The final conformation of the complete ectodomain of the HA2 subunit of influenza hemagglutinin can by itself drive low pH-dependent fusion

One of the best characterized fusion proteins, the influenza virus hemagglutinin (HA), mediates fusion between the viral envelope and the endosomal membrane during viral entry into the cell. In the initial conformation of HA, its fusogenic subunit, the transmembrane protein HA2, is locked in a metastable conformation by the receptor-binding HA1 subunit of HA. Acidification in the endosome triggers HA2 refolding toward the final lowest energy conformation. Is the fusion process driven by this final conformation or, as often suggested, by the energy released by protein restructuring? Here we explored structural properties as well as the fusogenic activity of the full sized trimeric HA2(1–185) (here called HA2*) that presents the final conformation of the HA2 ectodomain. We found HA2* to mediate fusion between lipid bilayers and between biological membranes in a low pH-dependent manner. Two mutations known to inhibit HA-mediated fusion strongly inhibited the fusogenic activity of HA2*. At surface densities similar to those of HA in the influenza virus particle, HA2* formed small fusion pores but did not expand them. Our results confirm that the HA1 subunit responsible for receptor binding as well as the transmembrane and cytosolic domains of HA2 is not required for fusion pore opening and substantiate the hypothesis that the final form of HA2 is more important for fusion than the conformational change that generates this form.     

Effect of lipid composition on the "membrane response" induced by a fusion peptide

To understand the initial stages of membrane destabilization induced by viral proteins, the factors important for binding of fusion peptides to cell membranes must be identified. In this study, effects of lipid composition on the mode of peptides' binding to membranes are explored via molecular dynamics (MD) simulations of the peptide E5, a water-soluble analogue of influenza hemagglutinin fusion peptide, in two full-atom hydrated lipid bilayers composed of dimyristoyl- and dipalmitoylphosphatidylcholine (DMPC and DPPC, respectively). The results show that, although the peptide has a common folding motif in both systems, it possesses different modes of binding. The peptide inserts obliquely into the DMPC membrane mainly with its N-terminal alpha helix, while in DPPC, the helix lies on the lipid/water interface, almost parallel to the membrane surface. The peptide seriously affects structural and dynamical parameters of surrounding lipids. Thus, it induces local thinning of both bilayers and disordering of acyl chains of lipids in close proximity to the binding site. The "membrane response" significantly depends upon lipid composition: distortions of DMPC bilayer are more pronounced than those in DPPC. Implications of the observed effects to molecular events on initial stages of membrane destabilization induced by fusion peptides are discussed.     

Peripherin-2: an intracellular analogy to viral fusion proteins

The C-terminus of the intracellular retinal rod outer segment disk protein peripherin-2 binds to membranes, adopts a helical conformation, and promotes membrane fusion, which suggests an analogy to the structure and function of viral envelope fusion proteins. Nuclear magnetic resonance (NMR) data and fluorescence data show that a 63-residue polypeptide comprising the C-terminus of bovine peripherin-2 (R284-G346) binds to the membrane mimetic, dodecylphosphocholine micelles. High-resolution NMR studies reveal that although this C-terminal fragment is unstructured in solution, the same fragment adopts helical structure when bound to the micelles. The C-terminus may be a member of the class of intrinsically unstructured protein domains. Using methods developed for the G-protein coupled receptor rhodopsin, a model for the structure of the transmembrane domain of peripherin-2 was constructed. Previously published data showed that both peripherin-2 and viral fusion proteins are transmembrane proteins that promote membrane fusion and have a fusion peptide sequence within the protein that independently promotes membrane fusion. Furthermore, the fusion-active sequence of peripherin-2 exhibits a sequence motif that matches the viral fusion peptide of influenza hemagglutinin (HA). These observations collectively suggest that the mechanism of intracellular membrane fusion induced by peripherin-2 and the mechanism of enveloped viral fusion may have features in common.     

Structure and plasticity of the human immunodeficiency virus gp41 fusion domain in lipid micelles and bilayers

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.     

