Novel requirements for HAP2/GCS1-mediated gamete fusion in Tetrahymena.

The ancestral gamete fusion protein, HAP2/GCS1, plays an essential role in fertilization in a broad range of taxa. To identify factors that may regulate HAP2/GCS1 activity, we screened mutants of the ciliate Tetrahymena thermophila for behaviors that mimic Δhap2/gcs1 knockout phenotypes in this species. Using this approach, we identified two new genes, GFU1 and GFU2, whose products are necessary for membrane pore formation following mating type recognition and adherence. GFU2 is predicted to be a single-pass transmembrane protein, while GFU1, though lacking obvious transmembrane domains, has the potential to interact directly with membrane phospholipids in the cytoplasm. Like Tetrahymena HAP2/GCS1, expression of GFU1 is required in both cells of a mating pair for efficient fusion to occur. To explain these bilateral requirements, we propose a model that invokes cooperativity between the fusion machinery on apposed membranes of mating cells and accounts for successful fertilization in Tetrahymena's multiple mating type system.

Thermal Degradation of Thaumatin at Low pH and Its Prevention Using Alkyl Gallates.

Thaumatin, a potent sweet tasting protein extracted from the Katemfe Plant, is emerging as a natural alternative to synthetic non-nutritive sweeteners and flavor enhancer. As a food additive, its stability within the food matrix during thermal processing is of great interest to the food industry. When heated under neutral or basic conditions, thaumatin was found to lose its sweetness due to protein aggregation caused by sulfhydryl catalyzed disulfide bond interchange. At lower pH, while thaumatin was also found to lose sweetness after heating, it does so at a slower rate and shows more resistance to sweetness loss. SDS-PAGE indicated that thaumatin fragmented into multiple smaller pieces under heating in acidic pH. Using BEMPO-3, a lipophilic spin trap, we were able to detect the presence of a free-radical within the hydrophobic region of the protein during heating. Protein carbonyl content, a byproduct of protein oxidation, also increased upon heating, providing additional evidence for protein cleavage by a radical pathway. Hexyl gallate successfully inhibited the radical generation as well as protein carbonyl formation of thaumatin during heating.

Membrane Binding Induces Distinct Structural Signatures in the Mouse Complexin-1C-Terminal Domain.

Complexins play a critical role in regulating SNARE-mediated exocytosis of synaptic vesicles. Evolutionary divergences in complexin function have complicated our understanding of the role these proteins play in inhibiting the spontaneous fusion of vesicles. Previous structural and functional characterizations of worm and mouse complexins have indicated the membrane curvature-sensing C-terminal domain of these proteins is responsible for differences in inhibitory function. We have characterized the structure and dynamics of the mCpx1 CTD in the absence and presence of membranes and membrane mimetics using NMR, ESR, and optical spectroscopies. In the absence of lipids, the mCpx1 CTD features a short helix near its N-terminus and is otherwise disordered. In the presence of micelles and small unilamellar vesicles, the mCpx1 CTD forms a discontinuous helical structure in its C-terminal 20 amino acids, with no preference for specific lipid compositions. In contrast, the mCpx1 CTD shows distinct compositional preferences in its interactions with large unilamellar vesicles. These studies identify structural divergences in the mCpx1 CTD relative to the wCpx1 CTD in regions that are known to be critical to the wCpx1 CTD's role in inhibiting spontaneous fusion of synaptic vesicles, suggesting a potential structural basis for evolutionary divergences in complexin function.1.

Negatively charged residues in the membrane ordering activity of SARS-CoV-1 and -2 fusion peptides.

Entry of coronaviruses into host cells is mediated by the viral spike protein. Previously, we identified the bona fide fusion peptides (FPs) for severe acute respiratory syndrome coronavirus ("SARS-1") and severe acute respiratory syndrome coronavirus-2 ("SARS-2") using electron spin resonance spectroscopy. We also found that their FPs induce membrane ordering in a Ca2+-dependent fashion. Here we study which negatively charged residues in SARS-1 FP are involved in this binding, to build a topological model and clarify the role of Ca2+. Our systematic mutation study on the SARS-1 FP shows that all six negatively charged residues contribute to the FP's membrane ordering activity, with D812 the dominant residue. The corresponding SARS-2 residue D830 plays an equivalent role. We provide a topological model of how the FP binds Ca2+ ions: its two segments FP1 and FP2 each bind one Ca2+. The binding of Ca2+, the folding of FP (both studied by isothermal titration calorimetry experiments), and the ordering activity correlate very well across the mutants, suggesting that the Ca2+ helps the folding of FP in membranes to enhance the ordering activity. Using a novel pseudotyped viral particle-liposome methodology, we monitored the membrane ordering induced by the FPs in the whole spike protein in its trimer form in real time. We found that the SARS-1 and SARS-2 pseudotyped viral particles also induce membrane ordering to the extent that separate FPs do, and mutations of the negatively charged residues also significantly suppress the membrane ordering activity. However, the slower kinetics of the FP ordering activity versus that of the pseudotyped viral particle suggest the need for initial trimerization of the FPs.

