Title: Role of Electrostatic Repulsion in Controlling pH-Dependent Conformational Changes of Viral Fusion Proteins
Abstract: •Viral fusion proteins undergo pH-dependent conformational changes•Electrostatic repulsion between side chains mediates structural transitions•Histidine-cation (HisCat) and anion-anion (AniAni) interactions are common•Ability of the virus to sense pH changes as it enters the cell plays an important role during the infection Viral fusion proteins undergo dramatic conformational transitions during membrane fusion. For viruses that enter through the endosome, these conformational rearrangements are typically pH sensitive. Here, we provide a comprehensive review of the molecular interactions that govern pH-dependent rearrangements and introduce a paradigm for electrostatic residue pairings that regulate progress through the viral fusion coordinate. Analysis of structural data demonstrates a significant role for side-chain protonation in triggering conformational change. To characterize this behavior, we identify two distinct residue pairings, which we define as Histidine-Cation (HisCat) and Anion-Anion (AniAni) interactions. These side-chain pairings destabilize a particular conformation via electrostatic repulsion through side-chain protonation. Furthermore, two energetic control mechanisms, thermodynamic and kinetic, regulate these structural transitions. This review expands on the current literature by identification of these residue clusters, discussion of data demonstrating their function, and speculation of how these residue pairings contribute to the energetic controls. Viral fusion proteins undergo dramatic conformational transitions during membrane fusion. For viruses that enter through the endosome, these conformational rearrangements are typically pH sensitive. Here, we provide a comprehensive review of the molecular interactions that govern pH-dependent rearrangements and introduce a paradigm for electrostatic residue pairings that regulate progress through the viral fusion coordinate. Analysis of structural data demonstrates a significant role for side-chain protonation in triggering conformational change. To characterize this behavior, we identify two distinct residue pairings, which we define as Histidine-Cation (HisCat) and Anion-Anion (AniAni) interactions. These side-chain pairings destabilize a particular conformation via electrostatic repulsion through side-chain protonation. Furthermore, two energetic control mechanisms, thermodynamic and kinetic, regulate these structural transitions. This review expands on the current literature by identification of these residue clusters, discussion of data demonstrating their function, and speculation of how these residue pairings contribute to the energetic controls. Both cells and enveloped viruses are surrounded by phospholipid bilayers that act as physical barriers between the cellular and viral genomes. Viruses have evolved efficient mechanisms to circumvent this barrier by fusing their membrane with that of the host (Kielian and Rey, 2006Kielian M. Rey F.A. Virus membrane-fusion proteins: more than one way to make a hairpin.Nat. Rev. Microbiol. 2006; 4: 67-76Crossref PubMed Scopus (442) Google Scholar; Weissenhorn et al., 2007Weissenhorn W. Hinz A. Gaudin Y. Virus membrane fusion.FEBS Lett. 2007; 581: 2150-2155Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar; Harrison, 2008Harrison S.C. Viral membrane fusion.Nat. Struct. Mol. Biol. 2008; 15: 690-698Crossref PubMed Scopus (934) Google Scholar; White et al., 2008White J.M. Delos S.E. Brecher M. Schornberg K. Structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme.Crit. Rev. Biochem. Mol. 