Abstract
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.
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- Bizebard T., Gigant B., Rigolet P., Rasmussen B., Diat O., Bösecke P., Wharton S. A., Skehel J. J., Knossow M. Structure of influenza virus haemagglutinin complexed with a neutralizing antibody. Nature. 1995 Jul 6;376(6535):92–94. doi: 10.1038/376092a0. [DOI] [PubMed] [Google Scholar]
- Blume A., Hübner W., Messner G. Fourier transform infrared spectroscopy of 13C = O-labeled phospholipids hydrogen bonding to carbonyl groups. Biochemistry. 1988 Oct 18;27(21):8239–8249. doi: 10.1021/bi00421a038. [DOI] [PubMed] [Google Scholar]
- Blumenthal R., Sarkar D. P., Durell S., Howard D. E., Morris S. J. Dilation of the influenza hemagglutinin fusion pore revealed by the kinetics of individual cell-cell fusion events. J Cell Biol. 1996 Oct;135(1):63–71. doi: 10.1083/jcb.135.1.63. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brand C. M., Skehel J. J. Crystalline antigen from the influenza virus envelope. Nat New Biol. 1972 Aug 2;238(83):145–147. doi: 10.1038/newbio238145a0. [DOI] [PubMed] [Google Scholar]
- Bullough P. A., Hughson F. M., Skehel J. J., Wiley D. C. Structure of influenza haemagglutinin at the pH of membrane fusion. Nature. 1994 Sep 1;371(6492):37–43. doi: 10.1038/371037a0. [DOI] [PubMed] [Google Scholar]
- Chan D. C., Fass D., Berger J. M., Kim P. S. Core structure of gp41 from the HIV envelope glycoprotein. Cell. 1997 Apr 18;89(2):263–273. doi: 10.1016/s0092-8674(00)80205-6. [DOI] [PubMed] [Google Scholar]
- Chen J., Wharton S. A., Weissenhorn W., Calder L. J., Hughson F. M., Skehel J. J., Wiley D. C. A soluble domain of the membrane-anchoring chain of influenza virus hemagglutinin (HA2) folds in Escherichia coli into the low-pH-induced conformation. Proc Natl Acad Sci U S A. 1995 Dec 19;92(26):12205–12209. doi: 10.1073/pnas.92.26.12205. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chernomordik L., Kozlov M. M., Zimmerberg J. Lipids in biological membrane fusion. J Membr Biol. 1995 Jul;146(1):1–14. doi: 10.1007/BF00232676. [DOI] [PubMed] [Google Scholar]
- Danieli T., Pelletier S. L., Henis Y. I., White J. M. Membrane fusion mediated by the influenza virus hemagglutinin requires the concerted action of at least three hemagglutinin trimers. J Cell Biol. 1996 May;133(3):559–569. doi: 10.1083/jcb.133.3.559. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Daniels R. S., Downie J. C., Hay A. J., Knossow M., Skehel J. J., Wang M. L., Wiley D. C. Fusion mutants of the influenza virus hemagglutinin glycoprotein. Cell. 1985 Feb;40(2):431–439. doi: 10.1016/0092-8674(85)90157-6. [DOI] [PubMed] [Google Scholar]
- Doms R. W., Helenius A., White J. Membrane fusion activity of the influenza virus hemagglutinin. The low pH-induced conformational change. J Biol Chem. 1985 Mar 10;260(5):2973–2981. [PubMed] [Google Scholar]
- Durrer P., Galli C., Hoenke S., Corti C., Glück R., Vorherr T., Brunner J. H+-induced membrane insertion of influenza virus hemagglutinin involves the HA2 amino-terminal fusion peptide but not the coiled coil region. J Biol Chem. 1996 Jun 7;271(23):13417–13421. doi: 10.1074/jbc.271.23.13417. [DOI] [PubMed] [Google Scholar]
- Englander S. W., Mayne L. Protein folding studied using hydrogen-exchange labeling and two-dimensional NMR. Annu Rev Biophys Biomol Struct. 1992;21:243–265. doi: 10.1146/annurev.bb.21.060192.001331. [DOI] [PubMed] [Google Scholar]
- Fass D., Harrison S. C., Kim P. S. Retrovirus envelope domain at 1.7 angstrom resolution. Nat Struct Biol. 1996 May;3(5):465–469. doi: 10.1038/nsb0596-465. [DOI] [PubMed] [Google Scholar]
