Engineered mimetics of the pre-hairpin intermediate and post-fusion six-helix bundle of HIV-1 gp41.

<p>(<i>A</i>) Domain organization of HIV-1 gp41 and sequences of the six-helix bundle (6-helix, core^S and core^SP) and pre-hairpin (5-helix and N35_CCG-N13) mimetics. (FP, fusion protein; FPPR, fusion peptide proximal region; N-HR, N-heptad repeat; IL, immune-dominant linker; C-HR, C-heptad repeat; MPER, membrane proximal external region; TM, transmembrane region; CT, intraviral C-terminal domain.) Three N35_CCG-N13 chains are linked covalently via intermolecular disulfide bridges (CCG, shown in purple) to form a stable helical trimer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104683#pone.0104683-Louis2" target="_blank">[19]</a>. N-HR, C-HR and linker residues are shown in green, orange and black (underlined), respectively. Numbering of N-HR and C-HR regions is according to their location in Env from HIV-1 (strain HXB2). Positions in the helical wheel (blue italic) of N-HR residues that are solvent accessible in the six-helix bundle conformation are indicated. (<i>B</i>) Interactions of Fab8066 with N-HR residues in the context of the six-helix bundle construct core^S mapped by Ala scanning mutagenesis and immunoblotting <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104683#pone.0104683-Gustchina2" target="_blank">[22]</a>. The core^S trimer is shown as a surface representation with N-HR and C-HR regions of gp41 in white and light orange, respectively. N-HR surface accessible residues (H564, W571, K574 and Q575) identified as sites of interaction with Fab8066 are shown in distinct colors. (<i>C</i>) Relative migration on SDS-PAGE of the gp41 mimetics used in this study. Molecular weights of constructs and markers (M) are indicated in kDa. N-HR and C-HR denote peptides which assemble to form the six-helix bundle conformation of core^SP. (<i>D</i>) Ribbon representations of the gp41 constructs. 5-helix, CCIZN36 and N35_CCG-N13 are pre-hairpin intermediate mimetics with one or more exposed N-HR helices, that are otherwise partially shielded in the six-helix bundle. The three N-HR peptide chains in N35_CCG-N13 and CCIZN36 are stabilized as disulfide-linked trimers by fusion with either a 13-residue repeat of the N-HR <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104683#pone.0104683-Louis2" target="_blank">[19]</a> or an N-terminal isoleucine zipper segment <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104683#pone.0104683-Bianchi1" target="_blank">[37]</a>, respectively. 6-helix and 5-helix are single chain polypeptides with the N-HR (N) and C-HR (C) regions connected by a six-residue linker in the order N-C-N-C-N-C and N-C-N-C-N, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104683#pone.0104683-Root1" target="_blank">[18]</a>. Core^S and core^SP also form a six-helix bundle but as a trimer consisting of 3 hairpin (N-linker-C) peptides and a hexamer consisting of 3 N-HR and 3 C-HR peptides, respectively (see panel A).