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This energetic barrier is overcome by glycoproteins embedded in the viral envelope. These proteins generally adopt at least three distinct conformational states during the membrane fusion process: (1) the prefusion state, (2) the extended intermediate state, and (3) the postfusion state (Figure 1) (Chernomordik and Kozlov, 2008Chernomordik L.V. Kozlov M.M. Mechanics of membrane fusion.Nat. Struct. Mol. Biol. 2008; 15: 675-683Crossref PubMed Scopus (729) Google Scholar; Harrison, 2008Harrison S.C. Viral membrane fusion.Nat. Struct. Mol. Biol. 2008; 15: 690-698Crossref PubMed Scopus (934) Google Scholar; Kielian and Rey, 2006Kielian M. Rey F.A. Virus membrane-fusion proteins: more than one way to make a hairpin.Nat. Rev. Microbiol. 2006; 4: 67-76Crossref PubMed Scopus (442) Google Scholar; White et al., 2008White J.M. Delos S.E. Brecher M. Schornberg K. Structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme.Crit. Rev. Biochem. Mol. 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Horvat B. et al.A general strategy to endow natural fusion-protein-derived peptides with potent antiviral activity.PLoS One. 2012; 7: e36833Crossref PubMed Scopus (59) Google Scholar). Two recent reports have provided direct information about the extended intermediates in paramyxoviruses (Kim et al., 2011Kim Y.H. Donald J.E. Grigoryan G. Leser G.P. Fadeev A.Y. Lamb R.A. DeGrado W.F. Capture and imaging of a prehairpin fusion intermediate of the paramyxovirus PIV5.Proc. Natl. Acad. Sci. USA. 2011; 108: 20992-20997Crossref PubMed Scopus (49) Google Scholar) and avian sarcoma leukosis virus (ASLV) (Cardone et al., 2012Cardone G. Brecher M. Fontana J. Winkler D.C. Butan C. White J.M. Steven A.C. Visualization of the two-step fusion process of the retrovirus avian sarcoma/leukosis virus by cryo-electron tomography.J. Virol. 2012; 86: 12129-12137Crossref PubMed Scopus (19) Google Scholar; Matsuyama et al., 2004Matsuyama S. Delos S.E. White J.M. Sequential roles of receptor binding and low pH in forming prehairpin and hairpin conformations of a retroviral envelope glycoprotein.J. Virol. 2004; 78: 8201-8209Crossref PubMed Scopus (51) Google Scholar). At present, no extended intermediate conformation from any virus has been characterized in high resolution. Many viruses enter the cell through the endocytic pathway where vesicle acidification triggers progression through the viral fusion cascade (Lozach et al., 2011Lozach P.Y. Huotari J. Helenius A. Late-penetrating viruses.Curr. Opin. Virol. 2011; 1: 35-43Crossref PubMed Scopus (85) Google Scholar; Mercer et al., 2010Mercer J. Schelhaas M. Helenius A. Virus entry by endocytosis.Annu. Rev. Biochem. 2010; 79: 803-833Crossref PubMed Scopus (721) Google Scholar). Thus, the viral envelope proteins function as pH sensors, sensing the pH decrease, to approximately pH 5, which is encountered as the endocytic vesicles mature (Huotari and Helenius, 2011Huotari J. Helenius A. Endosome maturation.EMBO J. 2011; 30: 3481-3500Crossref PubMed Scopus (1539) Google Scholar). From a chemical perspective, differential side-chain protonation likely triggers the conformational rearrangements. Of the functional groups in canonical proteins, only three amino acid side chains (Asp, Glu, and His) titrate in the necessary pH range to function as candidate sensors. Indeed, numerous structural studies have demonstrated the critical role of histidines in these conformational changes (Boo et al., 2012Boo I. teWierik K. Douam F. Lavillette D. Poumbourios P. Drummer H.E. Distinct roles in folding, CD81 receptor binding and viral entry for conserved histidine residues of hepatitis C virus glycoprotein E1 and E2.Biochem. J. 2012; 443: 85-94Crossref PubMed Scopus (38) Google Scholar; Carneiro et al., 2003Carneiro F.A. Stauffer F. Lima C.S. Juliano M.A. Juliano L. Da Poian A.T. Membrane fusion induced by vesicular stomatitis virus depends on histidine protonation.J. Biol. Chem. 2003; 278: 13789-13794Crossref PubMed Scopus (71) Google Scholar, Carneiro et al., 2006Carneiro F.A. Vandenbussche G. Juliano M.A. Juliano L. Ruysschaert J.M. Da Poian A.T. Charged residues are involved in membrane fusion mediated by a hydrophilic peptide located in vesicular stomatitis virus G protein.Mol. Membr. Biol. 2006; 23: 396-406Crossref PubMed Scopus (9) Google Scholar; Chanel-Vos and Kielian, 2004Chanel-Vos C. Kielian M. A conserved histidine in the ij loop of the Semliki Forest virus E1 protein plays an important role in membrane fusion.J. Virol. 