- Frey S., Tamm L. K. Orientation of melittin in phospholipid bilayers. A polarized attenuated total reflection infrared study. Biophys J. 1991 Oct;60(4):922–930. doi: 10.1016/S0006-3495(91)82126-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gething M. J., Doms R. W., York D., White J. Studies on the mechanism of membrane fusion: site-specific mutagenesis of the hemagglutinin of influenza virus. J Cell Biol. 1986 Jan;102(1):11–23. doi: 10.1083/jcb.102.1.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Godley L., Pfeifer J., Steinhauer D., Ely B., Shaw G., Kaufmann R., Suchanek E., Pabo C., Skehel J. J., Wiley D. C. Introduction of intersubunit disulfide bonds in the membrane-distal region of the influenza hemagglutinin abolishes membrane fusion activity. Cell. 1992 Feb 21;68(4):635–645. doi: 10.1016/0092-8674(92)90140-8. [DOI] [PubMed] [Google Scholar]
- Gray C., Tamm L. K. Structural studies on membrane-embedded influenza hemagglutinin and its fragments. Protein Sci. 1997 Sep;6(9):1993–2006. doi: 10.1002/pro.5560060920. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gray C., Tatulian S. A., Wharton S. A., Tamm L. K. Effect of the N-terminal glycine on the secondary structure, orientation, and interaction of the influenza hemagglutinin fusion peptide with lipid bilayers. Biophys J. 1996 May;70(5):2275–2286. doi: 10.1016/S0006-3495(96)79793-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Heimburg T., Schuenemann J., Weber K., Geisler N. Specific recognition of coiled coils by infrared spectroscopy: analysis of the three structural domains of type III intermediate filament proteins. Biochemistry. 1996 Feb 6;35(5):1375–1382. doi: 10.1021/bi9515883. [DOI] [PubMed] [Google Scholar]
- Hinterdorfer P., Baber G., Tamm L. K. Reconstitution of membrane fusion sites. A total internal reflection fluorescence microscopy study of influenza hemagglutinin-mediated membrane fusion. J Biol Chem. 1994 Aug 12;269(32):20360–20368. [PubMed] [Google Scholar]
- Jackson M., Mantsch H. H. The use and misuse of FTIR spectroscopy in the determination of protein structure. Crit Rev Biochem Mol Biol. 1995;30(2):95–120. doi: 10.3109/10409239509085140. [DOI] [PubMed] [Google Scholar]
- Kemble G. W., Bodian D. L., Rosé J., Wilson I. A., White J. M. Intermonomer disulfide bonds impair the fusion activity of influenza virus hemagglutinin. J Virol. 1992 Aug;66(8):4940–4950. doi: 10.1128/jvi.66.8.4940-4950.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kim C. H., Macosko J. C., Yu Y. G., Shin Y. K. On the dynamics and conformation of the HA2 domain of the influenza virus hemagglutinin. Biochemistry. 1996 Apr 30;35(17):5359–5365. doi: 10.1021/bi960332+. [DOI] [PubMed] [Google Scholar]
- Korte T., Ludwig K., Krumbiegel M., Zirwer D., Damaschun G., Herrmann A. Transient changes of the conformation of hemagglutinin of influenza virus at low pH detected by time-resolved circular dichroism spectroscopy. J Biol Chem. 1997 Apr 11;272(15):9764–9770. doi: 10.1074/jbc.272.15.9764. [DOI] [PubMed] [Google Scholar]
- Lee J., Lentz B. R. Evolution of lipidic structures during model membrane fusion and the relation of this process to cell membrane fusion. Biochemistry. 1997 May 27;36(21):6251–6259. doi: 10.1021/bi970404c. [DOI] [PubMed] [Google Scholar]
- Lewis R. N., McElhaney R. N., Pohle W., Mantsch H. H. Components of the carbonyl stretching band in the infrared spectra of hydrated 1,2-diacylglycerolipid bilayers: a reevaluation. Biophys J. 1994 Dec;67(6):2367–2375. doi: 10.1016/S0006-3495(94)80723-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Marsh D. Dichroic ratios in polarized Fourier transform infrared for nonaxial symmetry of beta-sheet structures. Biophys J. 1997 Jun;72(6):2710–2718. doi: 10.1016/S0006-3495(97)78914-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Qiao H., Pelletier S. L., Hoffman L., Hacker J., Armstrong R. T., White J. M. Specific single or double proline substitutions in the "spring-loaded" coiled-coil region of the influenza hemagglutinin impair or abolish membrane fusion activity. J Cell Biol. 1998 Jun 15;141(6):1335–1347. doi: 10.1083/jcb.141.6.1335. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Reisdorf W. C., Jr, Krimm S. Infrared amide I' band of the coiled coil. Biochemistry. 1996 Feb 6;35(5):1383–1386. doi: 10.1021/bi951589v. [DOI] [PubMed] [Google Scholar]
