Membrane-transferring Sequences of the HIV-1 Gp41 Ectodomain Assemble into an Immunogenic Complex Maier Lorizate 1 , María J. Gómara 1 , Beatriz G. de la Torre 2 David Andreu 2 and José L. Nieva 1 ⁎ 1 Biophysics Unit (CSIC-UPV/ EHU) and Biochemistry Department, University of the Basque Country, P.O. Box 644, 48080 Bilbao, Spain 2 Proteomics Unit, Pompeu Fabra University, Dr. Aiguader 80, 08003 Barcelona, Spain The membrane-proximal stem region of gp41 has been postulated to host the two conserved membrane-transferring domains that promote HIV-1 fusion: the amino-terminal fusion peptide (FP) and the highly aromatic pre- transmembrane sequence. Our results confirm that the hydrophobic FP and membrane-proximal sequences come into contact and form structurally de- fined complexes. These complexes are immunogenic and evoke responses in rabbits that compete with the recognition of native functional gp41 by the 2F5 monoclonal antibody. We conclude that co-assembly of the FP and the pre-transmembrane sequences might exert a constraint that helps maintain a gp41 stem region pre-fusion structure. © 2006 Elsevier Ltd. All rights reserved. *Corresponding author Keywords: HIV-1; gp41; HIV-1 fusion peptide; gp41 pre-transmembrane; 2F5 antibody Introduction Envelope protein-induced fusion of the viral and the plasma membranes enables the human immu- nodeficiency virus type-1 (HIV-1) to enter into the CD4 + target cell. 1–4 The fusogenic activity of the gp120/41 envelope glycoprotein is triggered by the sequential binding of the surface gp120 subunit to CD4 and to human chemokine receptors. 5,6 Subse- quently, and in conjunction with the formation of a low-energy six-helix bundle structure, the trans- membrane gp41 subunit promotes the merging of the lipid bilayers. 7–11 Mutational analyses indicate that the activity of gp41 is dependent on two hydrophobic ectodomain sequences: the free amino-terminal fusion peptide (FP), 12,13 which is not exposed to the solvent in the metastable structure primed for fusion, 2,3 and the highly aromatic pre-transmembrane region (preTM). 14 Given the hydrophobic character of FP and preTM, it has been postulated that during the process of fusion these sequences insert into the target cell and the virion membranes, respectively. 15 When com- pared with other sequences that insert into mem- branes, such as signal peptides or transmembrane domains, the FP and preTM sequences display an unusually high degree of conservation. 14–17 Al- though atomic structures of gp41 states prior to the six-helix bundle form are not elucidated, predictive work 18 and epitope-mapping 19 suggest that both membrane-transferring regions might be located at the same end of the ectodomain, proximal to the virion envelope, in the native state. Here, we have used the 2F5 monoclonal antibody (Mab2F5), 20,21 which recognizes pre-fusion stem structures, 22 to test the hypothesis that the amino-terminal of gp41 and the membrane-proximal hydrophobic regions as- semble into a defined complex. In support of this hypothesis, the FP increased recognition of 2F5 linear epitope, a fact that correlated with establishment of interactions mediated specifically by the canonical FLG tripeptide duplication. 16 The FP also stabilized distinct conformations and promoted self-oligo- merization of a sequence that combined the 2F5 linear epitope and the preTM domain. Consistent with a native-like epitope presentation, the com- plexes formed could elicit in rabbits immunoglo- bulins that competed efficiently for Mab2F5 epitope recognition. We conclude that FP may impart a native-like orientation/conformation onto the membrane-proximal gp41 residues. Our findings may help understanding pre-fusion gp41 structure–function relationships, and guide future Abbreviations used: HIV-1, human immunodeficiency virus type-1; FP, fusion peptide; preTM, pre-transmembrane region; HFIP, 1,1,1,3,3,3-hexafluoro-2- propanol; Mab, monoclonal antibody. E-mail address of the corresponding author: gbpniesj@lg.ehu.es doi:10.1016/j.jmb.2006.04.056 J. Mol. Biol. (2006) 360, 45–55 0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.