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

Human immunodeficiency virus (HIV) infection begins with fusion between viral and host cell membranes and is catalyzed by the HIV gp41 fusion protein. The ∼20 N-terminal apolar residues of gp41 are called the HIV fusion peptide (HFP), interact with the host cell membrane, and play a key role in fusion. In this study, the membrane location of peptides which contained the HFP sequence (AVGIGALFLGFLGAAGSTMGARS) was probed in samples containing either only phospholipids or phospholipids and cholesterol. Four HFPs were examined which each contained 13CO labeling at three sequential residues between G5 and G16. The 13CO chemical shifts indicated that HFP had predominant â strand conformation over the labeled residues in the samples. The internuclear distances between the HFP 13CO groups and the lipid 31P atoms were measured using solid-state nuclear magnetic resonance rotational-echo double-resonance experiments. The shortest 13CO-31P distances of 5-6 Å were observed for HFP labeled between A14 and G16 and correlated with intimate association of â strand HFP and membranes. These results were confirmed with measurements using HFPs singly labeled with 13CO at A6 or A14. To our knowledge, these data are the first measurements of distances between HIV fusion peptide nuclei and lipid P, and qualitative models of the membrane location of oligomeric â strand HFP which are consistent with the experimental data are presented. Observation of intimate contact between â strand HFP and membranes provides a rationale for further investigation of the relationship between structure and fusion activity for this conformation. The infection of enveloped viruses such as human immunodeficiency virus (HIV)1 begins with fusion between the viral and host cell membranes (1-4). Fusion may be catalyzed by fusion proteins, and several models of fusion protein catalysis have been proposed (2, 5-7). For HIV, fusion is catalyzed by a “gp160” glycoprotein complex which is incorporated in the virus membrane and is composed of two noncovalently associated subunits, “gp120” and “gp41”. The gp120 subunit lies outside the virus and binds to receptors in the target cell membrane, and the gp41 subunit contains a region inside HIV as well as a single-pass transmembrane domain (8, 9). The ∼170-residue ectodomain of gp41 lies outside HIV and is subdivided into a more C-terminal “soluble ectodomain” and an ∼20-residue Nterminal fusion peptide (HFP) which is apolar and fairly conserved. The HFP is believed to interact with the target cell membrane after gp120 binds to cellular receptors, and fusion is greatly disrupted by mutation or deletion of the HFP (10-13). Peptides with the HFP sequence catalyze vesicle fusion, and there are good correlations between the mutation-fusion activity relationships of HFP-induced vesicle fusion and HIV-target cell fusion (14-17). Studies of membraneassociated HFP should therefore provide useful information about some aspects of biological fusion. Both the conformation and membrane location of the HFP have been hypothesized to be significant structural factors for the catalysis of fusion by the HFP (16, 18). The conformation of the HFP has been investigated in detergent micelles and membranes using a variety of biophysical techniques. For HFP associated † This work was supported by NIH Grant AI47153 to D.P.W. * To whom correspondence should be addressed. Telephone: (517) 355-9715. Fax: (517) 353-1793. E-mail: [email protected]. ‡ Michigan State University. § Cleveland Clinic Foundation. 