- Rodionova N. A., Tatulian S. A., Surrey T., Jähnig F., Tamm L. K. Characterization of two membrane-bound forms of OmpA. Biochemistry. 1995 Feb 14;34(6):1921–1929. doi: 10.1021/bi00006a013. [DOI] [PubMed] [Google Scholar]
- Ruigrok R. W., Aitken A., Calder L. J., Martin S. R., Skehel J. J., Wharton S. A., Weis W., Wiley D. C. Studies on the structure of the influenza virus haemagglutinin at the pH of membrane fusion. J Gen Virol. 1988 Nov;69(Pt 11):2785–2795. doi: 10.1099/0022-1317-69-11-2785. [DOI] [PubMed] [Google Scholar]
- Ruigrok R. W., Wrigley N. G., Calder L. J., Cusack S., Wharton S. A., Brown E. B., Skehel J. J. Electron microscopy of the low pH structure of influenza virus haemagglutinin. EMBO J. 1986 Jan;5(1):41–49. doi: 10.1002/j.1460-2075.1986.tb04175.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Skehel J. J., Bayley P. M., Brown E. B., Martin S. R., Waterfield M. D., White J. M., Wilson I. A., Wiley D. C. Changes in the conformation of influenza virus hemagglutinin at the pH optimum of virus-mediated membrane fusion. Proc Natl Acad Sci U S A. 1982 Feb;79(4):968–972. doi: 10.1073/pnas.79.4.968. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Steinhauer D. A., Martín J., Lin Y. P., Wharton S. A., Oldstone M. B., Skehel J. J., Wiley D. C. Studies using double mutants of the conformational transitions in influenza hemagglutinin required for its membrane fusion activity. Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):12873–12878. doi: 10.1073/pnas.93.23.12873. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Straume M., Johnson M. L. Monte Carlo method for determining complete confidence probability distributions of estimated model parameters. Methods Enzymol. 1992;210:117–129. doi: 10.1016/0076-6879(92)10009-3. [DOI] [PubMed] [Google Scholar]
- Tamm L. K., Tatulian S. A. Infrared spectroscopy of proteins and peptides in lipid bilayers. Q Rev Biophys. 1997 Nov;30(4):365–429. doi: 10.1017/s0033583597003375. [DOI] [PubMed] [Google Scholar]
- Tamm L. K., Tatulian S. A. Orientation of functional and nonfunctional PTS permease signal sequences in lipid bilayers. A polarized attenuated total reflection infrared study. Biochemistry. 1993 Aug 3;32(30):7720–7726. doi: 10.1021/bi00081a017. [DOI] [PubMed] [Google Scholar]
- Tatulian S. A., Hinterdorfer P., Baber G., Tamm L. K. Influenza hemagglutinin assumes a tilted conformation during membrane fusion as determined by attenuated total reflection FTIR spectroscopy. EMBO J. 1995 Nov 15;14(22):5514–5523. doi: 10.1002/j.1460-2075.1995.tb00238.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tatulian S. A., Tamm L. K. Reversible pH-dependent conformational change of reconstituted influenza hemagglutinin. J Mol Biol. 1996 Jul 19;260(3):312–316. doi: 10.1006/jmbi.1996.0402. [DOI] [PubMed] [Google Scholar]
- Tsurudome M., Glück R., Graf R., Falchetto R., Schaller U., Brunner J. Lipid interactions of the hemagglutinin HA2 NH2-terminal segment during influenza virus-induced membrane fusion. J Biol Chem. 1992 Oct 5;267(28):20225–20232. [PubMed] [Google Scholar]
- Weissenhorn W., Dessen A., Harrison S. C., Skehel J. J., Wiley D. C. Atomic structure of the ectodomain from HIV-1 gp41. Nature. 1997 May 22;387(6631):426–430. doi: 10.1038/387426a0. [DOI] [PubMed] [Google Scholar]
- Wharton S. A., Ruigrok R. W., Martin S. R., Skehel J. J., Bayley P. M., Weis W., Wiley D. C. Conformational aspects of the acid-induced fusion mechanism of influenza virus hemagglutinin. Circular dichroism and fluorescence studies. J Biol Chem. 1988 Mar 25;263(9):4474–4480. [PubMed] [Google Scholar]
- White J. M., Wilson I. A. Anti-peptide antibodies detect steps in a protein conformational change: low-pH activation of the influenza virus hemagglutinin. J Cell Biol. 1987 Dec;105(6 Pt 2):2887–2896. doi: 10.1083/jcb.105.6.2887. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wilson I. A., Skehel J. J., Wiley D. C. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature. 1981 Jan 29;289(5796):366–373. doi: 10.1038/289366a0. [DOI] [PubMed] [Google Scholar]