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Reversible pH-dependent Conformational Change of Reconstituted Influenza Hemagglutinin
J. Mol. Biol. (1996) 260, 312–316
COMMUNICATION
Reversible pH-dependent Conformational Change of Reconstituted Influenza Hemagglutinin Suren A. Tatulian* and Lukas K. Tamm Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine Box 449, Charlottesville VA 22908, USA
Fusion between influenza virus and cell membranes is mediated by a major acid-induced conformational change of the spike glycoprotein of the viral envelope, hemagglutinin (HA). The conformational change of HA is commonly believed to be a kinetically controlled irreversible process, although the experimental evidence for this is controversial. Here we show by polarized infrared spectroscopy that the previously described acid-induced inclination of HA reconstituted in supported phospholipid bilayers is reversible in the absence, but irreversible in the presence, of bound target membranes. We also demonstrate reversible pH-dependent changes in the capability of reconstituted HA to bind target membranes. These results support a thermodynamically controlled mechanism of the conformational change of HA and provide new insight into the understanding of the energetics of influenza-mediated membrane fusion. 7 1996 Academic Press Limited
*Corresponding author
Keywords: influenza hemagglutinin; conformational change; fusion; reversibility; infrared spectroscopy
Influenza virus transfers its genome into target cells by endocytosis followed by fusion between viral and endosomal membranes (White, 1994; Stegmann & Helenius, 1993; Wiley & Skehel, 1987). Fusion is mediated by HA, a trimeric spike glycoprotein of the viral envelope, which inserts the N-terminal fusion peptide of the HA2 subunit into the target membrane as a result of a low pHinduced conformational change (Tsurudome et al., 1992; Stegmann et al., 1991). Although the X-ray structures of HA at neutral and acidic pH revealed a major refolding of the secondary and tertiary structure of the molecule (Wilson et al., 1981; Bullough et al., 1994), the molecular mechanism of HA-mediated membrane fusion is still poorly understood. In order to comprehend the energetic basis and the sequence of transitions in this process, information on the reversibility of the pH-dependent conformational change of HA is required (Siegel, 1993; Baker & Agard, 1994). The conformational change of HA is believed to be irreversible based on several lines of evidence: (1) purified HA or its water-soluble ectodomain become and remain Abbreviations used: HA, hemagglutinin; ATR attenuated total reflection; FTIR, Fourier transform infrared; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; POPG, 1-palmitoyl-2-oleoylphosphatidylglycerol; CL, cardiolipin. 0022–2836/96/280312–05 $18.00/0
susceptible to proteinase K upon acid treatment and reneutralization (Gething et al., 1986; Doms & Helenius, 1986; Stegmann et al., 1987); (2) binding of antibodies to some of the antigenic sites of HA demonstrated an irreversible conformational change in HA (Yewdell et al., 1983; White & Wilson, 1987; Stegmann et al., 1990); and (3) after acid treatment and reneutralization, the HA spikes on the virus surface become disorganized (Doms et al., 1985). In the presence of bound target membranes, the sequence of structural changes is more complex; acidification converts the HA to a fusion-active intermediate which becomes inactivated only in a subsequent step (Stegmann et al., 1990; Krumbiegel et al., 1994). A number of observations indicate that a reversible component may be involved in the transition of HA to the fusogenic state: (1) the low pH-initiated fusion activity of influenza virus is inhibited by neutralization and is recovered upon reacidification (Stegmann et al., 1990; RomalhoSantos et al., 1993); (2) fluorescence experiments revealed a reversible conformational change of HA when pH was lowered to 6 (Sato et al., 1983); after exposure to pH 4.9 reversibility is detected at short times but is lost after prolonged acid treatment (Krumbiegel et al., 1994); (3) hemagglutination activity of influenza virus is completely retained upon incubation at pH 5 for up to 90 minutes and reneutralization (Yewdell et al., 1983). It is also 7 1996 Academic Press Limited
Communication
