2002, 41, 12687-12696 10.1021/bi026261y CCC: $22.00 © 2002 American Chemical Society Published on Web 09/27/2002