Critical Negatively Charged Residues Are Important for the Activity of SARS-CoV-1 and SARS-CoV-2 Fusion Peptides.

Coronaviruses are a major infectious disease threat, and include the human pathogens of zoonotic origin SARS-CoV ("SARS-1"), SARS-CoV-2 ("SARS-2") and MERS-CoV ("MERS"). Entry of coronaviruses into host cells is mediated by the viral spike (S) protein. Previously, we identified that the domain immediately downstream of the S2' cleavage site is the bona fide FP (amino acids 798-835) for SARS-1 using ESR spectroscopy technology. We also found that the SARS-1 FP induces membrane ordering in a Ca 2+ dependent fashion. In this study, we want to know which residues are involved in this Ca 2+ binding, to build a topological model and to understand the role of the Ca2+. We performed a systematic mutation study on the negatively charged residues on the SARS-1 FP. While all six negatively charged residues contributes to the membrane ordering activity of the FP to some extent, D812 is the most important residue. We provided a topological model of how the FP binds Ca 2+ ions: both FP1 and FP2 bind one Ca 2+ ion, and there are two binding sites in FP1 and three in FP2. We also found that the corresponding residue D830 in the SARS-2 FP plays a similar critical role. ITC experiments show that the binding energies between the FP and Ca 2+ as well as between the FP and membranes also decreases for all mutants. The binding of Ca 2+ , the folding of FP and the ordering activity correlated very well across the mutants, suggesting that the function of the Ca 2+ is to help to folding of FP in membranes to enhance its activity. Using a novel pseudotyped virus particle (PP)-liposome methodology, we monitored the membrane ordering induced by the FPs in the whole S proteins in its trimer form in real time. We found that the SARS-1 and SARS-2 PPs also induce membrane ordering as the separate FPs do, and the mutations of the negatively charged residues also greatly reduce the membrane ordering activity. However, the difference in kinetic between the PP and FP indicates a possible role of FP trimerization. This finding could lead to therapeutic solutions that either target the FP-calcium interaction or block the Ca 2+ channel to combat the ongoing COVID-19 pandemic.

SARS-CoV-2 Fusion Peptide has a Greater Membrane Perturbating Effect than SARS-CoV with Highly Specific Dependence on Ca2.

Coronaviruses are a major infectious disease threat, and include the zoonotic-origin human pathogens SARS-CoV-2, SARS-CoV, and MERS-CoV (SARS-2, SARS-1, and MERS). Entry of coronaviruses into host cells is mediated by the spike (S) protein. In our previous ESR studies, the local membrane ordering effect of the fusion peptide (FP) of various viral glycoproteins including the S of SARS-1 and MERS has been consistently observed. We previously determined that the sequence immediately downstream from the S2' cleavage site is the bona fide SARS-1 FP. In this study, we used sequence alignment to identify the SARS-2 FP, and studied its membrane ordering effect. Although there are only three residue differences, SARS-2 FP induces even greater membrane ordering than SARS-1 FP, possibly due to its greater hydrophobicity. This may be a reason that SARS-2 is better able to infect host cells. In addition, the membrane binding enthalpy for SARS-2 is greater. Both the membrane ordering of SARS-2 and SARS-1 FPs are dependent on Ca2+, but that of SARS-2 shows a greater response to the presence of Ca2+. Both FPs bind two Ca2+ ions as does SARS-1 FP, but the two Ca2+ binding sites of SARS-2 exhibit greater cooperativity. This Ca2+ dependence by the SARS-2 FP is very ion-specific. These results show that Ca2+ is an important regulator that interacts with the SARS-2 FP and thus plays a significant role in SARS-2 viral entry. This could lead to therapeutic solutions that either target the FP-calcium interaction or block the Ca2+ channel.