noteworthy that the fusion proteins of vesicular stomatitis and rabies viruses, which share many common structural and functional features with influenza HA, undergo reversible acid-induced conformational changes (Clague et al., 1990; Gaudin et al., 1993). Recently we functionally reconstituted HA in substrate-supported phospholipid bilayers (Hinterdorfer et al., 1994) and demonstrated that the reconstituted HA tilts towards the membrane surface at low pH (Tatulian et al., 1995). Here we present polarized attenuated total reflection (ATR) Fourier transform infrared (FTIR) results which indicate that this pH-dependent inclination of HA is reversible. We further detect reversible pH-dependent changes in the membrane-binding capacity of reconstituted HA. These findings support a reversible, thermodynamically controlled mechanism of the pH-dependent conformational change of influenza HA. The ATR dichroic ratio (R ATR ) of the amide I band of HA at pH 7.4 was 1.90(20.04) (Figure 1b and Table 1). The fact that the two largest a-helices of HA (53 and 19 residues) are parallel to each other (Wilson et al., 1981) allowed us to calculate a helical order parameter (see the legend to Figure 1), SH = 0.95(20.19), which is close to the theoretical limit of 1.0 corresponding to an orientation perpendicular to the plane of the membrane. At pH 5 R ATR decreases to 1.68(20.04) (Figure 1c and Table 1). Based on the low pH crystal structure of TBHA2, which basically comprises the HA2 chain without the transmembrane and fusion peptides (Bullough et al., 1994), we calculate SH = 0 to −0.64, corresponding to tilt angles of 54 to 90°: Since the longest helices of HA at neutral and acidic pH are nearly parallel to the trimer axis (166 and 169°), the observed decrease in R ATR indicates a profound tilt of the HA trimers toward the membrane surface. Reneutralization resulted in an increase in R ATR to 1.83(20.05), indicating restoration of the initial vertical orientation of HA to 50 to 100% (Figure 1d and Table 1). Injection of negatively charged vesicles into reconstituted HA at pH 7.4 increased the lipid absorbance bands, indicating binding of vesicles (Figure 1a). The amide I dichroic ratio decreased to 1.82 and 1.79 for 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) vesicles containing 40% 1palmitoyl-2-oleoylphosphatidylglycerol (POPG) or 20% cardiolipin (CL), respectively (Figure 1e and Table 1). Lowering the pH to 5 in the presence of bound vesicles caused a further decrease in R ATR to 1.66 to 1.70, which yields helical tilt angles of 57 to 82° (Figure 1f and Table 1). Thus, the binding of negatively charged membranes induces a small inclination of HA trimers and acidification in the presence of bound vesicles results in a strongly tilted conformation of HA which does not revert after reneutralization (Figure 1g and Table 1). Because of a 3 to 5 cm−1 shift between parallel and perpendicular polarized amide I bands (Figure 1, see also Reisdorf & Krimm, 1995), and because of
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Figure 1. Polarized ATR-FTIR specra of reconstituted HA (strain A/PR/8/34). a, Perpendicular polarized spectra without and with bound vesicles composed of POPC and CL at 4:1 molar ratio. The absorbance bands at 02922 and 02852 cm−1 are due to the antisymmetric and symmetric CH2 stretching vibrations of the lipid acyl chains; the bands at 01735 and 01647 cm−1 result from the lipid carbonyl groups and the amide I vibration of HA, respectively. b to g, Amide I bands at parallel (continuous lines) and perpendicular (shaded areas) polarizations of infrared light without (b, c, d) and with (e, f, g) bound POPC/CL vesicles. The initial pH was 7.4 (b, e), then changed to 5 (c, f) and readjusted to 7.4 (d, g). HA was purified and reconstituted in supported phospholipid bilayers as described (Hinterdorfer et al., 1994; Tatulian et al., 1995). The membranes were flushed with 2H2 O buffers of pH 7.4 or 5 (5 mM Hepes, 10 mM Mes, 135 mM NaCl, pH adjusted with NaOH without corrections for the isotope effect). The ATR dichroic ratio of HA was determined as the ratio of the heights of parallel and perpendicular polarized amide I bands: R ATR = A> /A_ . The helical order parameter was determined as SH = B/fH Sa (B − 3Ez2 ), where B = Ex2 − R ATREy2 + Ez2 (Ex , Ey , and Ez are the electric vector components of the infrared light at the germanium/membrane interface), fH is the fraction of residues in helices of similar orientation, Sa = (3 cos2a − 1)/2, and a is the angle between the transition dipole moment and the helical axis (39°). The angles of orientation of the major helices relative to the membrane normal (u) were evaluated from: SH = (3cos2u − 1)/2. All experiments were carried out at room temperature and the system was kept for 35 minutes at each experimental condition.
different orientations of individual secondary structure elements, the dichroic ratio varies within the amide I region (Figure 2). The dichroic spectra of Figure 2a demonstrate that acidification in the absence of vesicles decreases the amide I R ATR of
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Table 1. ATR dichroic ratios of the amide I bands of reconstituted influenza hemagglutinin under different experimental conditions R ATR pH 7.4 7.4 (flush) 5.0 7.4
No vesicles
POPC/POPG
POPC/CL
1.9020.04 1.9020.04 1.6820.04 1.8320.05
1.8220.04 1.8220.03 1.7020.03 1.7120.03
1.7920.05 1.7820.04 1.6620.06 1.6720.05
The experimental conditions were changed from top to bottom.
