Conformational Flexibility and Strand Arrangements of the Membrane-Associated HIV Fusion Peptide Trimer Probed by Solid-State NMR Spectroscopy

Abstract

The human immunodeficiency virus (HIV) fusion peptide (HFP) is the N-terminal apolar region of the HIV gp41 fusion protein and interacts with target cell membranes and promotes membrane fusion. The free peptide catalyzes vesicle fusion at least to the lipid mixing stage and serves as a useful model fusion system. For gp41 constructs which lack the HFP, high-resolution structures show trimeric protein and suggest that at least three HFPs interact with the membrane with their C-termini in close proximity. In addition, previous studies have demonstrated that HFPs which are cross-linked at their C-termini to form trimers (HFPtr) catalyze fusion at a rate which is 15−40 times greater than noncross-linked HFP. In the present study, the structure of membrane-associated HFPtr was probed with solid-state nuclear magnetic resonance (NMR) methods. Chemical shift and intramolecular 13 CO-15 N distance measurements show that the conformation of the Leu-7 to Phe-11 region of HFPtr has predominant helical conformation in membranes without cholesterol and β strand conformation in membranes containing ∼30 mol% cholesterol. Interstrand 13 CO-13 CO and 13 CO-15 N distance measurements were not consistent with an in-register parallel strand arrangement but were consistent with either: (1) parallel arrangement with adjacent strands tworesidues out-of-register; or (2) antiparallel arrangement with adjacent strand crossing between Phe-8 and Leu-9. Arrangement (1) could support the rapid fusion rate of HFPtr because of placement of the apolar N-terminal regions of all strands on the same side of the oligomer while arrangement (2) could support the assembly of multiple fusion protein trimers. KeywordsHIV; fusion peptide; cholesterol; membranes; NMR; trimer Enveloped viruses such as human immunodeficiency virus (HIV) are surrounded by a membrane and infect cells through fusion between the viral membrane and the target cell membrane (1,2). This process is mediated by envelope proteins which traverse the viral membrane (3). For HIV, the gp41 envelope protein has a ∼170-residue ectodomain region † This work was supported by NIH award AI47153 to D. P. W. * To whom correspondence should be addressed. Telephone: 517−355−9715. Fax: 517−353−1793. Email: weliky@chemistry.msu.edu.. SUPPORTING INFORMATION AVAILABLE Equations are derived for determination of (ΔS/S 0 ) cor for REDOR and fpCTDQBU analyses. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 October 20. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript which lies outside the viral membrane and contains a N-terminal ∼20-residue apolar "fusion peptide" (HFP). The HFP interacts with the target cell membrane and plays an essential role in membrane fusion (4). Peptides composed of the HFP sequence induce fusion between large unilamellar vesicles (LUVs) and between erythrocytes (5,6). There are similar mutation/fusion activity relationships...

Solid-State Nuclear Magnetic Resonance Measurements of HIV Fusion Peptide to Lipid Distances Reveal the Intimate Contact of β Strand Peptide with Membranes and the Proximity of the Ala-14−Gly-16 Region with Lipid Headgroups

“…For the HFP3-8 FLG and HFP2-14 AAG samples associated with PC:PG, there were shoulders at ~178 and 179 ppm, respectively, which correlated with helical conformation of Ala, Leu, and Phe residues. These results were consistent with previous studies of the conformation of membrane-associated HFP with peptide:lipid ~ 0.04 and with previous observations of greater preference for β strand conformation in cholesterol-containing membranes (27,28,35,55,56,59,70). The data demonstrated that samples containing HFP2-14 AAG have qualitatively larger (ΔS/ S 0 ) exp than do samples containing HFP labeled at other residues.…”

“…This result correlated with previous studies which supported the following structural features: (1) β strand HFP was fully extended between A1 and G16; (2) β strand HFP formed hydrogen bonded oligomers or aggregates; and (3) a significant fraction of the oligomers have an antiparallel arrangement with adjacent strand crossing between F8 and L9 (25,(27)(28)(29)35,37,85,86). Some of these studies also supported conformational disorder at A21 (27,28).…”

“…There may be a distinct membrane location of the cross-linked HFPs which correlates with their fast fusion rate. It is also known that the cross-linked HFP trimer will form helical or β strand conformation in membranes without or with cholesterol, respectively, so that studies of the trimer in different membrane compositions can provide information about the conformational dependence of peptide location in membranes (35).…”

“…For a 13 CO-31 P spin pair, r = 23.05/ d 1/3 where r and d are in units of Å and Hz, respectively. The upper limit of REDOR detection of r is ~10 Å (d ≥ 10 Hz) (35).…”

“…Oligomerization/aggregation has also been detected by other biophysical methods (15,30). There is evidence that at least the lipid mixing step of membrane fusion can occur with the HFP in either helical or β strand conformation although there is some controversy in the literature about this conclusion (14,16,18,(31)(32)(33)(34)(35).HFP location in membranes has been primarily probed using a HFP-F8W mutant and by variation of the tryptophan fluorescence of this mutant with changes in environment (36,37). Key results have included: (1) fluorescence was higher for membrane-associated HFP-F8W than for HFP-F8W in buffered saline solution; (2) greater fluorescence quenching by acrylamide was observed for a soluble tryptophan analog than for membrane-associated HFP-F8W; and (3) similar fluorescence quenching of membrane-associated HFP-F8W was observed in samples containing either 1-palmitoyl-2-stearoyl-phosphocholine brominated at the 6, 7 carbons of the stearoyl chain or the corresponding lipid brominated at the 11, 12 carbons of the chain.…”