Properties and structures of the influenza and HIV fusion peptides on lipid membranes: implications for a role in fusion

The fusion peptides of HIV and influenza virus are crucial for viral entry into a host cell. We report the membrane-perturbing and structural properties of fusion peptides from the HA fusion protein of influenza virus and the gp41 fusion protein of HIV. Our goals were to determine: 1), how fusion peptides alter structure within the bilayers of fusogenic and nonfusogenic lipid vesicles and 2), how fusion peptide structure is related to the ability to promote fusion. Fluorescent probes revealed that neither peptide had a significant effect on bilayer packing at the water-membrane interface, but both increased acyl chain order in both fusogenic and nonfusogenic vesicles. Both also reduced free volume within the bilayer as indicated by partitioning of a lipophilic fluorophore into membranes. These membrane ordering effects were smaller for the gp41 peptide than for the HA peptide at low peptide/lipid ratio, suggesting that the two peptides assume different structures on membranes. The influenza peptide was predominantly helical, and the gp41 peptide was predominantly antiparallel beta-sheet when membrane bound, however, the depths of penetration of Trps of both peptides into neutral membranes were similar and independent of membrane composition. We previously demonstrated: 1), the abilities of both peptides to promote fusion but not initial intermediate formation during PEG-mediated fusion and 2), the ability of hexadecane to compete with this effect of the fusion peptides. Taken together, our current and past results suggest a hypothesis for a common mechanism by which these two viral fusion peptides promote fusion.     

The aromatic domain of the coronavirus class I viral fusion protein induces membrane permeabilization: putative role during viral entry

Coronavirus (CoV) entry is mediated by the viral spike (S) glycoprotein, a class I viral fusion protein. During viral and target cell membrane fusion, the heptad repeat (HR) regions of the S2 subunit assume a trimer-of-hairpins structure, positioning the fusion peptide in close proximity to the C-terminal region of the ectodomain. The formation of this structure appears to drive apposition and subsequent fusion of viral and target cell membranes; however, the exact mechanism is unclear. Here, we characterize an aromatic amino acid rich region within the ectodomain of the S2 subunit that both partitions into lipid membranes and has the capacity to perturb lipid vesicle integrity. Circular dichroism analysis indicated that peptides analogous to the aromatic domains of the severe acute respiratory syndrome (SARS)-CoV, mouse hepatitis virus (MHV) and the human CoV OC43 S2 subunits, did not have a propensity for a defined secondary structure. These peptides strongly partitioned into lipid membranes and induced lipid vesicle permeabilization at peptide/lipid ratios of 1:100 in two independent leakage assays. Thus, partitioning of the peptides into the lipid interface is sufficient to disorganize membrane integrity. Our study of the S2 aromatic domain of three CoVs provides supportive evidence for a functional role of this region. We propose that, when aligned with the fusion peptide and transmembrane domains during membrane apposition, the aromatic domain of the CoV S protein functions to perturb the target cell membrane and provides a continuous track of hydrophobic surface, resulting in lipid-membrane fusion and subsequent viral nucleocapsid entry.     

Structural characterizations of fusion peptide analogs of influenza virus hemagglutinin. Implication of the necessity of a helix-hinge-helix motif in fusion activity

Infection by enveloped viruses initially involves membrane fusion between viral and host cell membranes. The fusion peptide plays a crucial role in triggering this reaction. To clarify how the fusion peptide exerts this specific function, we carried out biophysical studies of three fusion peptide analogs of influenza virus hemagglutinin HA2, namely E5, G13L, and L17A. E5 exhibits an activity similar to the native fusion peptide, whereas G13L and L17A, which are two point mutants of the E5 analog, possess much less fusion activity. Our CD data showed that the conformations of these three analogs in SDS micelles are pH-dependent, with higher alpha-helical contents at acidic pH. Tryptophan fluorescence emission experiments indicated that these three analogs insert deeper into lipid bilayers at acidic pH. The three-dimensional structure of the E5 analog in SDS micelles at pH 4.0 revealed that two segments, Leu(2)-Glu(11) and Trp(14)-Ile(18), form amphipathic helical conformations, with Gly(12)-Gly(13) forming a hinge. The hydrophobic residues in the N- and C-terminal helices form a hydrophobic cluster. At neutral pH, however, the C-terminal helix of Trp(14)-Ile(18) reduces dramatically, and the hydrophobic core observed at acidic pH is severely disrupted. We suggest that the disruption of the C-terminal helix renders the E5 analog fusion-inactive at neutral pH. Furthermore, the decrease of the hinge and the reduction of fusion activity in G13L reveal the importance of the hinge in fusion activity. Also, the decrease in the C-terminal helix and the reduction of fusion activity in L17A demonstrates the importance of the C-terminal helix in fusion activity. Based on these biophysical studies, we propose a model that illustrates the structural change of the HA2 fusion peptide analog and explains how the analog interacts with the lipid bilayer at different pH values.     