Ca2+ Ions Promote Fusion of Middle East Respiratory Syndrome Coronavirus with Host Cells and Increase Infectivity.

Fusion with, and subsequent entry into, the host cell is one of the critical steps in the life cycle of enveloped viruses. For Middle East respiratory syndrome coronavirus (MERS-CoV), the spike (S) protein is the main determinant of viral entry. Proteolytic cleavage of the S protein exposes its fusion peptide (FP), which initiates the process of membrane fusion. Previous studies on the related severe acute respiratory syndrome coronavirus (SARS-CoV) FP have shown that calcium ions (Ca2+) play an important role in fusogenic activity via a Ca2+ binding pocket with conserved glutamic acid (E) and aspartic acid (D) residues. SARS-CoV and MERS-CoV FPs share a high sequence homology, and here, we investigated whether Ca2+ is required for MERS-CoV fusion by screening a mutant array in which E and D residues in the MERS-CoV FP were substituted with neutrally charged alanines (A). Upon verifying mutant cell surface expression and proteolytic cleavage, we tested their ability to mediate pseudoparticle (PP) infection of host cells in modulating Ca2+ environments. Our results demonstrate that intracellular Ca2+ enhances MERS-CoV wild-type (WT) PP infection by approximately 2-fold and that E891 is a crucial residue for Ca2+ interaction. Subsequent electron spin resonance (ESR) experiments revealed that this enhancement could be attributed to Ca2+ increasing MERS-CoV FP fusion-relevant membrane ordering. Intriguingly, isothermal calorimetry showed an approximate 1:1 MERS-CoV FP to Ca2+ ratio, as opposed to an 1:2 SARS-CoV FP to Ca2+ ratio, suggesting significant differences in FP Ca2+ interactions of MERS-CoV and SARS-CoV FP despite their high sequence similarity.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is a major emerging infectious disease with zoonotic potential and has reservoirs in dromedary camels and bats. Since its first outbreak in 2012, the virus has repeatedly transmitted from camels to humans, with 2,468 confirmed cases causing 851 deaths. To date, there are no efficacious drugs and vaccines against MERS-CoV, increasing its potential to cause a public health emergency. In order to develop novel drugs and vaccines, it is important to understand the molecular mechanisms that enable the virus to infect host cells. Our data have found that calcium is an important regulator of viral fusion by interacting with negatively charged residues in the MERS-CoV FP region. This information can guide therapeutic solutions to block this calcium interaction and also repurpose already approved drugs for this use for a fast response to MERS-CoV outbreaks.

Calcium Ions Directly Interact with the Ebola Virus Fusion Peptide To Promote Structure-Function Changes That Enhance Infection.

Ebola virus disease is a serious global health concern given its periodic occurrence, high lethality, and the lack of approved therapeutics. Certain drugs that alter intracellular calcium, particularly in endolysosomes, have been shown to inhibit Ebola virus infection; however, the underlying mechanism is unknown. Here, we provide evidence that Zaire ebolavirus (EBOV) infection is promoted in the presence of calcium as a result of the direct interaction of calcium with the EBOV fusion peptide (FP). We identify the glycoprotein residues D522 and E540 in the FP as functionally critical to EBOV's interaction with calcium. We show using spectroscopic and biophysical assays that interactions of the fusion peptide with Ca2+ ions lead to lipid ordering in the host membrane during membrane fusion, and these changes are promoted at low pH and can be correlated with infectivity. We further demonstrate using circular dichroism spectroscopy that calcium interaction with the fusion peptide promotes α-helical structure of the fusion peptide, a conformational change that enhances membrane fusion, as validated using functional assays of membrane fusion. This study shows that calcium directly targets the Ebola virus fusion peptide and influences its conformation. As these residues are highly conserved across the Filoviridae, calcium's impact on fusion, and subsequently infectivity, is a key interaction that can be leveraged for developing strategies to defend against Ebola infection. This mechanistic insight provides a rationale for the use of calcium-interfering drugs already approved by the FDA as therapeutics against Ebola and enables further development of novel drugs to combat the virus.

The SARS-CoV Fusion Peptide Forms an Extended Bipartite Fusion Platform that Perturbs Membrane Order in a Calcium-Dependent Manner.