2004; 78: 13543-13552Crossref PubMed Scopus (55) Google Scholar; Huang et al., 2002Huang Q. Opitz R. Knapp E.W. Herrmann A. Protonation and stability of the globular domain of influenza virus hemagglutinin.Biophys. J. 2002; 82: 1050-1058Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar; Kampmann et al., 2006Kampmann T. Mueller D.S. Mark A.E. Young P.R. Kobe B. The Role of histidine residues in low-pH-mediated viral membrane fusion.Structure. 2006; 14: 1481-1487Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar; Liu and Kielian, 2009Liu C.Y. Kielian M. E1 mutants identify a critical region in the trimer interface of the Semliki forest virus fusion protein.J. Virol. 2009; 83: 11298-11306Crossref PubMed Scopus (22) Google Scholar; Mueller et al., 2008Mueller D.S. Kampmann T. Yennamalli R. Young P.R. Kobe B. Mark A.E. Histidine protonation and the activation of viral fusion proteins.Biochem. Soc. Trans. 2008; 36: 43-45Crossref PubMed Scopus (52) Google Scholar; Qin et al., 2009Qin Z.L. Zheng Y. Kielian M. Role of conserved histidine residues in the low-pH dependence of the Semliki Forest virus fusion protein.J. Virol. 2009; 83: 4670-4677Crossref PubMed Scopus (60) Google Scholar; Schowalter et al., 2009Schowalter R.M. Chang A. Robach J.G. Buchholz U.J. Dutch R.E. Low-pH triggering of human metapneumovirus fusion: essential residues and importance in entry.J. Virol. 2009; 83: 1511-1522Crossref PubMed Scopus (69) Google Scholar; Stauffer et al., 2007Stauffer F. De Miranda J. Schechter M.C. Carneiro F.A. Salgado L.T. Machado G.F. Da Poian A.T. Inactivation of vesicular stomatitis virus through inhibition of membrane fusion by chemical modification of the viral glycoprotein.Antiviral Res. 2007; 73: 31-39Crossref PubMed Scopus (11) Google Scholar), whereas the role of anionic side chains has only recently been elucidated (Chang et al., 2012Chang A. Hackett B. Winter C.C. Buchholz U.J. Dutch R.E. Potential electrostatic interactions in multiple regions affect HMPV F-mediated membrane fusion.J. Virol. 2012; 86: 9843-9853Crossref PubMed Scopus (15) Google Scholar; Harrison et al., 2011Harrison J.S. Higgins C.D. Chandran K. Lai J.R. Designed protein mimics of the Ebola virus glycoprotein GP2 α-helical bundle: stability and pH effects.Protein Sci. 2011; 20: 1587-1596Crossref PubMed Scopus (38) Google Scholar, Harrison et al., 2012Harrison J.S. Koellhoffer J.F. Chandran K. Lai J.R. Marburg virus glycoprotein GP2: pH-dependent stability of the ectodomain α-helical bundle.Biochemistry. 2012; 51: 2515-2525Crossref PubMed Scopus (31) Google Scholar; Liu and Kielian, 2009Liu C.Y. Kielian M. E1 mutants identify a critical region in the trimer interface of the Semliki forest virus fusion protein.J. Virol. 2009; 83: 11298-11306Crossref PubMed Scopus (22) Google Scholar). Therefore, electrostatic changes provide some of the forces behind these conformational changes. Note that in contrast, the hydrophobic effect—not electrostatics—is the dominant stabilizing force for protein folding (Baldwin, 2007Baldwin R.L. Energetics of protein folding.J. Mol. Biol. 2007; 371: 283-301Crossref PubMed Scopus (226) Google Scholar; Dill, 1990Dill K.A. Dominant forces in protein folding.Biochemistry. 