A thorough understanding of the structure of fusion domains of enveloped viruses in changing lipid environments helps us to formulate mechanistic models on how they might function in mediating viral entry by membrane fusion. We have expressed the N-terminal fusion domain of HIV-1 gp41 as a construct that is water-soluble in the absence of membranes, but that also binds with high affinity to lipid micelles and bilayers in their presence. We have solved the structure and studied the dynamics of this domain bound to dodecylphosphocholine micelles by homo- and heteronuclear NMR spectroscopy. The fusion peptide forms a stable hydrophobic helix from Ile(4) to Ala(14), but is increasingly more disordered and dynamic in a segment of intermediate polarity that stretches from Ala(15) to Ser(23). When bound to lipid bilayers at low concentration, the HIV fusion domain is also largely alpha-helical, as determined by CD and FTIR spectroscopy. However, at higher protein/lipid ratios, the domain is partially converted to form beta-structures in lipid bilayers. Controlled lipid mixing occurs at concentrations that support the alpha-helical, but not the beta-strand conformation.

Influenza virus hemagglutinin (HA)-mediated membrane fusion is initiated by a conformational change that releases a V-shaped hydrophobic fusion domain, the fusion peptide, into the lipid bilayer of the target membrane. The most N-terminal residue of this domain, a glycine, is highly conserved and is particularly critical for HA function; G1S and G1V mutant HAs cause hemifusion and abolish fusion, respectively. We have determined the atomic resolution structures of the G1S and G1V mutant fusion domains in membrane environments. G1S forms a V with a disrupted "glycine edge" on its N-terminal arm and G1V adopts a slightly tilted linear helical structure in membranes. Abolishment of the kink in G1V results in reduced hydrophobic penetration of the lipid bilayer and an increased propensity to form ␤-structures at the membrane surface. These results underline the functional importance of the kink in the fusion peptide and suggest a structural role for the N-terminal glycine ridge in viral membrane fusion.Enveloped viruses enter and infect animal cells by fusing their own membrane with the plasma or an internal membrane of target cells after appropriate receptor recognition. Highly specialized viral membrane proteins catalyze the recognition and fusion process, and the structures of the soluble domains of many viral fusion proteins and fragments of fusion proteins have been solved by X-ray crystallography (17,22,41,45,46). Viral fusion proteins can be classified according to their structures into classes I and II. Class I fusion proteins are characterized by trimeric helical bundles, whereas class II fusion proteins form lattices of dimers of ␤-sheet-rich proteins on the viral surfaces.The structurally and functionally best-characterized class I fusion protein is the hemagglutinin (HA) of influenza virus. Therefore, it has become the prototypic system to study mechanisms of viral membrane fusion (16, 31). Influenza virus enters cells by receptor-mediated endocytosis and subsequent fusion of the viral and endosomal membranes triggered by the pH ϳ5 environment in the endosome. Influenza virus HA is a complex of six polypeptide chains with the stoichiometry (HA1/HA2) 3 . The HA2 transmembrane subunits bear the major responsibility for membrane fusion. Upon exposure to pH 5, HA2 undergoes a massive conformational change (6, 52), which results in exposure of the hydrophobic "fusion domain" at the N terminus. Because energy is released, this conformational change has been described as spring-loaded (8). A second conformational change reverses the direction of the C terminus and brings it into close proximity to the N terminus of the postfusion structure of the ectodomain (9).Although structural studies of the soluble domains of HA2 have yielded many insights into the "engine" that drives membrane fusion, they provided little information on how the released energy is transmitted into the membrane and how the "handles" of this machine shape the membranes into fusioncompetent structures. This task falls to the fusion and transm...

The HIV gp41 fusion domain plays a critical role in membrane fusion during viral entry. A thorough understanding of the relationship between the structure and activity of the fusion domain in different lipid environments helps to formulate mechanistic models on how it might function in mediating membrane fusion. The secondary structure of the fusion domain in small liposomes composed of different lipid mixtures was investigated by circular dichroism spectroscopy. In membranes containing less than 30 mol% cholesterol the fusion domain formed an α-helix and in membranes containing equal to or more than 30 mol% cholesterol the fusion domain formed β-sheet secondary structure. EPR spectra of spin-labeled fusion domains also indicated different conformations in membranes with and without cholesterol. Power saturation EPR data were further used to determine the orientation and depth of α-helical fusion domains in lipid bilayers. Fusion and membrane perturbation activities of the gp41 fusion domain were measured by lipid mixing and contents leakage. The fusion domain fused membranes in both its helical and β-sheet forms. High cholesterol, which induced β-sheet, promoted fusion, but acidic lipids, which promoted relatively deep membrane insertion as an α-helix, also induced fusion. The results indicate that the structure of the HIV gp41 fusion domain is plastic and depends critically on the lipid environment. Provided their membrane insertion is deep, α-helical and β-sheet conformations contribute to membrane fusion.

The fusion peptides of viral membrane fusion proteins play a key role in the mechanism of viral spike glycoprotein mediated membrane fusion. These peptides insert into the lipid bilayers of cellular target membranes where they adopt mostly helical secondary structures. To better understand how membranes may be converted to high-energy intermediates during fusion, it is of interest to know how much energy, enthalpy and entropy, is provided by the insertion of fusion peptides into lipid bilayers. Here, we describe a detailed thermodynamic analysis of the binding of analogues of the influenza hemagglutinin fusion peptide of different lengths and amino acid compositions. In small unilamellar vesicles, the interaction of these peptides with lipid bilayers is driven by enthalpy (-16.5 kcal/mol) and opposed by entropy (-30 cal mol(-1) K(-1)). Most of the driving force (deltaG = -7.6 kcal/mol) comes from the enthalpy of peptide insertion deep into the lipid bilayer. Enthalpic gains and entropic losses of peptide folding in the lipid bilayer cancel to a large extent and account for only about 40% of the total binding free energy. The major folding event occurs in the N-terminal segment of the fusion peptide. The C-terminal segment mainly serves to drive the N-terminus deep into the membrane. The fusion-defective mutations G1S, which causes hemifusion, and particularly G1V, which blocks fusion, have major structural and thermodynamic consequences on the insertion of fusion peptides into lipid bilayers. The magnitudes of the enthalpies and entropies of binding of these mutant peptides are reduced, their helix contents are reduced, but their energies of self-association at the membrane surface are increased compared to the wild-type fusion peptide.

Membrane fusion peptides are highly conserved hydrophobic domains of fusion proteins that insert into membranes during membrane fusion. Recent success with solving the structures of the influenza hemagglutinin fusion peptide and some critical mutants of this peptide in membrane environments at high resolution has led to a new understanding of the mechanism of membrane fusion. This review highlights the structures that have been solved and summarizes recent thermodynamic and spectroscopic studies on the interactions of this interesting class of peptides with lipid bilayers.