Coronaviruses (CoVs) are a major infectious disease threat and include the pathogenic human pathogens of zoonotic origin: severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV). Entry of CoVs into host cells is mediated by the viral spike (S) protein, which is structurally categorized as a class I viral fusion protein, within the same group as influenza virus and HIV. However, S proteins have two distinct cleavage sites that can be activated by a much wider range of proteases. The exact location of the CoV fusion peptide (FP) has been disputed. However, most evidence suggests that the domain immediately downstream of the S2' cleavage site is the FP (amino acids 798-818 SFIEDLLFNKVTLADAGFMKQY for SARS-CoV, FP1). In our previous electron spin resonance spectroscopic studies, the membrane-ordering effect of influenza virus, HIV, and Dengue virus FPs has been consistently observed. In this study, we used this effect as a criterion to identify and characterize the bona fide SARS-CoV FP. Our results indicate that both FP1 and the region immediately downstream (amino acids 816-835 KQYGECLGDINARDLICAQKF, FP2) induce significant membrane ordering. Furthermore, their effects are calcium dependent, which is consistent with in vivo data showing that calcium is required for SARS-CoV S-mediated fusion. Isothermal titration calorimetry showed a direct interaction between calcium cations and both FPs. This Ca2+-dependency membrane ordering was not observed with influenza FP, indicating that the CoV FP exhibits a mechanistically different behavior. Membrane-ordering effects are greater and penetrate deeper into membranes when FP1 and FP2 act in a concerted manner, suggesting that they form an extended fusion "platform."

Unique Structural Features of Membrane-Bound C-Terminal Domain Motifs Modulate Complexin Inhibitory Function.

Complexin is a small soluble presynaptic protein that interacts with neuronal SNARE proteins in order to regulate synaptic vesicle exocytosis. While the SNARE-binding central helix of complexin is required for both the inhibition of spontaneous fusion and the facilitation of synchronous fusion, the disordered C-terminal domain (CTD) of complexin is specifically required for its inhibitory function. The CTD of worm complexin binds to membranes via two distinct motifs, one of which undergoes a membrane curvature dependent structural transition that is required for efficient inhibition of neurotransmitter release, but the conformations of the membrane-bound motifs remain poorly characterized. Visualizing these conformations is required to clarify the mechanisms by which complexin membrane interactions regulate its function. Here, we employ optical and magnetic resonance spectroscopy to precisely define the boundaries of the two CTD membrane-binding motifs and to characterize their conformations. We show that the curvature dependent amphipathic helical motif features an irregular element of helical structure, likely a pi-bulge, and that this feature is important for complexin inhibitory function in vivo.

Key features of an Hsp70 chaperone allosteric landscape revealed by ion-mobility native mass spectrometry and double electron-electron resonance.

Proteins are dynamic entities that populate conformational ensembles, and most functions of proteins depend on their dynamic character. Allostery, in particular, relies on ligand-modulated shifts in these conformational ensembles. Hsp70s are allosteric molecular chaperones with conformational landscapes that involve large rearrangements of their two domains (viz. the nucleotide-binding domain and substrate-binding domain) in response to adenine nucleotides and substrates. However, it remains unclear how the Hsp70 conformational ensemble is populated at each point of the allosteric cycle and how ligands control these populations. We have mapped the conformational species present under different ligand-binding conditions throughout the allosteric cycle of the Escherichia coli Hsp70 DnaK by two complementary methods, ion-mobility mass spectrometry and double electron-electron resonance. Our results obtained under biologically relevant ligand-bound conditions confirm the current picture derived from NMR and crystallographic data of domain docking upon ATP binding and undocking in response to ADP and substrate. Additionally, we find that the helical lid of DnaK is a highly dynamic unit of the structure in all ligand-bound states. Importantly, we demonstrate that DnaK populates a partially docked state in the presence of ATP and substrate and that this state represents an energy minimum on the DnaK allosteric landscape. Because Hsp70s are emerging as potential drug targets for many diseases, fully mapping an allosteric landscape of a molecular chaperone like DnaK will facilitate the development of small molecules that modulate Hsp70 function via allosteric mechanisms.

Structure-Function Studies Link Class II Viral Fusogens with the Ancestral Gamete Fusion Protein HAP2.

