The gp41 protein of the Human Immunodeficiency Virus (HIV) catalyzes fusion between HIV and host cell membranes. The ~180-residue ectodomain of gp41 is outside the virion and is the most important gp41 region for membrane fusion. The ectodomain consists of an apolar fusion peptide (FP) region followed by N-heptad repeat (NHR), loop, and C-heptad repeat (CHR) regions. The FP is critical for fusion and is hypothesized to bind to the host cell membrane. Large ectodomain constructs either with or without the FP are highly aggregated at physiologic pH but soluble in the pH 3–4 range with hyperthermostable hairpin structure with antiparallel NHR and CHR helices. The present study focuses on the large gp41 ectodomain constructs “Hairpin” (HP) containing NHR+loop+CHR and “FP-Hairpin” (FP-HP) containing FP+NHR+loop+CHR. Both proteins induce rapid and extensive fusion of anionic vesicles at pH 4 where the protein is positively-charged but do not induce fusion at pH 7 where the protein is negatively charged. This observation, along with lack of fusion of neutral vesicles at either pH supports the significance of attractive protein/membrane electrostatics in fusion. The functional role of the hydrophobic FP is supported by increases in the rate and extent of fusion for FP-HP relative to HP. There are two kinetically distinct fusion processes at pH 4: (1) a faster ~100 ms−1 process with rate strongly positively correlated with vesicle charge; and (2) a slower ~5 ms−1 process with extent strongly inversely correlated with this charge. The faster charge-dependent process is likely related to the electrostatic energy released upon initial monomer protein binding to the vesicle. After dissipation of this energy, the subsequent slower process is likely due to the equilibrium membrane-associated structure of the protein. The slower process may be more physiologically relevant because HIV/host cell fusion occurs at physiologic pH with gp41 restricted to the narrow region between the two membranes. Previous solid-state NMR (SSNMR) of membrane-associated FP-HP has supported protein oligomers with FP’s in an intermolecular antiparallel β sheet. There was an additional population of molecules with α helical FPs and the samples likely contained a mixture of membrane-bound and -unbound protein. For the present study, samples were prepared with fully membrane-bound FP-HP and subsequent SSNMR showed dominant β FP conformation at both low and neutral pH. The fusogenic structure is likely β for the slow and perhaps the fast process. SSNMR also showed close contact of the FP with the lipid headgroups at both low and neutral pH whereas the NHR+CHR regions had contact at low pH and were more distant at neutral pH, consistent with the protein/membrane electrostatics.

Influenza virus is a Class I enveloped virus which is initially endocytosed into a host respiratory epithelial cell. Subsequent reduction of the pH to the 5–6 range triggers a structural change of the viral hemagglutinin II (HA2) protein, fusion of the viral and endosomal membranes, and release of the viral nucleocapsid into the cytoplasm. HA2 contains fusion peptide (FP), soluble ectodomain (SE), transmembrane (TM), and intraviral domains with respective lengths of ~25, ~160, ~25, and ~10 residues. The present work provides a straightforward protocol for producing and purifying mg quantities of full-length HA2 from expression in bacteria. Biophysical and structural comparisons are made between full-length HA2 and shorter constructs including SHA2 ≡ SE, FHA2 ≡ FP + SE, and SHA2-TM ≡ SE + TM constructs. The constructs are helical in detergent at pH 7.4 and the dominant trimer species. The proteins are highly thermostable in decylmaltoside detergent with Tm > 90 °C for HA2 with stabilization provided by the SE, FP, and TM domains. The proteins are likely in a trimer-of-hairpins structure, the final protein state during fusion. All constructs induce fusion of negatively-charged vesicles at pH 5.0 with much less fusion at pH 7.4. Attractive protein/vesicle electrostatics play a role in fusion, as the proteins are positively-charged at pH 5.0 and negatively-charged at pH 7.4 and the pH-dependence of fusion is reversed for positively-charged vesicles. Comparison of fusion between constructs supports significant contributions to fusion from the SE and the FP with little effect from the TM.

