The HIV gp41 protein catalyzes fusion between viral and host cell membranes and its apolar N-terminal region or “fusion peptide” binds to the host cell membrane and plays a key role in fusion. “HFP” is a construct containing the fusion peptide sequence, induces membrane vesicle fusion, and is an important fusion model system. Earlier solid-state NMR (SSNMR) studies showed that when HFP is associated with membranes with ~30 mol% cholesterol, the first sixteen residues have predominant β strand secondary structure and a fraction of the strands form antiparallel β sheet structure with residue 16→1/1→16 or 17→1/1→17 registries of adjacent strands. In some contrast, other SSNMR and infrared studies have been interpreted to support a large fraction of approximately in-register parallel registry of adjacent strands. However, the samples had extensive isotopic labeling and other structural models were also consistent with the data. The present SSNMR study uses sparse labeling schemes that reduce ambiguity in the determination of the fraction of HFP molecules with parallel β registry. Quantitative analysis of the data shows that the parallel fraction is at most 0.15 with a much greater fraction of antiparallel 16→1/1→16 and 17→1/1→17 registries. These data strongly support a model of HFP-induced vesicle fusion caused by antiparallel rather than parallel registries and provide insight into the arrangement of gp41 molecules during HIV/host cell fusion. This study is an example of quantitative determination of a complex structural distribution by SSNMR including experimentally-validated inclusion of natural abundance contributions to the SSNMR data.

Fusion of the HIV membrane and the host cell membrane is an initial step of infection of the host cell. Fusion is catalyzed by gp41 which is an integral membrane protein of HIV. The fusion peptide (FP) is the ~25 N-terminal residues of gp41 and is a domain of gp41 that plays a key role in fusion catalysis likely through interaction with the host cell membrane. Much of our understanding of the FP domain has been accomplished with studies of “HFP”, ie. a ~25-residue peptide composed of the FP sequence but lacking the rest of gp41. HFP catalyzes fusion between membrane vesicles and serves as a model system to understand fusion catalysis. HFP binds to membranes and the membrane location of HFP is likely a significant determinant of fusion catalysis perhaps because the consequent membrane perturbation reduces the fusion activation energy. In the present study, many HFPs were synthesized and differed in the residue position that was 13CO backbone labeled. Samples were then prepared that each contained a singly 13CO labeled HFP incorporated into membranes which lacked cholesterol. HFP had distinct molecular populations with either α helical or oligomeric β sheet structure. Proximity between the HFP 13CO nuclei and 31P nuclei in the membrane headgroups was probed by solid-state NMR (SSNMR) rotational-echo double-resonance (REDOR) measurements. For many samples, there were distinct 13CO shifts for the α helical and β sheet structures so that the proximities to 31P nuclei could be determined for each structure. Data from several differently labeled HFPs were then incorporated into a membrane location model for the particular structure. In addition to the 13CO labeled residue position, the HFPs also differed in sequence and/or chemical structure. “HFPmn” was a linear peptide that contained the 23 N-terminal residues of gp41. “HFPmn_V2E” contained the V2E mutation which for HIV leads to greatly reduced extent of fusion and infection. The present study shows that HFPmn_V2E induces much less vesicle fusion than HFPmn. “HFPtr” contained three strands with HFPmn sequence that were chemically cross-linked near their C-termini. HFPtr mimics the trimeric topology of gp41 and induces much more rapid and extensive vesicle fusion than HFPmn. For HFPmn and HFPtr, well-resolved α and β peaks were observed for A6-, L9-, and L12-labeled samples. For each of these samples, there were similar HFP 13CO to lipid 31P proximities in the α and β structures which evidenced comparable membrane locations of the HFP in either structure including insertion into a single membrane leaflet. The data were also consistent with deeper insertion of HFPtr relative to HFPmn in both the α and β structures. The results supported a strong correlation between the membrane insertion depth of the HFP and its fusogenicity. More generally, the results supported membrane location of the HFP as an important determinant of its fusogenicity. The deep insertion of HFPtr in both the α and β structures provides the most relevant membrane location of the FP for HIV gp41-cat...

Residue-specific location of peptides in the hydrophobic core of membranes was examined using 13C-2H REDOR and samples in which the lipids were selectively deuterated. The transmembrane topology of the KALP peptide was validated with this approach with substantial dephasing observed for deuteration in the bilayer center and reduced or no dephasing for deuteration closer to the headgroups. Insertion of β sheet HIV and helical and β sheet influenza virus fusion peptides into the hydrophobic core of the membrane was validated in samples with extensively deuterated lipids.

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