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.
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.
Lay
Chan-Sien
CS
St. Vincent's Institute of Medical Research, and Department of Microbiology and Immunology, University of Melbourne, Victoria, Australia.
Wilson
Kirilee A
KA
Kobe
Bostjan
B
Kemp
Bruce E
BE
Drummer
Heidi E
HE
Poumbourios
Pantelis
P
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics; D002850:Chromatography, Gel; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D015700:HIV Envelope Protein gp41 / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D015498:HIV-2 / Q000737:chemistry*; D006358:Hot Temperature; D006801:Humans; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D021241:Spectrometry, Mass, Electrospray Ionization; D014461:Ultracentrifugation / Q000379:methods
D002352:Carrier Proteins; D015700:HIV Envelope Protein gp41; D058619:Maltose-Binding Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D003545:Cysteine