HA by 0.2 to 0.3 unit and reneutralization results in a restoration of R ATR to a large extent. The R ATR decreases upon the binding of target membranes at neutral pH and acidification with bound membranes further decreases R ATR, which remains at this low level after reneutralization (Figure 2b). The acid-induced tilting motion of HA trimers which are bound to both (viral and target) membranes by the transmembrane and fusion peptides may bring the membranes to a close contact and thus serve as a critical step in membrane fusion. The orientational change of HA described here is likely related to the pH-dependent conformational change of HA. An acid-induced loop-helix transition of residues 56 to 75 of the HA2 chain moves the fusion peptide to the top of the trimer (Bullough et al., 1994; Carr & Kim, 1993). This causes a transition to a loop of a segment of the longest a-helix (residues 106 to 112 at the base of HA2), whose helical structure at neutral pH is stabilized by extensive interactions with the fusion peptide, resulting in an inversion of the most C-terminal part of the long helix (Bullough et al., 1994). We
Figure 2. Infrared dichroic spectra of reconstituted HA in the amide I region in the absence (a) and presence (b) of bound POPC/CL vesicles (4:1, mol/mol). The pH initially was 7.4 (broken lines), then was shifted to 5 (dotted lines) and readjusted to 7.4 (continuous lines). Unilamellar vesicles of 100 nm diameter were prepared as described (Tatulian et al., 1995). The dichroic spectra were obtained by dividing the parallel polarized absorbance spectra by the perpendicular polarized ones.
Figure 3. Binding of phospholipid vesicles to reconstituted HA in planar supported membranes as measured by the integrated absorbance due to the stretching vibrations of the lipid acyl chain CH2 groups (2990 to 2820 cm−1 ). Three different sequential pH protocols were adopted: (a) vesicles were injected into reconstituted HA at pH 7.4, the cell was flushed with pH 7.4 buffer, the pH was decreased to 5 and readjusted to 7.4; (b) the pH was changed from 7.4 to 5, vesicles were added, and the cell was flushed with pH 5 and then with pH 7.4 buffers; (c) reconstituted HA was preincubated at pH 5, vesicles were added at pH 7.4, and the cell was flushed with pH 7.4 buffer. Squares, triangles, and circles correspond to 100% POPC, 60% POPC + 40% POPG, and 80% POPC + 20% CL vesicles, respectively. Vesicles were prepared in 2H2 O buffers at the pH of injection. All experiments were carried out at room temperature and the system was kept for 35 minutes at each experimental condition.
hypothesize that this segment may serve as a flexible hinge in the tilting motion of HA. A synthetic peptide corresponding to the loop56-75 and a fraction of the long helix of HA2 was shown to undergo a reversible acid-induced change from a monomeric irregular structure to a trimeric helical coiled coil (Yu et al., 1994). Therefore, one might speculate that the pH-dependent structural change of this stretch could be reversible even in the full-length HA, resulting in a partial sequestering of the fusion peptide and possibly other hydrophobic parts of HA upon reneutralization. Exposure and seclusion of the fusion peptide are expected to change the amphiphilic character and hence the lipid-binding capability of HA. When vesicles of POPC/CL, POPC/POPG, and pure POPC were bound to reconstituted HA at pH 7.4, the integrated absorbance due to the lipid acyl chain CH2 groups increased 5-, 3.5-, and 2.5-fold, respectively (Figure 3a). This binding is likely mediated by electrostatic interaction between the cationic globular head of HA and negatively charged membranes and van der Waals attraction. The difference between the binding of POPC/CL (4:1) and POPC/POPG (3:2) vesicles, which have similar surface charge density, indicates that additional (e.g. steric) factors are involved in vesicle-HA interactions. At pH 5, the capacity of reconstituted HA to bind POPC/CL vesicles was substantially increased (Figure 3b), evidently due to hydrophobic interactions between the exposed fusion peptide and the membranes (Harter et al.,