1990; 29: 7133-7155Crossref PubMed Scopus (3341) Google Scholar). Mutational data from many pH-dependent viral fusion systems have implicated two discrete residue pairings as potential pH-sensitive elements. These residue pairs function by destabilizing a conformation at a particular pH through electrostatic repulsion. Undoubtedly, other intermolecular interactions can contribute to pH-sensitive conformational rearrangements, such as hydrogen bonds and salt bridges, but neither of these forces can provide a destabilizing force like repulsion. Moreover, the concept of repulsion influencing pH-dependent rearrangements provides a plausible explanation for free energy changes between the prefusion and postfusion conformation, discussed later. The first pair, histidine-cation (HisCat), consists of an interaction between a histidine residue and another cationic residue: Lys, Arg, or His (Figure 2). The pKa of free histidine is 6.5; however, in the context of a folded protein’s microenvironment, this value is commonly altered. As the pH decreases, the imidazole ring accepts a proton rendering this residue cationic (Figure 2). In HisCat pairs, histidine residues are found in close proximity, usually less then 7 Å, to another His or a basic residue (Arg or Lys). When the His is protonated, these clusters acquire cationic charge and repel, destabilizing the prefusion state, contributing to the formation of the extended intermediate. Recently, a HisCat interaction that is critical for pH triggering in human metapneumovirus was dissected with mutagenesis (Chang et al., 2012Chang A. Hackett B. Winter C.C. Buchholz U.J. Dutch R.E. Potential electrostatic interactions in multiple regions affect HMPV F-mediated membrane fusion.J. Virol. 2012; 86: 9843-9853Crossref PubMed Scopus (15) Google Scholar). His435 is found clustered with basic residues, Lys295, Arg396, and Lys438, and variation of this histidine to an arginine resulted in a hyperfusogenic glycoprotein (Chang et al., 2012Chang A. Hackett B. Winter C.C. Buchholz U.J. Dutch R.E. Potential electrostatic interactions in multiple regions affect HMPV F-mediated membrane fusion.J. Virol. 2012; 86: 9843-9853Crossref PubMed Scopus (15) Google Scholar). Intriguingly, many HisCat pairs cluster at the interfaces between domains or subunits that undergo large spatial rearrangements during the fusion coordinate. For example, mutation of His3 from the Semliki Forest virus glycoprotein, which is found at the trimer interface and contacts the cognate residue in the other subunits, markedly decreased the pH requirement for membrane fusion (Qin et al., 2009Qin Z.L. Zheng Y. Kielian M. Role of conserved histidine residues in the low-pH dependence of the Semliki Forest virus fusion protein.J. Virol. 2009; 83: 4670-4677Crossref PubMed Scopus (60) Google Scholar). The rapid change in electrostatic potential upon protonation may be a contributing force to spatial reorganization in these proteins. The second pH-dependent interaction occurs between the side chains of two anionic residues (anion-anion [AniAni]), Asp or Glu, in close proximity, often below 4 Å and as close as 2.5 Å (Figure 2). Asp and Glu side chains have pKa values of 3.9 and 4.2, respectively, but the pKa values of these residues are often elevated in the context of a folded protein’s microenvironment (Harms et al., 2009Harms M.J. Castañeda C.A. Schlessman J.L. Sue G.R. Isom D.G. Cannon B.R. García-Moreno E B. The pK(a) values of acidic and basic residues buried at the same internal location in a protein are governed by different factors.J. Mol. Biol. 2009; 389: 34-47Crossref PubMed Scopus (101) Google Scholar). At neutral pH, Asp and Glu are negatively charged; as the pH decreases, they are protonated forming the conjugate acid. The proximity of these anionic side chains in the postfusion conformation disfavors its formation at neutral pH (Figure 2). However, as the pH decreases, this repulsion is relieved, thereby increasing the stability of the postfusion conformation. For example, variation of Asp188, which is buried in the trimer core of Semliki Forest virus, has profound effects on the pH dependence of membrane fusion (Liu and Kielian, 2009Liu C.Y. Kielian M. E1 mutants identify a critical region in the trimer interface of the Semliki forest virus fusion protein.J. Virol. 2009; 83: 11298-11306Crossref PubMed Scopus (22) Google Scholar). AniAni interactions may provide a stabilizing force in the postfusion conformation because they fulfill the theoretical definition of low-barrier hydrogen bond partners (Cleland, 2000Cleland W.W. Low-barrier hydrogen bonds and enzymatic catalysis.Arch. Biochem. Biophys. 