Structural studies on membrane-embedded influenza hemagglutinin and its fragments.

The mechanism of influenza virus hemagglutinin (HA)-mediated membrane fusion has been inferred in part from studies examining pH-induced structural changes in soluble HA derivatives lacking the viral membrane anchor and, sometimes, the fusion peptide (the C- and N-terminal residues of the HA2 chain, respectively). To reconcile structure-based mechanisms of HA-mediated membrane fusion with structural implications of functional studies performed on membrane-embedded HA, we have undertaken attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic analyses of membrane-embedded HA (strain X:31) and its fragments reconstituted into supported lipid bilayers. The fragments correspond to proteolytic products with the majority of the HA1 chain and, in some cases, the fusion peptide removed (THA2 and THA2F-, respectively). In combination with R18 fluorescence dequenching to monitor the functional implications of HA1 subunit removal, we have assessed the influence of pH and target membrane presentation on the secondary structures, orientations relative to the membrane, and dynamics of these molecules. We find that X:31 HA is more tilted towards the plane of the membrane under fusion than under resting conditions, that the fitting of HA depends on the presence of the HA1 chain, that the residues connecting the membrane-inserted fusion peptide with the crystallographically determined coiled coil probably adopt an alpha-helical conformation, and that several changes in the secondary structure and the amide H/D exchange kinetics occur as a result of acidification and target membrane presentation, which can be interpreted as small changes and a release of strain in the static and dynamic structure of membrane-bound HA. THA2 mediatcs fusion, but less efficiently and with less pH-selectivity than HA.     

Thermodynamics of fusion peptide-membrane interactions

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.     

The Interaction between Influenza HA Fusion Peptide and Transmembrane Domain Affects Membrane Structure

Viral glycoproteins, such as influenza hemagglutinin (HA) and human immunodeficiency virus gp41, are anchored by a single helical segment transmembrane domain (TMD) on the viral envelope membrane. The fusion peptides (FP) of the glycoproteins insert into the host membrane and initiate membrane fusion. Our previous study showed that the FP or TMD alone perturbs membrane structure. Interaction between the influenza HA FP and TMD has previously been shown, but its role is unclear. We used PC spin labels dipalmitoylphospatidyl-tempo-choline (on the headgroup), 5PC and 14PC (5-C and 14-C positions on the acyl chain) to detect the combined effect of FP-TMD interaction by titrating HA FP to TMD-reconstituted 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phospho-(1’-rac-glycerol)/cholesterol lipid bilayers using electron spin resonance. We found that the FP-TMD increases the lipid order at all positions, which has a greater lipid ordering effect than the sum of the FP or TMD alone, and this effect reaches deeper into the membranes. Although HA-mediated membrane fusion is pH dependent, this combined effect is observed at both pH 5 and pH 7. In addition to increasing lipid order, multiple components are found for 5PC at increased concentration of FP-TMD, indicating that distinct domains are induced. However, the mutation of Gly1 in the FP and L187 in the TMD eliminates the perturbations, consistent with their fusogenic phenotypes. Electron spin resonance on spin-labeled peptides confirms these observations. We suggest that this interaction may provide a driving force in different stages of membrane fusion: initialization, transition from hemifusion stalk to transmembrane contact, and fusion pore formation.     