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1989) and additional protonation of HA. The amount of bound vesicles stays at the same level upon reneutralization, implying that the electrostatic effects play only a minor part in the acid-induced increase in vesicle binding to HA. Strikingly, when the vesicles were injected at pH 7.4 after HA was subjected to a pH cycle from 7.4 to 5 to 7.4, the vesicle binding was similar to that when they were injected at pH 7.4 without preincubation of HA at low pH (Figure 3c), indicating that the exposure of the fusion peptide and/or other amphiphilic structures at low pH in the absence of target membranes is reversed upon reneutralization. The data of Figure 3 are consistent with our results indicating that the acid-induced inclination of HA is reversible in the absence, but irreversible in the presence, of bound target membranes. The results of Figure 3 imply that after acid treatment for 35 minutes HA is not inactivated in terms of its lipid binding properties. Inactivation of influenza HA at low pH, in the absence of target membranes, is strongly dependent on temperature and on the virus strain. Treatment of X-47 virus at pH 5.2 for 15 minutes inactivated the virus above 30°C but had no effect at or below 20°C (Junankar & Cherry, 1986). The half-time of the inactivation of the strain PR8 virus at pH 5.2 is 30 seconds at 37°C and ten minutes at 0°C (Sato et al., 1983). After incubation of this virus for 35 minutes at room temperature, still 025% of the hemolytic activity is retained (Yewdell et al., 1983). On the other hand, the inactivation is believed to be caused by immobilization of HA due to its lateral aggregation, which requires the HA molecules to be mobile in the viral membrane (Junankar & Cherry, 1986; Gutman et al., 1993). Earlier work in this laboratory, using fluorescence recovery after photobleaching, showed that HA is immobile and therefore unable to undergo lateral aggregation in supported bilayers (P. Hinterdorfer, unpublished results). We also demonstrated that the fusion activity of PR8 HA in supported bilayers, as measured by the spread of lipid dye, declines only slightly when it is exposed to pH 5 buffer at room temperature over a time period of 60 minutes (L. Tamm, unpublished results; Hinterdorfer et al., 1994). These considerations imply that under our experimental conditions, when HA is reconstituted in supported membranes at room temperature, it can largely escape inactivation at low pH. Reversible components of the conformational change of HA are more readily detected in supported membranes than in suspensions of the virus with or without target membranes because the HA molecules are confined to a single membrane and their interaction with other membranes is prevented. Our findings lead to a new understanding of the energetics of the structural change of HA. According to the irreversible kinetic mechanism, which is currently more widely accepted, the native HA is kinetically prevented from adopting the more stable low pH structure;
lowering the pH reduces the energy barrier between the two states and thereby triggers the structural change. The alternative thermodynamic concept suggests that changes in pH switch the energy levels of the two states, resulting in a reversible mechanism of the conformational change (Baker & Agard, 1994). Our results show that the conformational change of HA per se, i.e. in the absence of target or viral membranes, is reversible and therefore thermodynamically controlled. The apparent irreversibility of the structural change in the presence of membranes is evidently due to mechanical obstructions and hydrophobic interactions between HA molecules and bound membranes. It should be noted that we have not yet shown unequivocally that the transmembrane domain of HA properly spans the supported membrane in our experiments. Formally, it is possible that HA is inserted into the supported membrane with a C-terminal hairpin loop. If this is the case, the reversible components we are observing may apply to the reactions leading to hemi-fusion rather than to those resulting in the final fusion product (Kemble et al., 1994). Finally, our results may be of epidemiological and immunological significance because the reversibility in the absence of target membranes would allow the virions to survive under diverse environmental conditions, and the HA molecules are less likely digested by the proteases of the host immune system before fusion and transfer of the viral genome into the cytosol take place.