The conserved transmembrane protein, HAP2/GCS1, has been linked to fertility in a wide range of taxa and is hypothesized to be an ancient gamete fusogen. Using template-based structural homology modeling, we now show that the ectodomain of HAP2 orthologs from Tetrahymena thermophila and other species adopt a protein fold remarkably similar to the dengue virus E glycoprotein and related class II viral fusogens. To test the functional significance of this predicted structure, we developed a flow-cytometry-based assay that measures cytosolic exchange across the conjugation junction to rapidly probe the effects of HAP2 mutations in the Tetrahymena system. Using this assay, alterations to a region in and around a predicted "fusion loop" in T. thermophila HAP2 were found to abrogate membrane pore formation in mating cells. Consistent with this, a synthetic peptide corresponding to the HAP2 fusion loop was found to interact directly with model membranes in a variety of biophysical assays. These results raise interesting questions regarding the evolutionary relationships of class II membrane fusogens and harken back to a long-held argument that eukaryotic sex arose as the byproduct of selection for the horizontal transfer of a "selfish" genetic element from cell to cell via membrane fusion.

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.

HIV gp41 fusion peptide increases membrane ordering in a cholesterol-dependent fashion.

Fusion between viral envelopes and host cell membranes, which is mediated by special glycoproteins anchored on the viral membrane, is required for HIV viral entry and infection. The HIV gp41 fusion peptide (FP), which initiates membrane fusion, adopts either an α-helical or ß-sheeted structure depending on the cholesterol concentration. We used phosphocholine spin labels on the lipid headgroup and different positions on the acyl chain to detect its perturbation on lipid bilayers containing different cholesterol concentrations by electron-spin resonance. Our findings were as follows. 1), gp41 FP affects the lipid order in the same manner as previously shown for influenza hemagglutinin FP, i.e., it has a cooperative effect versus the peptide/lipid ratio, supporting our hypothesis that membrane ordering is a common prerequisite for viral membrane fusion. 2), gp41 FP induces membrane ordering in all lipid compositions studied, whereas a nonfusion mutant FP perturbs lipid order to a significantly smaller extent. 3), In high-cholesterol-containing lipid bilayers, where gp41 FP is in the ß-aggregation conformation, its effect on the lipid ordering reaches deeper into the bilayer. The different extent to which the two conformers perturb is correlated with their fusogenicity. The possible role of the two conformers in membrane fusion is discussed.

Fusion activity of HIV gp41 fusion domain is related to its secondary structure and depth of membrane insertion in a cholesterol-dependent fashion.

The human immunodeficiency virus (HIV) gp41 fusion domain plays a critical role in membrane fusion during viral entry. A thorough understanding of the relationship between the structure and the 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.  The fusion domain formed an α-helix in membranes containing less than 30 mol% cholesterol and  formed ß-sheet secondary structure in membranes containing ≥30 mol% cholesterol. 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 form and its ß-sheet form. High cholesterol, which induced ß-sheets, promoted fusion; however, 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 that their membrane insertion is deep, α-helical and ß-sheet conformations contribute to membrane fusion.

Synaptotagmin 1 modulates lipid acyl chain order in lipid bilayers by demixing phosphatidylserine.

Synaptotagmin 1 (syt1) functions as the Ca(2+) sensor in neuronal exocytosis, and it has been proposed to act by modulating lipid bilayer curvature. Here we examine the effect of the two C2 domains (C2A and C2B) of syt1 on membrane lipid order and lateral organization. In mixtures of phosphatidylcholine and phosphatidylserine (PS), attenuated total internal reflection Fourier transform infrared spectroscopy indicates that a fragment containing both domains (C2AB) or C2B alone disorders the lipid acyl chains, whereas the C2A domain has little effect upon chain order. Two observations suggest that these changes reflect a demixing of PS. First, the changes in acyl chain order are reversed at higher protein concentration; second, selective lipid deuteration demonstrates that the changes in lipid order are associated only with the PS component of the bilayer. Independent evidence for lipid demixing is obtained from fluorescence self-quenching of labeled lipid and from natural abundance (13)C NMR, where heteronuclear single quantum correlation spectra reveal Ca(2+)-dependent chemical shift changes for PS, but not for phosphatidylcholine, in the presence of the syt1 C2 domains. The ability of syt1 to demix PS is observed in a range of lipid mixtures that includes cholesterol, phosphatidylethanolamine, and varied PS content. These data suggest that syt1 might facilitate SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors)-mediated membrane fusion by phase separating PS, a process that is expected to locally buckle bilayers and disorder lipids due to the curvature tendencies of PS.