Meta-stability of the hemifusion intermediate induced by glycosylphosphatidylinositol-anchored influenza hemagglutinin.

Fusion between influenza virus and target membranes is mediated by the viral glycoprotein hemagglutinin (HA). Replacement of the transmembrane domain of HA with a glycosylphosphatidylinositol (GPI) membrane anchor allows lipid mixing but not the establishment of cytoplasmic continuity. This observation led to the proposal that the fusion mechanism passes through an intermediate stage corresponding to hemifusion between outer monolayers. We have used confocal fluorescence microscopy to study the movement of probes for specific bilayer leaflets of erythrocytes fusing with HA-expressing cells. N-Rh-PE and NBD-PC were used for specific labeling of the outer and inner membrane leaflet, respectively. In the case of GPI-HA-induced fusion, different behaviors of lipid transfer were observed, which include 1) exclusive movement of N-Rh-PE (hemifusion), 2) preferential movement of N-Rh-PE relative to NBD-PC, and 3) equal movement of both lipid analogs. The relative population of these intermediate states was dependent on the time after application of a low pH trigger for fusion. At early time points, hemifusion was more common and full redistribution of both bilayers was rare, whereas later full redistribution of both probes was frequently observed. In contrast to wild-type HA, the latter was not accompanied by mixing of the cytoplasmic marker Lucifer Yellow. We conclude that 1) the GPI-HA-mediated hemifusion intermediate is meta-stable and 2) expansion of an aqueous fusion pore requires the transmembrane and/or cytoplasmic domain of HA.     

pH-induced conformational changes of membrane-bound influenza hemagglutinin and its effect on target lipid bilayers.

Influenza virus hemagglutinin (HA) has served as a paradigm for both pH-dependent and -independent viral membrane fusion. Although large conformational changes were observed by X-ray crystallography when soluble fragments of HA were subjected to fusion-pH conditions, it is not clear whether the same changes occur in membrane-bound HA, what the spatial relationship is between the conformationally changed HA and the target and viral membranes, and in what way HA perturbs the target membrane at low pH. We have taken a spectroscopic approach using an array of recently developed FTIR techniques to address these questions. Difference attenuated total reflection FTIR spectroscopy was employed to reveal reversible and irreversible components of the pH-induced conformational change of the membrane-bound bromelain fragment of HA, BHA. Additional proteolytic fragments of BHA were produced which permitted a tentative assignment of the observed changes to the HA1 and HA2 subunits, respectively. The membrane-bound HA1 subunit undergoes a reversible conformational change, which most likely involves the loss of a small proportion of beta-sheet at low pH. BHA was found to undergo a partially reversible tilting motion relative to the target membrane upon exposure to pH 5, indicating a previously undescribed hinge near the anchoring point to the target membrane. Time-resolved amide H/D exchange experiments revealed a more dynamic (tertiary) structure of membrane-bound BHA and its HA2, but not its HA1, subunit. Finally BHA and, to a lesser degree, HA1 perturbed the lipid bilayer of the target membrane at the interface, as assessed by spectral changes of the lipid ester carbonyl groups. These results are discussed in the context of a complementary study of HA that was bound to viral membranes through its transmembrane peptide (Gray C, Tamm LK, 1997, Protein Sci 6:1993-2006). A distinctive role for the HA1 subunit in the conformational change of HA becomes apparent from these combined studies.     

Lipid interactions of the hemagglutinin HA2 NH2-terminal segment during influenza virus-induced membrane fusion.