References Baker, D. & Agard, D. A. (1994). Influenza hemagglutinin: kinetic control of protein function. Structure, 2, 907–910. Bullough, P. A., Hughson, F. M., Skehel, J. J. & Wiley, D. C. (1994). Structure of influenza hemagglutinin at the pH of membrane fusion. Nature, 371, 37–43. Carr, C. M. & Kim, P. S. (1993). A spring-loaded mechanism for the conformational change of influenza hemagglutinin. Cell, 73, 823–832. Clague, M. J., Schoch, C., Zech, L. & Blumenthal, R. (1990). Gating kinetics of pH-activated membrane fusion of vesicular stomatitis virus with cells: stopped-flow measurements by dequenching of octadecylrhodamine fluorescence. Biochemistry, 29, 1303–1308. Doms, R. W. & Helenius, A. (1986). Quaternary structure of influenza virus hemagglutinin after acid treatment. J. Virol. 60, 833–839. Doms, R. W., Helenius, A. & White, J. (1985). Membrane fusion activity of the influenza virus hemagglutinin. The low pH-induced conformational change. J. Biol. Chem. 260, 2973–2981. Gaudin, Y., Ruigrok, R. W. H., Knossow, M. & Flamand, A. (1993). Low-pH conformational changes of rabies virus glycoprotein and their role in membrane fusion. J. Virol. 67, 1365–1372. Gething, M.-J., Doms, R. W., York, D. & White, J. (1986). Studies on the mechanism of membrane fusion: site-specific mutagenesis of the hemagglutinin of influenza virus. J. Cell Biol. 102, 11–23.
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Gutman, O., Danieli, T., White, J. M. & Henis, Y. I. (1993) Effects of exposure to low pH on the lateral mobility of influenza hemagglutinin expressed at the cell surface: correlation between mobility inhibition and inactivation. Biochemistry, 32, 101–106. Harter, C., James, P., Ba¨chi, T., Semezna, G. & Brunner, J. (1989). Hydrophobic binding of the ectodomain of influenza hemagglutinin to membranes accurs through the ‘‘fusion peptide’’. J. Biol. Chem. 264, 6459–6464. Hinterdorfer, P., Baber, G. & Tamm, L. K. (1994). Reconstitution of membrane fusion sites. A total internal reflection fluorescence microscopy study of influenza hemagglutinin-mediated membrane fusion. J. Biol. Chem. 269, 20360–20368. Junankar, P. R. & Cherry, R. J. (1986). Temperature and pH dependence of the haemolytic activity of influenza virus and of the rotational mobility of the spike glycoproteins. Biochim. Biophys. Acta, 854, 198–206. Kemble, G. W., Danieli, T. & White, J. M. (1994) Lipid-anchored influenza hemagglutinin promotes hemifusion, not complete fusion. Cell, 76, 383–391. Krumbiegel, M., Herrmann, A. & Blumenthal, R. (1994). Kinetics of the low pH-induced conformational changes and fusogenic activity of influenza hemagglutinin. Biophys. J. 67, 2355–2360. Reisdorf, W. C., Jr & Krimm, S. (1995). Infrared dichroism of amide I and amide II modes of aI- and aII-helix segments in membrane proteins. Biophys. J. 69, 271–273. Romalho-Santos, J., Nir, S., Du¨zgu¨nes, N., Carvalho, A. P. & Lima, M. P. C. (1993). A common mechanism for influenza virus fusion activity and inactivation. Biochemistry, 32, 2771–2779. Sato, S. B., Kawasaki, K. & Ohnishi, S. (1983). Hemolytic activity of influenza virus hemagglutinin glycoproteins activated in mildly acidic environments. Proc. Natl Acad. Sci. USA, 80, 3153–3157. Siegel, D. P. (1993). Energetics of intermediates in membrane fusion: comparison of stalk and inverted micellar intermediate mechanisms. Biophys. J. 65, 2124–2140. Stegmann, T. & Helenius, A. (1993). Influenza virus fusion: from models towards a mechanism. In Viral Fusion Mechanisms (Bentz, J., ed.), pp. 89–111, CRC Press, Boca Raton, FL.