2000; 382: 1-5Crossref PubMed Scopus (136) Google Scholar). This type of interaction has been observed between Asp residues in aspartic acid proteases (Northrop, 2001Northrop D.B. Follow the protons: a low-barrier hydrogen bond unifies the mechanisms of the aspartic proteases.Acc. Chem. Res. 2001; 34: 790-797Crossref PubMed Scopus (177) Google Scholar). This phenomenon may contribute to the variation of the side-chain pKa, although more extensive studies are necessary to demonstrate this effect. To analyze the distribution of HisCat and AniAni interactions in different conformations of viral fusion proteins, we used the Rosetta3 Scientific Benchmarking ChargeCharge feature reporter (Leaver-Fay et al., 2013Leaver-Fay A. O’Meara M.J. Tyka M. Jacak R. Song Y. Kellogg E.H. Thompson J. Davis I.W. Pache R.A. Lyskov S. et al.Scientific benchmarks for guiding macromolecular energy function improvement.Methods Enzymol. 2013; 523: 109-143Crossref PubMed Scopus (159) Google Scholar) to measure interatomic distances between chemical moieties on charged residues’ side chains in both the pre- and postfusion conformations of three model viral fusion systems discussed in detail herein: hemagglutinin from influenza A (HA), protein G from vesicular stomatitis virus (VSV-G), and GP from filoviruses (GP). As a control, we analyzed a reference homotrimer set assembled from the 3D complex database with viral fusion proteins removed (Levy et al., 2006Levy E.D. Pereira-Leal J.B. Chothia C. Teichmann S.A. 3D complex: a structural classification of protein complexes.PLoS Compt. Biol. 2006; 2: e155Crossref PubMed Scopus (265) Google Scholar). All of these data are normalized to report the number of pairs contained within 100 amino acids, to account for differences in the total number of residues in the various structures. The Protein Data Bank (PDB) structures that were analyzed are indicated in the legend for Figure 3. This analysis confirmed that HisCat pairs are prevalent in the prefusion conformation of HA and VSV-G and decrease as the postfusion conformation is assumed (Figure 3). Because HisCat interactions are tolerated at neutral pH but not at low pH, one explanation for their enrichment in prefusion conformations relative to postfusion conformations is that they are required for destabilizing the prefusion state as the pH decreases. HisCat interactions are also found in the reference sets because these interactions are tolerated at neutral pH. Interestingly, HisCat pairs are rare in the prefusion state of filoviruses, which is consistent with recent evidence indicating that formation of the extended intermediate is pH independent (Carette et al., 2011Carette J.E. Raaben M. Wong A.C. Herbert A.S. Obernosterer G. Mulherkar N. Kuehne A.I. Kranzusch P.J. Griffin A.M. Ruthel G. et al.Ebola virus entry requires the cholesterol transporter Niemann-Pick C1.Nature. 2011; 477: 340-343Crossref PubMed Scopus (900) Google Scholar). Therefore, low pH may not play a direct role in destabilizing the prefusion conformation of GP by direct side-chain protonation. Our analysis revealed that AniAni pairs are significantly enriched in the postfusion state of the viral fusion proteins. These residue pairs are very rarely found in both the reference data set and the prefusion conformations of the viral fusion proteins. One potential explanation for this observation is that AniAni interactions are destabilizing at neutral pH and thus critical to preventing formation of the postfusion state under neutral conditions. Furthermore, AniAni interactions may be involved in regulating the precise timing of the collapse of the extended intermediate (Figure 3). HisCat and AniAni pairs have a similar, though opposite, contribution to the free energy of folding of a protein: at one pH, the pairing is tolerated, and at another pH, the pair is repulsive. It is difficult to calculate the energetic contribution that electrostatic interactions have to the ΔG of a protein fold, due to uncertainties about desolvation energies, local dielectric constants, and variations in side-chain pKa values. Studies have found that electrostatic interactions can contribute as much as −7 kcal/mol to ΔG, and as a consequence of Coulomb’s law, repulsive forces are equally destabilizing (Kumar and Nussinov, 2001Kumar S. Nussinov R. Fluctuations in ion pairs and their stabilities in proteins.Proteins. 