A host–guest system to study structure–function relationships of membrane fusion peptides

Abstract

We designed a host-guest fusion peptide system, which is completely soluble in water and has a high affinity for biological and lipid model membranes. The guest sequences are those of the fusion peptides of influenza hemagglutinin, which are solubilized by a highly charged unstructured C-terminal host sequence. These peptides partition to the surface of negatively charged liposomes or erythrocytes and elicit membrane fusion or hemolysis. They undergo a conformational change from random coil to an obliquely inserted (Ϸ33°from the surface) ␣-helix on binding to model membranes. Partition coefficients for membrane insertion were measured for influenza fusion peptides of increasing lengths (n ‫؍‬ 8, 13, 16, and 20). The hydrophobic contribution to the free energy of binding of the 20-residue fusion peptide at pH 5.0 is ؊7.6 kcal͞mol (1 cal ‫؍‬ 4.18 J). This energy is sufficient to stabilize a ''stalk'' intermediate if a typical number of fusion peptides assemble at the site of membrane fusion. The fusion activity of the fusion peptides increases with each increment in length, and this increase strictly correlates with the hydrophobic binding energy and the angle of insertion.F usion peptides play a central role in facilitating membrane fusion. Fusion peptides are highly conserved and quite hydrophobic sequences of fusion proteins that reside in viral envelope membranes or in the plasma membranes of sperm cells (1). These peptides are thought to be the only segments of the fusion proteins that insert deeply into the lipid bilayer of the target membrane at an early stage of membrane fusion (2). Despite this prominent role and numerous biophysical studies, no precise structure of a fusion peptide in lipid bilayers has yet been determined. Also, the binding of fusion peptides to lipid bilayers could not be studied previously, because these peptides are too hydrophobic to measure their partitioning between aqueous and membrane phases. Obtaining thermodynamic parameters of fusion peptide binding to lipid bilayers would be extremely helpful for the formulation of physically relevant models of energy coupling in membrane fusion. Most previous physical studies of fusion peptide-bilayer interactions were conducted with samples in which peptides and lipids were mixed in organic solvents and hydrated or added from organic solvent to preformed liposomes before spectroscopic analysis. A notable exception was a spin-label EPR spectroscopic study in which an expressed fragment of influenza hemagglutinin (HA) was used to assess the depth of the fusion peptide in lipid bilayers (3). The conformation of hydrophobic peptides in lipid bilayers often depends on the solvent that was used in the reconstitution procedure. Gramicidin A, for example, adopts many different conformations depending on how the samples are prepared (4). Therefore, the current procedures to reconstitute fusion peptide-bilayer complexes may not be adequate for higher-resolution structural studies of fusion peptides in lipid bilayers. To alleviate these problems, we ...

Structure and Mechanism of the Saposin-like Domain of a Plant Aspartic Protease

“…Interestingly, influenza A hemagglutinin fusion peptide is inaccessible to membranes at neutral pH; however, a drop of the pH inside the endosome below a critical threshold induces a large conformational change in the parent protein and is subsequently activated by a protease (plasmin) that cleaves the precursor polypeptide (76) into two disulfide-linked polypeptides and a fusion peptide (77). The hemagglutinin fusion peptide has a slightly higher helix content at pH 5 than at pH 7.4, as revealed by comparison of CD spectra (61,74), which are remarkably similar to those for StAP PSI in the present study (Fig. 2) in terms of overall shape (dominant helix content) as well as their x intercepts and superimposed relative spectra (gain of helical structure upon acidification).…”

“…These associations of ␣-helices contain one hydrophobic face, an arrangement similar to StAP PSI with its five helices, hydrophobic inner cavity, and N terminus. An important fusase fusion peptide for virus-cell fusion is the N-terminal portion of influenza A hemagglutinin, which adopts an ␣-helical conformation in lipid bilayers (74), constitutes an autonomous folding unit in the membrane, and catalyzes lipid exchange between juxtaposed membranes (61).…”

Application of REDOR subtraction for filtered MAS observation of labeled backbone carbons of membrane-bound fusion peptides

“…HFP-KKK-UF8L9G10 (sequence AVGIGALFLGFLGAAGSTMGARSKKK) was synthesized with uniformly 13 C, 15 N-labeled amino acids at Phe-8, Leu-9, and Gly-10. Because of the three C-terminal lysines, HFP-KKK dissolved more quickly in aqueous solution than HFP [65].…”

“…The IFP-KKK peptide corresponds to the 20 N-terminal residues of the Influenza A hemagglutinin fusion protein plus three C-terminal lysine residues to improve aqueous solubility [65]. There is also a glycine linker in IFP-GKKK.…”

Solid-State Nuclear Magnetic Resonance Evidence for Parallel and Antiparallel Strand Arrangements in the Membrane-Associated HIV-1 Fusion Peptide

“…In each of these soluble ectodomain structures, the protein is trimeric, and its three N-termini (corresponding to about residue 30 in the whole envelope protein) are in close proximity at the end of an in-register helical coiled-coil. These structures end several residues C-terminal of the fusion peptide, and it has therefore been hypothesized that during viral/target cell fusion, at least three fusion peptides insert into the target cell membrane with their C-termini in close proximity.A variety of experimental methods have shown that both the HIV-1 and influenza fusion peptides can assume helical or nonhelical structures in their membrane-associated forms (6,9,10,14,15,17,18,(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50) 1,phosphatidylethanolamine; N-Rh-PE, N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine; PDB, Protein Data Bank; PI, phosphatidylinositol; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPE, 1-palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine; POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine; rf, radio frequency; RET, resonance energy transfer; REDOR, rotational-echo double resonance; RFDR, radio frequencydriven dipolar recoupling; SEDRA, simple excitation for the dephasing of rotational-echo amplitudes; SIMPSON, simulation program for solidstate NMR spectroscopy; TFA, trifluoroacetic acid; TPPM, two pulse phase modulation; 1D, one dimensional; 2D, two dimensional. …”