Stegmann, T., Booy, F. P. & Wilschut, J. (1987). Effect of low pH on influenza virus. Activation and inactivation of the membrane fusion capacity of the hemagglutinin. J. Biol. Chem. 262, 17744– 17749. Stegmann, T., White, J. M. & Helenius, A. (1990). Intermediates in influenza induced membrane fusion. EMBO J. 9, 4231–4241. Stegmann, T., Delfino, J. M., Richards, F. M. & Helenius, A. (1991). The HA2 subunit of influenza hemagglutinin inserts into the target membrane prior to fusion. J. Biol. Chem. 266, 18404–18410. Tatulian, S. A., Hinterdorfer, P., Baber, G. & Tamm, L. K. (1995). Influenza hemagglutinin assumes a tilted conformation during memebrane fusion as detected by attenuated total reflection FTIR spectroscopy. EMBO J. 14, 5514–5523. Tsurudome, M., Glu¨ck, R., Graf, R., Falchetto, R., Schaller, U. & Brunner, J. (1992). Lipid interactions of the hemagglutinin HA2 NH2-terminal segment during influenza virus-induced membrane fusion. J. Biol. Chem. 267, 20225–20232. White, J. M. (1994). Fusion of influenza virus in endosomes: role of the hemagglutinin. In Cellular Receptors for Animal Viruses (Wimmer, E., ed.), pp. 281–301, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. White, J. M. & Wilson, I. A. (1987). Anti-peptide antibodies detect steps in a protein conformational change: low-pH activation of the influenza virus hemagglutinin. J. Cell Biol. 105, 2887–2896. Wiley, D. C. & Skehel, J. J. (1987) The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. Annu. Rev. Biochem. 56, 365–394. Wilson, I. A., Skehel, J. J. & Wiley, D. C. (1981). Structure of the hemagglutinin membrane glycoprotein of ˚ resolution. Nature, 289, influenza virus at 3 A 366–373. Yewdell, J. W., Gerhard, W. & Ba¨chi, T. (1983). Monoclonal anti-hemagglutinin antibodies detect irreversible antigenic alterations that coincide with the acid activation of influenza A/PR/834-mediated hemolysis. J. Virol. 48, 239–248. Yu, Y. G., King, D. S. & Shin, Y.-K. (1994). Insertion of a coiled-coil peptide from influenza virus hemagglutinin into membranes. Science, 266, 274–276.
Edited by R. Huber (Received 22 November 1995; received in revised form 25 April 1996; accepted 1 May 1996)
COMMUNICATION
Reversible pH-dependent Conformational Change of Reconstituted Influenza Hemagglutinin Suren A. Tatulian* and Lukas K. Tamm Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine Box 449, Charlottesville VA 22908, USA
Fusion between influenza virus and cell membranes is mediated by a major acid-induced conformational change of the spike glycoprotein of the viral envelope, hemagglutinin (HA). The conformational change of HA is commonly believed to be a kinetically controlled irreversible process, although the experimental evidence for this is controversial. Here we show by polarized infrared spectroscopy that the previously described acid-induced inclination of HA reconstituted in supported phospholipid bilayers is reversible in the absence, but irreversible in the presence, of bound target membranes. We also demonstrate reversible pH-dependent changes in the capability of reconstituted HA to bind target membranes. These results support a thermodynamically controlled mechanism of the conformational change of HA and provide new insight into the understanding of the energetics of influenza-mediated membrane fusion. 7 1996 Academic Press Limited
*Corresponding author
Keywords: influenza hemagglutinin; conformational change; fusion; reversibility; infrared spectroscopy
Influenza virus transfers its genome into target cells by endocytosis followed by fusion between viral and endosomal membranes (White, 1994; Stegmann & Helenius, 1993; Wiley & Skehel, 1987). Fusion is mediated by HA, a trimeric spike glycoprotein of the viral envelope, which inserts the N-terminal fusion peptide of the HA2 subunit into the target membrane as a result of a low pHinduced conformational change (Tsurudome et al., 1992; Stegmann et al., 1991). Although the X-ray structures of HA at neutral and acidic pH revealed a major refolding of the secondary and tertiary structure of the molecule (Wilson et al., 1981; Bullough et al., 1994), the molecular mechanism of HA-mediated membrane fusion is still poorly understood. In order to comprehend the energetic basis and the sequence of transitions in this process, information on the reversibility of the pH-dependent conformational change of HA is required (Siegel, 1993; Baker & Agard, 1994). The conformational change of HA is believed to be irreversible based on several lines of evidence: (1) purified HA or its water-soluble ectodomain become and remain Abbreviations used: HA, hemagglutinin; ATR attenuated total reflection; FTIR, Fourier transform infrared; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; POPG, 1-palmitoyl-2-oleoylphosphatidylglycerol; CL, cardiolipin. 0022–2836/96/280312–05 $18.00/0