Fusion of influenza viruses with target membranes is induced by acid and involves complex changes in the viral fusion protein hemagglutinin (HA) and in the contact sites between viruses and target membranes (Stegmann, T., White, J. M., and Helenius, A. (1990) EMBO J. 9, 4231-4241). At 0 degrees C, in a first, kinetically distinct step, target membranes irreversibly adhere to the viruses. Fusion itself starts only after a lag-phase of several minutes (X-31 strain viruses) or after raising the temperature (PR8/34 strain viruses). We now provide evidence that the initial conformational change resulting in virus-target membrane adhesion is restricted to a (minor) subpopulation of the HA molecules. These molecules become susceptible to bromelain digestion, and they could be labeled with the photoactivatable reagent [3H]PTPC/11, a nonexchangeable lipid present in the target lipid bilayer (Harter, C., B?chi, T., Semenza, G., and Brunner, J. (1988) Biochemistry 27, 1856-1864). Only the HA2 subunit was labeled, and analyses of 2-nitro-5-thio-cyanobenzoic acid fragments derived thereof indicate that the HA2 NH2-terminal segment (fusion peptide) inserted into the target membrane bilayer. When the temperature was raised to trigger fusion of PR8/34 viruses, labeling of HA2 increased by a factor of 130. Most (74%) of that label was incorporated into the COOH-terminal membrane anchor region, but there was also a strong increase (about 30-fold) of NH2-terminal fusion peptide labeling. This suggests that fusion is preceded., or accompanied, by further changes in HA which lead to additional extensive lipid insertions of HA2 fusion peptides.     

Membrane structures of the hemifusion-inducing fusion peptide mutant G1S and the fusion-blocking mutant G1V of influenza virus hemagglutinin suggest a mechanism for pore opening in membrane fusion

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 beta-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.     

Membrane perturbation and fusion pore formation in influenza hemagglutinin-mediated membrane fusion. A new model for fusion

Low pH-induced fusion mediated by the hemagglutinin (HA) of influenza virus involves conformational changes in the protein that lead to the insertion of a "fusion peptide" domain of this protein into the target membrane and is thought to perturb the membrane, triggering fusion. By using whole virus, purified HA, or HA ectodomains, we found that shortly after insertion, pores of less than 26 A in diameter were formed in liposomal membranes. As measured by a novel assay, these pores stay open, or continue to close and open, for minutes to hours and persist after pH neutralization. With virus and purified HA, larger pores, allowing the leakage of dextrans, were seen at times well after insertion. For virus, dextran leakage was simultaneous with lipid mixing and the formation of "fusion pores," allowing the transfer of dextrans from the liposomal to the viral interior or vice versa. Pores did not form in the viral membrane in the absence of a target membrane. Based on these data, we propose a new model for fusion, in which HA initially forms a proteinaceous pore in the target, but not in the viral membrane, before a lipidic hemifusion intermediate is formed.     

Fusion-competent state induced by a C-terminal HIV-1 fusion peptide in cholesterol-rich membranes

The replicative cycle of the human immunodeficiency virus type-1 begins after fusion of the viral and target-cell membranes. The envelope glycoprotein gp41 transmembrane subunit contains conserved hydrophobic domains that engage and perturb the merging lipid bilayers. In this work, we have characterized the fusion-committed state generated in vesicles by CpreTM, a synthetic peptide derived from the sequence connecting the membrane-proximal external region (MPER) and the transmembrane domain (TMD) of gp41. Pre-loading cholesterol-rich vesicles with CpreTM rendered them competent for subsequent lipid-mixing with fluorescently-labeled target vesicles. Highlighting the physiological relevance of the lasting fusion-competent state, the broadly neutralizing antibody 4E10 bound to the CpreTM-primed vesicles and inhibited lipid-mixing. Heterotypic fusion assays disclosed dependence on the lipid composition of the vesicles that acted either as virus or cell membrane surrogates. Lipid-mixing exhibited above all a critical dependence on the cholesterol content in those experiments. We infer that the fusion-competent state described herein resembles bona-fide perturbations generated by the pre-hairpin MPER–TMD connection within the viral membrane.     

Peptide models for the membrane destabilizing actions of viral fusion proteins.

The fusion of enveloped viruses to target membranes is promoted by certain viral fusion proteins. However, many other proteins and peptides stabilize bilayer membranes and inhibit membrane fusion. We have evaluated some characteristics of the interaction of peptides that are models of segments of measles and influenza fusion proteins with membranes. Our results indicate that these models of the fusogenic domains of viral fusion proteins promote conversion of model membrane bilayers to nonbilayer phases. This is opposite to the effects of peptides and proteins that inhibit viral fusion. A peptide model for the fusion segment of the HA protein of influenza increased membrane leakage as well as promoted the formation of nonbilayer phases upon acidification from pH 7-5. We analyze the gross conformational features of the peptides, and speculate on how these conformational features relate to the structures of the intact proteins and to their role in promoting membrane fusion.     

