Abstract
The human and simian immunodeficiency viruses (HIV and SIV) envelope glycoprotein consists of a trimer of two noncovalently and weakly associated subunits, gp120 and gp41. Upon binding of gp120 to cellular receptors, this labile native envelope complex undergoes conformational changes, resulting in a stable trimer-of-hairpins structure in gp41. Formation of the hairpin structure is thought to mediate membrane fusion by placing the viral and cellular membranes in close proximity. An in vitro-derived variant of SIVmac251, denoted CPmac, has acquired an unusually stable virion-associated gp120-gp41 complex. This unique phenotype is conferred by five amino acid substitutions in the gp41 ectodomain. Here we characterize the structural and physicochemical properties of the N40(L6)C38 model of the CPmac gp41 core. The 1.7-A resolution crystal structure of N40(L6)C38 is very similar to the six-helix bundle structure present in the parent SIVmac251 gp41. In both structures, three N40 peptides form a central three-stranded coiled coil, and three C38 peptides pack in an antiparallel orientation into hydrophobic grooves on the coiled-coil surface. Thermal unfolding studies show that the CPmac mutations destabilize the SIVmac251 six-helix bundle by 15 kJ/mol. Our results suggest that the formation of the gp41 trimer-of-hairpins structure is thermodynamically coupled to the conformational stability of the native envelope glycoprotein and raise the intriguing possibility that introduction of mutations to destabilize the six-helix bundle may lead to the stabilization of the trimeric gp120-gp41 complex. This study suggests a potential strategy for the production of stably folded envelope protein immunogens for HIV vaccine development.
Highlights
For human and simian immunodeficiency viruses (HIV1 and SIV), infection of cells is initiated by fusion of the viral and
Our results suggest that the formation of the gp41 trimer-of-hairpins structure is thermodynamically coupled to the conformational stability of the native envelope glycoprotein and raise the intriguing possibility that introduction of mutations to destabilize the six-helix bundle may lead to the stabilization of the trimeric gp120-gp41 complex
This membrane fusion process is mediated by the viral envelope glycoprotein and receptors on the target cell
Summary
Protein Expression and Purification—The gp ectodomain segment (representing residues 555– 674 of full-length gp160) from the env gene of the SIVmac239 isolate [61] was amplified using primers containing NdeI and BamHI restriction sites and ligated into the NdeI and BamHI sites of the pAED4 vector [62] to make pSIVegp. The soluble fraction, containing denatured N40(L6)C38, was dialyzed into 5% acetic acid overnight at room temperature. For most of the proteins with a melting temperature of 100 °C or higher in TBS, thermal denaturation experiments were performed in TBS in the presence of 2 or 3 M GdmCl. Sedimentation Equilibrium—Sedimentation equilibrium measurements were performed on a Beckman XL-A (Beckman Coulter) analytical ultracentrifuge equipped with an An-60 Ti rotor (Beckman Coulter). Crystallization, Data Collection, and Structure Determination— Crystals were obtained using the handing drop method of vapor diffusion by equilibrating 2-l drops (protein solution mixed 1:1 with reservoir solution) against a reservoir solution at room temperature. The refined coordinates for NCmac-N40(L6)C38 (entry 1JPX) and CPmac-N40(L6)C38 (entry 1JQ0) have been deposited in the Research Collaboratory for Structural Bioinformatics Protein Data Bank
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