2001; 43: 433-454Crossref PubMed Scopus (44) Google Scholar). In general, protein folds are typically only stabilized by ΔG values between −5 and −20 kcal/mol (Dill, 1990Dill K.A. Dominant forces in protein folding.Biochemistry. 1990; 29: 7133-7155Crossref PubMed Scopus (3341) Google Scholar). What is clear is that repulsive interactions can substantially destabilize a protein fold because there are often multiple repulsive interactions found in each protein subunit. The reaction coordinates of viral fusion proteins are controlled by two distinct energetic mechanisms: (1) kinetic control, where there is a large activation barrier between the two conformations, and this barrier decreases as a result of pH changes; or (2) thermodynamic control, where a protein can exist in two distinct conformations, and the energy minima are dictated by the pH (Figure 4) (Baker and Agard, 1994Baker D. Agard D.A. Influenza hemagglutinin: kinetic control of protein function.Structure. 1994; 2: 907-910Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Manifestations of these two mechanisms can be observed experimentally, and these characteristics help to classify which energetic paradigm controls the conformational change. Fusion proteins regulated by thermodynamic control can undergo reversible conformational changes (Yao et al., 2003Yao Y. Ghosh K. Epand R.F. Epand R.M. Ghosh H.P. Membrane fusion activity of vesicular stomatitis virus glycoprotein G is induced by low pH but not by heat or denaturant.Virology. 2003; 310: 319-332Crossref PubMed Scopus (35) Google Scholar), whereas systems regulated by kinetic control undergo irreversible conformational changes (Ruigrok et al., 1986Ruigrok R.W. Martin S.R. Wharton S.A. Skehel J.J. Bayley P.M. Wiley D.C. Conformational changes in the hemagglutinin of influenza virus which accompany heat-induced fusion of virus with liposomes.Virology. 1986; 155: 484-497Crossref PubMed Scopus (138) Google Scholar). The physical properties of HisCat and AniAni pairs contribute to these control mechanisms. Kinetic control relies on destabilization of the prefusion state, thereby decreasing the kinetic barrier that traps viral fusion proteins in the prefusion state (Figure 4). Thermodynamic control, on the other hand, relies upon destabilizing the postfusion state at neutral pH and the prefusion state at low pH, allowing these proteins to function as a reversible switch. Here, there is a prominent role for both HisCat and AniAni interactions to destabilize the respective states. Exactly how these pairings contribute to kinetic and thermodynamic controls is still unclear though, and further studies are warranted. Below, we examine how HisCat and AniAni interactions function in three systems: HA under kinetic control, VSV-G under thermodynamic control, and GP for which the mechanism is uncertain. Influenza A is a member of the Orthomyxoviridae family and causes respiratory tract infection in mammals and birds (Beigel et al., 2005Beigel J.H. Farrar J. Han A.M. Hayden F.G. Hyer R. de Jong M.D. Lochindarat S. Nguyen T.K.T. Nguyen T.H. Tran T.H. et al.Writing Committee of the World Health Organization (WHO) Consultation on Human Influenza A/H5Avian influenza A (H5N1) infection in humans.N. Engl. J. Med. 2005; 353: 1374-1385Crossref PubMed Scopus (1165) Google Scholar). Influenza A contains two glycoprotein subunits, HA1 (surface) and HA2 (transmembrane), which facilitate viral entry. These proteins exist as a trimer of heterodimers (HA1/HA2) in the prefusion conformation. HA1 contains a glycan binding site that recognizes the influenza receptor, sialic acid, whereas HA2 is embedded in the membrane and contains the fusogenic subunit that promotes membrane fusion (Wilson et al., 1981Wilson I.A. Skehel J.J. Wiley D.C. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution.Nature. 1981; 289: 366-373Crossref PubMed Scopus (1980) Google Scholar). Unlike other viral proteins, receptor binding yields little conformational change and instead promotes viral endocytosis (Ha et al., 2001Ha Y. Stevens D.J. Skehel J.J. Wiley D.C. X-ray structures of H5 avian and H9 swine influenza virus hemagglutinins bound to avian and human receptor analogs.Proc. Natl. Acad. Sci. USA. 2001; 98: 11181-11186Crossref PubMed Scopus (388) Google Scholar). Once inside the endosome, HA2 undergoes a dramatic reorganization (Doms et al., 1985Doms R.W. Helenius A. White J. Membrane fusion activity of the influenza virus hemagglutinin. The low pH-induced conformational change.J. Biol. Chem. 1985; 260: 2973-2981Abstract Full Text PDF PubMed Google Scholar). Upon exposure to acidic pH, HA2 forms a highly stable extended α helix, with a particularly impressive loop-to-helix transition in the hinge of the central stalk (Figure 6) (Bullough et al., 1994Bullough P.A. Hughson F.M. Skehel J.J. Wiley D.C. Structure of influenza haemagglutinin at the pH of membrane fusion.Nature. 1994; 371: 37-43Crossref PubMed Scopus (1378) Google Scholar). In the prefusion state, HA2 is a compacted α helix kinetically trapped by its association with HA1. HA2 conformational changes can be triggered in the absence of low pH by the addition of mild denaturants or heat, providing further support for the kinetic model (Ruigrok et al., 1986Ruigrok R.W. Martin S.R. Wharton S.A. Skehel J.J. Bayley P.M. Wiley D.C. Conformational changes in the hemagglutinin of influenza virus which accompany heat-induced fusion of virus with liposomes.Virology. 1986; 155: 484-497Crossref PubMed Scopus (138) Google Scholar). Moreover, HA2 cannot adopt the prefusion conformation in the absence of HA1, again consistent with the kinetic model (Swalley et al., 2004Swalley S.E. Baker B.M. Calder L.J. Harrison S.C. Skehel J.J. Wiley D.C. Full-length influenza hemagglutinin HA2 refolds into the trimeric low-pH-induced conformation.Biochemistry. 2004; 43: 5902-5911Crossref PubMed Scopus (34) Google Scholar). A consequence of the kinetic mechanism is that formation of the postfusion state is irreversible; therefore; it has been proposed that pH change predominantly destabilizes the HA1 interactions with itself and with HA2 (Huang et al., 2002Huang Q. Opitz R. Knapp E.W. Herrmann A. Protonation and stability of the globular domain of influenza virus hemagglutinin.Biophys. J. 2002; 82: 1050-1058Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Indeed, as the virus enters the endosome and the vesicle matures, HA1 is predicted to acquire a greater cationic charge, destabilizing the HA1/HA1 interface and the interactions with HA2 (Kampmann et al., 2006Kampmann T. Mueller D.S. Mark A.E. Young P.R. Kobe B. The Role of histidine residues in low-pH-mediated viral membrane fusion.Structure. 2006; 14: 1481-1487Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar; Mueller et al., 2008Mueller D.S. Kampmann T. Yennamalli R. Young P.R. Kobe B. Mark A.E. Histidine protonation and the activation of viral fusion proteins.Biochem. Soc. Trans. 2008; 36: 43-45Crossref PubMed Scopus (52) Google Scholar). Identifying precise residues that destabilize HA1 is challenging because of sequence drift in this highly mutable virus and limited structural data for each HA subtype. There are 17 known subtypes of influenza A HA, and these subtypes can be divided into four clades based on evolutionary similarity. We generated consensus sequences for each HA subtype by aligning all available sequences in the Influenza Virus Resource (Bao et al., 2008Bao Y. Bolotov P. Dernovoy D. Kiryutin B. Zaslavsky L. Tatusova T. Ostell J. Lipman D. The influenza virus resource at the National Center for Biotechnology I