1 Abbreviations: 14AAG, residues A14, A15, and G16; 13CO, 13Clabeled carbonyl; d, magnitude of 13C-31P dipolar coupling; DPPC13C, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine with 13C labeling at both carbonyl sites; DTPC, 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine; DTPG, 1,2-di-O-tetradecyl-sn-glycero-3-[phospho-rac-(1glycerol)]; f, fraction of 13CO groups close to 31P atoms; FMI, full membrane insertion; 5GALFLGFLG, residues G5, A6, L7, F8, L9, G10, F11, L12, and G13; HEPES, N-(2-hydroxyethyl)piperazine-N′-2ethanesulfonic acid; HFP, HIV fusion peptide; HFP1, AVGIGALFLGFLGAAGSTMGARS-NH2; HFP1-8FLG, HFP1 labeled with 13CO at F8, L9, and G10; HFP2, AVGIGALFLGFLGAAGSTMGARSKKKNH2; HFP2-5GAL, HFP2 labeled with 13CO at G5, A6, and L7; HFP211FLG, HFP2 labeled with 13CO at F11, L12, and G13; HFP2-14AAG, HFP2 labeled with 13CO at A14, A15, and G16; HFP3, AVGIGALFLGFLGAAGSTMGARSKKKAâ; HFP3-8FLG, HFP3 labeled with 13CO at F8, L9, and G10; HFP4, AVGIGALFLGFLGAAGSTMGARSWKKKKKKAâ; HFP4-6A, HFP4 labeled with 13CO at A6; HFP4-14A, HFP4 labeled with 13CO at A14; HIV, human immunodeficiency virus; IR, infrared; 7LFLGFL, residues L7, F8, L9, G10, F11, and L12; MAS, magic angle spinning; NMR, nuclear magnetic resonance; PC:PG, 4:1 DTPC/DTPG mixture; PC:PG:CHOL, 8:2:5 DTPC/DTPG/cholesterol mixture; PMI, partial membrane insertion; r, 13C-31P internuclear distance; REDOR, rotational-echo doubleresonance; τ, duration of the dephasing period; TFA, trifluoroacetic acid. 4997 Biochemistry 2007, 46, 4997-5008 10.1021/bi6024808 CCC: $37.00 © 2007 American Chemical Society Published on Web 04/07/2007 with negatively charged sodium dodecyl sulfate micelles, one liquid-state nuclear magnetic resonance (NMR) study showed that there was uninterrupted R helical structure from I4 to M19, while another study showed a helix from I4 to A14 followed by a â turn (19, 20). For HFP associated with neutral dodecylphosphocholine micelles, helical structure was detected from I4 to L12 (21; C. M. Gabrys and D. P. Weliky, unpublished data). There is not yet a consensus for the micelle location of HFP, and there are distinct models based on experiment and simulation of either predominant micelle surface location or micelle traversal by HFP (19-23). In one NMR study, residues I4-A15 were found to be fully shielded from solvent and residues G3 and G16 were at the micelle-solvent interface (20). The conformation of membrane-associated HFP has been investigated with different lipid components and different peptide:lipid ratios. A greater fraction of HFPs adopted helical structure at low peptide:lipid ratios, while nonhelical structure became more favored at higher ratios (24). Helical structure was also promoted by negatively charged lipids, while a higher fraction of â strand structure was adopted with neutral lipids or with bound Ca2+ (16, 24-26). Solidstate NMR provided residue-specific conformational information about HFP associated with membranes whose lipid headgroup and cholesterol composition were comparable to those of host cells of the virus. A â strand conformation was observed for residues A1-G16, while A21 appeared to be unstructured (27, 28). Formation of â strand oligomers or aggregates was supported by detection of short distances between labeled 13CO groups on one HFP and labeled 15N on an adjacent HFP (29). Oligomerization and aggregation have 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 â strand conformation, although there is some controversy in the literature about this conclusion (14, 16, 18, 31-35). HFP location in membranes has been primarily probed using an HFP-F8W mutant and by variation of the tryptophan fluorescence of this mutant with changes in environment (36, 37). Key results have included the following. (1) Fluorescence was higher for membrane-associated HFP-F8W than for HFP-F8W in a buffered saline solution. (2) Greater fluorescence quenching by acrylamide was observed for a soluble tryptophan analogue than for membrane-associated HFP-F8W. (3) Similar fluorescence quenching of membraneassociated HFP-F8W was observed in samples containing either 1-palmitoyl-2-stearoylphosphocholine brominated at the 6 and 7 carbons of the stearoyl chain or the corresponding lipid brominated at the 11 and 12 carbons of the chain. The