Synaptotagmin 1 and SNAREs form a complex that is structurally heterogeneous.

Synaptotagmin 1 (syt1) functions as a Ca(2+)-sensor for neuronal exocytosis. Here, site-directed spin labeling was used to examine the complex formed between a soluble fragment of syt1, which contains its two C2 domains, and the neuronal core soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Changes in electron paramagnetic resonance lineshape and accessibility for spin-labeled syt1 mutants indicate that in solution, the assembled core SNARE complex contacts syt1 in several regions. For the C2B domain, contact occurs in the polybasic face and sites opposite the Ca(2+)-binding loops. For the C2A domain, contact is seen with the SNARE complex in a region near loop 2. Double electron-electron resonance was used to estimate distances between the two C2 domains of syt1. These distances have broad distributions in solution, which do not significantly change when syt1 is fully associated with the core SNARE complex. The broad distance distributions indicate that syt1 is structurally heterogeneous when bound to the SNAREs and does not assume a well-defined structure. Simulated annealing using electron paramagnetic resonance-derived distance restraints produces a family of syt1 structures where the Ca(2+)-binding regions of each domain face in roughly opposite directions. The results suggest that when associated with the SNAREs, syt1 is configured to bind opposing bilayers, but that the syt1/SNARE complex samples multiple conformational states.

Shallow boomerang-shaped influenza hemagglutinin G13A mutant structure promotes leaky membrane fusion.

Our previous studies showed that an angled boomerang-shaped structure of the influenza hemagglutinin (HA) fusion domain is critical for virus entry into host cells by membrane fusion. Because the acute angle of ∼105° of the wild-type fusion domain promotes efficient non-leaky membrane fusion, we asked whether different angles would still support fusion and thus facilitate virus entry. Here, we show that the G13A fusion domain mutant produces a new leaky fusion phenotype. The mutant fusion domain structure was solved by NMR spectroscopy in a lipid environment at fusion pH. The mutant adopted a boomerang structure similar to that of wild type but with a shallower kink angle of ∼150°. G13A perturbed the structure of model membranes to a lesser degree than wild type but to a greater degree than non-fusogenic fusion domain mutants. The strength of G13A binding to lipid bilayers was also intermediate between that of wild type and non-fusogenic mutants. These membrane interactions provide a clear link between structure and function of influenza fusion domains: an acute angle is required to promote clean non-leaky fusion suitable for virus entry presumably by interaction of the fusion domain with the transmembrane domain deep in the lipid bilayer. A shallower angle perturbs the bilayer of the target membrane so that it becomes leaky and unable to form a clean fusion pore. Mutants with no fixed boomerang angle interacted with bilayers weakly and did not promote any fusion or membrane perturbation.

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.

Locking the kink in the influenza hemagglutinin fusion domain structure.

We have previously identified Trp(14) as a critical residue that stabilizes the kink in the boomerang structure of the influenza fusion domain and found that cells expressing hemagglutinin with a Trp(14) to Ala mutation cannot fuse with red blood cells. However, mutating another aromatic residue, Phe(9), on the other side of the kink did not have a significant effect on fusion or the ability of the mutant fusion peptide to bind to or perturb the bilayer structure of lipid model membranes. We reasoned that Phe is not as potent to contribute to the kink as the larger Trp and that the cooperation of Phe(9) and Ile(10) might be needed to elicit the same effect. Indeed, the double mutant F9A/I10A diminished cell-cell fusion and the ability of the fusion domain to bind to and perturb lipid bilayers in a similar fashion as the W14A mutant. A structure determination of F9A in lipid micelles by solution NMR shows that F9A adopts a similarly kinked structure as wild type. Distances between the two arms of the boomerang structure of wild type, F9A, W14A, and F9A/I10A in lipid bilayers were measured by double electron-electron resonance spectroscopy and showed that the kinks of W14A and F9A/I10A are more flexible than those of wild type and F9A. These results underscore the importance of large hydrophobic residues on both sides of the kink region of the influenza hemagglutinin fusion domain to fix the angle of the boomerang structure and thereby confer fusion function to this critical domain.