Abstract
To investigate the structural context of the fusion peptide region in human T-cell leukemia virus type 1 gp21, maltose-binding protein (MBP) was used as an N-terminal solubilization partner for the entire gp21 ectodomain (residues 313-445) and C-terminally truncated ectodomain fragments. The bacterial expression of the MBP/gp21 chimeras resulted in soluble trimers containing intramonomer disulfide bonds. Detergents blocked the proteolytic cleavage of fusion peptide residues in the MBP/gp21-(313-425) chimera, indicating that the fusion peptide is available for interaction with detergent despite the presence of an N-terminal MBP domain. Limited proteolysis experiments indicated that the transmembrane domain proximal sequence Thr(425)-Ala(439) protects fusion peptide residues from chymotrypsin. MBP/gp21 chimera stability therefore depends on a functional interaction between N-terminal and transmembrane domain proximal regions in a gp21 helical hairpin structure. In addition, thermal aggregation experiments indicated that the Thr(425)-Ser(436) sequence confers stability to the fusion peptide-containing MBP/gp21 chimeras. The functional role of the transmembrane domain proximal sequence was assessed by alanine-scanning mutagenesis of the full-length envelope glycoprotein, with 11 of 12 single alanine substitutions resulting in 1.5- to 4.5-fold enhancements in cell-cell fusion activity. By contrast, single alanine substitutions in MBP/gp21 did not significantly alter chimera stability, indicating that multiple residues within the transmembrane domain proximal region and the fusion peptide and adjacent glycine-rich segment contribute to stability, thereby mitigating the potential effects of the substitutions. The fusion-enhancing effects of the substitutions are therefore likely to be caused by alteration of the prefusion complex. Our observations suggest that the function of the transmembrane domain proximal sequence in the prefusion envelope glycoprotein is distinct from its role in stabilizing the fusion peptide region in the fusion-activated helical hairpin conformation of gp21.
Highlights
Retroviral envelope glycoproteins (Env)1 are synthesized as inactive precursors that are processed in the Golgi apparatus to yield a functional hetero-oligomeric complex
The functional role of the transmembrane domain proximal sequence was assessed by alanine-scanning mutagenesis of the full-length envelope glycoprotein, with 11 of 12 single alanine substitutions resulting in 1.5- to 4.5-fold enhancements in cell-cell fusion activity
Our observations suggest that the function of the transmembrane domain proximal sequence in the prefusion envelope glycoprotein is distinct from its role in stabilizing the fusion peptide region in the fusion-activated helical hairpin conformation of gp21
Summary
Envelope glycoprotein; MBP, maltose-binding protein; SU, surface-exposed envelope glycoprotein; TM, transmembrane; HTLV-1, human T-cell leukemia virus type 1; TMD, transmembrane domain; C12E8, octaethyleneglycol mono-n-dodecyl ether; C12E9, nonaethyleneglycol mono-n-dodecyl ether; Deoxy BigChap, N,N-bis(3-D-gluconamidopropyl)-deoxycholamine; CYMAL-3, cyclohexyl-propyl--D-maltoside; CYMAL-5, cyclohexyl-pentyl--D-maltoside; 4-VP, 4-vinylpyridine; mAb, monoclonal antibody. A striking feature of the low pH-induced HA2 conformational change is a loop-to-helix transition that extends the N terminus of the central coiled-coil by ϳ100 Å [21, 25] This helical extension would translocate the fusion peptide from the HA2 core to the tip of the helical hairpin rod for membrane insertion. A similar mode of fusion peptide insertion was observed for a trimeric N-terminal HA2 fragment, where fusion peptide residues 1–10 enter the outer bilayer leaflet as monomeric ␣-helices at an oblique angle [32]. The function of the TMD proximal sequence in the prefusion Env complex may be distinct from its role in stabilizing the fusion peptide and adjacent glycine-rich region in the fusion-activated gp helical hairpin
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