first two results indicated that the level of solvent exposure of the HFP-F8W tryptophan is reduced with membrane association, and the third result indicated that the membrane location of the tryptophan indole group is centered near the carbon 9 position of the brominated lipid stearoyl chain, i.e., ∼8.5 Å from the bilayer center and ∼10 Å from the lipid phosphorus. Infrared (IR) and solid-state NMR spectra of membrane-associated HFP suggested that HFP-F8W had predominant â strand conformation under the conditions of the fluorescence experiments (16, 27, 36, 37). In a different set of experiments, electron spin resonance spectra showed that chromium oxalate in the aqueous phase quenched the signal of membrane-associated HFP which was spin-labeled at M19 but did not quench HFP spin-labeled at A1 (30). These data indicated a location for M19 close to the aqueous interface of the membrane and a location for A1 away from this interface. Models for HFP location in membranes have also been developed from simulations of a single HFP molecule in membranes and have shown either partial insertion or traversal of the membrane. The HFP always adopted predominant R helical conformation and in one simulation was generally near the membrane surface with the F8 backbone and side chain nuclei 4 and 6 Å deeper than the phosphorus longitude, respectively (38). For a different simulation, HFP traversed the membrane and the backbone and side chain F8 nuclei were at the bilayer center, i.e., ∼19 Å from the phosphorus longitude (39). This paper includes solid-state NMR measurements of distances between 13C-labeled carbonyl (13CO) nuclei in HFP and lipid 31P nuclei. These studies provide information about the location of specific HFP residues relative to the phosphorus headgroups and are complementary to other solidstate NMR methods of probing membrane location of peptides and proteins (40-50). The 13CO-31P distance approach has previously been used to probe the locations of antimicrobial peptides, antibiotics, and sterols in membranes (51-53). Measurements were taken both on HFP associated with membranes containing only phospholipids and on HFP associated with membranes which contained both phospholipids and cholesterol. The potential significance of cholesterolcontaining membranes is suggested both by the cholesterol: phospholipid molar ratios of ∼0.5 and 0.8 for HIV host cell and HIV membranes, respectively, and by the observation that â strand conformation of HFP is promoted by membrane cholesterol (27, 35, 54-57). The 13CO-31P distances (r) were probed with the rotationalecho double-resonance (REDOR) technique which is a solidstate NMR method for measuring magnitudes of dipolar couplings (d) between spin /2 heteronuclei such as 13C and 31P (58). For a 13CO-31P spin pair, r ) 23.05/d1/3, where r and d are in units of angstroms and hertz, respectively. The upper limit of REDOR detection of r is ∼10 Å (d g 10 Hz) (35). MATERIALS AND METHODS Materials. Resins and 9-fluorenylmethoxycarbonyl (FMOC) amino acids were obtained from Peptides International (Louisville, KY). Amino acids isotopically labeled with 13CO were obtained from Cambridge Isotope Laboratories (Andover, MA). The lipids 1,2-di-O-tetradecyl-sn-glycero3-phosphocholine (DTPC), 1,2-di-O-tetradecyl-sn-glycero3-[phospho-rac-(1-glycerol)] (DTPG), and [1-13C]-1,2dipalmitoyl-sn-glycero-3-phosphocholine (DPPC-13C) were obtained from Avanti Polar Lipids (Alabaster, AL). The 5 mM HEPES buffer at pH 7 contained 0.01% (w/v) NaN3 preservative. Peptides. All peptides contained the sequence AVGIGALFLGFLGAAGSTMGARS which is the 23 N-terminal residues of HIV-1 gp41, LAV1a strain. A set of peptides for probing peptide-lipid headgroup distances between G5 and G16 were synthesized. “HFP1-8FLG” had the sequence AVGIGALFLGFLGAAGSTMGARS-NH2 and was labeled with 13CO at F8, L9, and G10. “HFP2-5GAL”, “HFP2-114998 Biochemistry, Vol. 46, No. 17, 2007 Qiang et al.