Expression and biochemical analysis of the entire HIV‐2 gp41 ectodomain: determinants of stability map to N‐ and C‐terminal sequences outside the 6‐helix bundle core

Abstract

The folding of HIV gp41 into a 6-helix bundle drives virus-cell membrane fusion. To examine the structural relationship between the 6-helix bundle core domain and other regions of gp41, we expressed in Escherichia coli, the entire ectodomain of HIV-2 ST gp41 as a soluble, trimeric maltose-binding protein (MBP)/gp41 chimera. Limiting proteolysis indicated that the Cys-591-Cys-597 disulfide-bonded region is outside a core domain comprising two peptides, Thr-529-Trp-589 and Val-604-Ser-666. A biochemical examination of MBP/gp41 chimeras encompassing these core peptides indicated that the N-terminal polar segment, 521-528, and C-terminal membrane-proximal segment, 658-666, cooperate in stabilizing the ectodomain. A functional interaction between sequences outside the gp41 core may contribute energy to membrane fusion.

Functional Links between the Fusion Peptide-proximal Polar Segment and Membrane-proximal Region of Human Immunodeficiency Virus gp41 in Distinct Phases of Membrane Fusion

“…By contrast, these sequences would become apposed at the membrane-interactive end of the fusion-activated trimer of hairpins by formation of the 6HB. Consistent with this idea, extension of the 6HB core to include residues 528 -535 of the polar segment and residues 669 -677 of the MPR confers stability to a trimer of hairpins model protein (53).…”

“…Alanine Scan of the Fusion Peptide-proximal and Membraneproximal Regions of HIV-1 AD8 gp41-Previously, we found that extension of the 6-helix bundle core of gp41 to include Ala 528 -Leu 535 , of the N-terminal polar segment, and Phe 669 -Ser 677 , within the MPR, conferred stability to a model of the HIV-2 ST gp41 trimer of hairpins, MBP/gp41 (53). In this paper, we examined the functional relevance of these sequences following alanine replacement of component amino acids that are conserved in HIV-1, HIV-2, and SIV ( Fig.…”

“…Segment and MPR-Our previous study with the MBP/gp41 chimeric model of the HIV-2 gp41 trimer of hairpins suggested that the polar segment and MPR confer trimer stability in a synergistic manner (53). Whereas these terminal sequences are likely to be brought together in late fusion forms of gp41 by 6-helix bundle formation, they appear to be spatially separate in the prefusion gp120-gp41 complex with the polar segment located in the interior of the trimer, whereas the MPR is external (17,20,21).…”

“…DNA sequences were confirmed using ABI prism BigDye terminator (Applied Biosystems, Foster City, CA). MBP/gp41 chimeras were induced in Escherichia coli strain BL21(DE3) and purified by amylose-agarose affinity chromatography (New England Biolabs) and gel filtration as described (53). MBP/gp41 trimers were proteolyzed with sequencing grade chymotrypsin (Roche) and analyzed by SDS-PAGE in 12-17% polyacrylamide gradient gels as described (53).…”

The Conserved Glycine-Rich Segment Linking the N-Terminal Fusion Peptide to the Coiled Coil of Human T-Cell Leukemia Virus Type 1 Transmembrane Glycoprotein gp21 Is a Determinant of Membrane Fusion Function

“…1), suggesting that hydrophobic contact between the glycine-rich segment and other TM regions is a conserved feature of fusion function. For example, the homologous N-terminal sequence of HIV-2 gp41 cooperates with the membrane-proximal segment in stabilizing the trimer of hairpins (32). The membrane-proximal segment of gp41 contains an aromatic cluster that may interact with hydrophobic residues of the glycine-rich segment in a late fusion intermediate to drive the pore expansion phase of fusion (40,44 Fusion function was also blocked by the five Gly-to-Pro substitutions with position-dependent effects on the various phases of membrane fusion.…”