Cellular entry of influenza virus is mediated by the viral protein hemagglutinin (HA), which forms an initial complex of three HA1 and three HA2 subunits. Each HA2 includes a fusion peptide (FP), a soluble ectodomain (SE), and a transmembrane domain. HA1 binds to cellular sialic acids, followed by virus endocytosis, pH reduction, dissociation of HA1, and structural rearrangement of HA2 into a final trimer-of-SE hairpins. A decrease in pH also triggers HA2-mediated virus/endosome membrane fusion. SE hairpins have an interior parallel helical bundle and C-terminal strands in the grooves of the exterior of the bundle. FPs are separate helical hairpins. This study compares wild-type HA2 (WT-HA2) with G1E(FP) and I173E(SE strand) mutants. WT-HA2 induces vesicle fusion at pH 5.0, whereas the extent of fusion is greatly reduced for both mutants. Circular dichroism for HA2 and FHA2≡FP+SE constructs shows dramatic losses of stability for the mutants, including a T reduced by 40 °C for I173E-FHA2. This is evidence of destabilization of SE hairpins via dissociation of strands from the helical bundle, which is also supported by larger monomer fractions for mutant versus WT proteins. The G1E mutant may have disrupted FP hairpins, with consequent non-native FP binding to dissociated SE strands. It is commonly proposed that free energy released by the HA2 structural rearrangement catalyzes HA-mediated fusion. This study supports an alternate mechanistic model in which fusion is preceded by FP insertion in the target membrane and formation of the final SE hairpin. Less fusion by the mutants is due to the loss of hairpin stability and consequent reduced level of membrane apposition of the virus and target membranes.

Human immunodeficiency virus (HIV) is membrane-enveloped, and an initial infection step is joining/fusion of viral and cell membranes. This step is catalyzed by gp41, which is a single-pass integral viral membrane protein. The protein contains an ∼170-residue ectodomain located outside the virus that is important for fusion and includes the fusion peptide (FP), N-helix, loop, C-helix, and viral membrane-proximal external region (MPER). The virion initially has noncovalent complexes between three gp41 ectodomains and three gp120 proteins. A gp120 contains ∼500 residues and functions to identify target T-cells and macrophages via binding to specific protein receptors of the target cell membrane. gp120 moves away from the gp41 ectodomain, and the ectodomain is thought to bind to the target cell membrane and mediate membrane fusion. The secondary and tertiary structures of the ectodomain are different in the initial complex with gp120 and the final state without gp120. There is not yet imaging of gp41 during fusion, so the temporal relationship between the gp41 and membrane structures is not known. This study describes biophysical and functional characterization of large gp41 constructs that include the ectodomain and transmembrane domain (TM). Significant fusion is observed of both neutral and anionic vesicles at neutral pH, which reflects the expected conditions of HIV/cell fusion. Fusion is enhanced by the FP, which in HIV/cell fusion likely contacts the host membrane, and the MPER and TM, which respectively interfacially contact and traverse the HIV membrane. Initial contact with vesicles is made by protein trimers that are in a native oligomeric state that reflects the initial complex with gp120 and also is commonly observed for the ectodomain without gp120. Circular dichroism data support helical structure for the N-helix, C-helix, and MPER and nonhelical structure for the FP and loop. Distributions of monomer, trimer, and hexamer states are observed by size-exclusion chromatography (SEC), with dependences on solubilizing detergent and construct. These SEC and other data are integrated into a refined working model of HIV/cell fusion that includes dissociation of the ectodomain into gp41 monomers followed by folding into hairpins that appose the two membranes, and subsequent fusion catalysis by trimers and hexamers of hairpins. The monomer and oligomer gp41 states may therefore satisfy dual requirements for HIV entry of membrane apposition and fusion.

The influenza virus hemagglutinin (HA) protein has HA1 and HA2 subunits, which form an initial complex. HA1's bind host cell sialic acids which triggers endocytosis, HA1/HA2 separation, and HA2-mediated fusion between virus and endosome membranes. We report hydrogen-deuterium exchange-mass spectrometry(HDX-MS) on the HA2 subunit without HA1. HA2 contains fusion peptide(FP), soluble ectodomain(SE), transmembrane domain(TM) and endodomain. FP is a monomer by itself, while SE is a trimer-of-hairpins that includes an interior bundle of residue 38-105 helices, turns, and 154-178 strands packed antiparallel to the bundle. FP and TM extend from the same side of the SE hairpin, and fusion models often depict a FP/TM complex with membrane traversal of both domains that is important for membrane pore expansion. The HDX-MS data of the present study do not support this complex, and instead support independent FP and TM with respective membrane interfacial and traversal locations. The data also show low aqueous exposure of the 22-38 segment, consistent with retention of the 23-35 antiparallel β sheet observed in the initial HA1/HA2 complex. We propose the β sheet as a semi-rigid connector between FP and SE that enables close membrane apposition prior to fusion. The I173E mutant exhibits greater exchange for 22-69 and 150-191, consistent with dissociation of SE C-terminal strands from interior N-helices. Similar trends are observed for G1E mutant, as well as lower exchange for G1E FP. Fusion is highly-impaired with either mutant, which correlates with reduced membrane apposition, and for G1E, FP binding to SE rather than target membrane.