Structure and orientation of pardaxin determined by NMR experiments in model membranes

Pardaxins are a class of ichthyotoxic peptides isolated from fish mucous glands. Pardaxins physically interact with cell membranes by forming pores or voltage-gated ion channels that disrupt cellular functions. Here we report the high-resolution structure of synthetic pardaxin Pa4 in sodium dodecylphosphocholine micelles, as determined by (1)H solution NMR spectroscopy. The peptide adopts a bend-helix-bend-helix motif with an angle between the two structure helices of 122 +/- 9 degrees , making this structure substantially different from the one previously determined in organic solvents. In addition, paramagnetic solution NMR experiments on Pa4 in micelles reveal that except for the C terminus, the peptide is not solvent-exposed. These results are complemented by solid-state NMR experiments on Pa4 in lipid bilayers. In particular, (13)C-(15)N rotational echo double-resonance experiments in multilamellar vesicles support the helical conformation of the C-terminal segment, whereas (2)H NMR experiments show that the peptide induces considerable disorder in both the head-groups and the hydrophobic core of the bilayers. These solid-state NMR studies indicate that the C-terminal helix has a transmembrane orientation in DMPC bilayers, whereas in POPC bilayers, this domain is heterogeneously oriented on the lipid surface and undergoes slow motion on the NMR time scale. These new data help explain how the non-covalent interactions of Pa4 with lipid membranes induce a stable secondary structure and provide an atomic view of the membrane insertion process of Pa4.     

The transmembrane homotrimer of ADAM 1 in model lipid bilayers

Fertilin is a transmembrane protein heterodimer formed by the two subunits fertilin alpha and fertilin beta that plays an important role in sperm-egg fusion. Fertilin alpha and beta are members of the ADAM family, and contain each one transmembrane alpha-helix, and are termed ADAM 1 and ADAM 2, respectively. ADAM 1 is the subunit that contains a putative fusion peptide, and we have explored the possibility that the transmembrane alpha-helical domain of ADAM 1 forms homotrimers, in common with other viral fusion proteins. Although this peptide was found to form various homooligomers in SDS, the infrared dichroic data obtained with the isotopically labeled peptide at specific positions is consistent with the presence of only one species in DMPC or POPC lipid bilayers. Comparison of the experimental orientational data with molecular dynamics simulations performed with sequence homologues of ADAM 1 show that the species present in lipid bilayers is only consistent with an evolutionarily conserved homotrimeric model for which we provide a backbone structure. These results support a model where ADAM 1 forms homotrimers as a step to create a fusion active intermediate.     

Combined NMR and EPR spectroscopy to determine structures of viral fusion domains in membranes

Methods are described to determine the structures of viral membrane fusion domains in detergent micelles by NMR and in lipid bilayers by site-directed spin labeling and EPR spectroscopy. Since in favorable cases, the lower-resolution spin label data obtained in lipid bilayers fully support the higher-resolution structures obtained by solution NMR, it is possible to graft the NMR structural coordinates into membranes using the EPR-derived distance restraints to the lipid bilayer. Electron paramagnetic dynamics and distance measurements in bilayers support conclusions drawn from NMR in detergent micelles. When these methods are applied to a structure determination of the influenza virus fusion domain and four point mutations with different functional phenotypes, it is evident that a fixed-angle boomerang structure with a glycine edge on the outside of the N-terminal arm is both necessary and sufficient to support membrane fusion. The human immunodeficiency virus fusion domain forms a straight helix with a flexible C-terminus. While EPR data for this fusion domain are not yet available, it is tentatively speculated that, because of its higher hydrophobicity, a critically tilted insertion may occur even in the absence of a kinked boomerang structure in this case.