susceptible to proteinase K upon acid treatment and reneutralization (Gething et al., 1986; Doms & Helenius, 1986; Stegmann et al., 1987); (2) binding of antibodies to some of the antigenic sites of HA demonstrated an irreversible conformational change in HA (Yewdell et al., 1983; White & Wilson, 1987; Stegmann et al., 1990); and (3) after acid treatment and reneutralization, the HA spikes on the virus surface become disorganized (Doms et al., 1985). In the presence of bound target membranes, the sequence of structural changes is more complex; acidification converts the HA to a fusion-active intermediate which becomes inactivated only in a subsequent step (Stegmann et al., 1990; Krumbiegel et al., 1994). A number of observations indicate that a reversible component may be involved in the transition of HA to the fusogenic state: (1) the low pH-initiated fusion activity of influenza virus is inhibited by neutralization and is recovered upon reacidification (Stegmann et al., 1990; RomalhoSantos et al., 1993); (2) fluorescence experiments revealed a reversible conformational change of HA when pH was lowered to 6 (Sato et al., 1983); after exposure to pH 4.9 reversibility is detected at short times but is lost after prolonged acid treatment (Krumbiegel et al., 1994); (3) hemagglutination activity of influenza virus is completely retained upon incubation at pH 5 for up to 90 minutes and reneutralization (Yewdell et al., 1983). It is also 7 1996 Academic Press Limited
Communication
noteworthy that the fusion proteins of vesicular stomatitis and rabies viruses, which share many common structural and functional features with influenza HA, undergo reversible acid-induced conformational changes (Clague et al., 1990; Gaudin et al., 1993). Recently we functionally reconstituted HA in substrate-supported phospholipid bilayers (Hinterdorfer et al., 1994) and demonstrated that the reconstituted HA tilts towards the membrane surface at low pH (Tatulian et al., 1995). Here we present polarized attenuated total reflection (ATR) Fourier transform infrared (FTIR) results which indicate that this pH-dependent inclination of HA is reversible. We further detect reversible pH-dependent changes in the membrane-binding capacity of reconstituted HA. These findings support a reversible, thermodynamically controlled mechanism of the pH-dependent conformational change of influenza HA. The ATR dichroic ratio (R ATR ) of the amide I band of HA at pH 7.4 was 1.90(20.04) (Figure 1b and Table 1). The fact that the two largest a-helices of HA (53 and 19 residues) are parallel to each other (Wilson et al., 1981) allowed us to calculate a helical order parameter (see the legend to Figure 1), SH = 0.95(20.19), which is close to the theoretical limit of 1.0 corresponding to an orientation perpendicular to the plane of the membrane. At pH 5 R ATR decreases to 1.68(20.04) (Figure 1c and Table 1). Based on the low pH crystal structure of TBHA2, which basically comprises the HA2 chain without the transmembrane and fusion peptides (Bullough et al., 1994), we calculate SH = 0 to −0.64, corresponding to tilt angles of 54 to 90°: Since the longest helices of HA at neutral and acidic pH are nearly parallel to the trimer axis (166 and 169°), the observed decrease in R ATR indicates a profound tilt of the HA trimers toward the membrane surface. Reneutralization resulted in an increase in R ATR to 1.83(20.05), indicating restoration of the initial vertical orientation of HA to 50 to 100% (Figure 1d and Table 1). Injection of negatively charged vesicles into reconstituted HA at pH 7.4 increased the lipid absorbance bands, indicating binding of vesicles (Figure 1a). The amide I dichroic ratio decreased to 1.82 and 1.79 for 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) vesicles containing 40% 1palmitoyl-2-oleoylphosphatidylglycerol (POPG) or 20% cardiolipin (CL), respectively (Figure 1e and Table 1). Lowering the pH to 5 in the presence of bound vesicles caused a further decrease in R ATR to 1.66 to 1.70, which yields helical tilt angles of 57 to 82° (Figure 1f and Table 1). Thus, the binding of negatively charged membranes induces a small inclination of HA trimers and acidification in the presence of bound vesicles results in a strongly tilted conformation of HA which does not revert after reneutralization (Figure 1g and Table 1). Because of a 3 to 5 cm−1 shift between parallel and perpendicular polarized amide I bands (Figure 1, see also Reisdorf & Krimm, 1995), and because of
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Figure 1. Polarized ATR-FTIR specra of reconstituted HA (strain A/PR/8/34). a, Perpendicular polarized spectra without and with bound vesicles composed of POPC and CL at 4:1 molar ratio. The absorbance bands at 02922 and 02852 cm−1 are due to the antisymmetric and symmetric CH2 stretching vibrations of the lipid acyl chains; the bands at 01735 and 01647 cm−1 result from the lipid carbonyl groups and the amide I vibration of HA, respectively. b to g, Amide I bands at parallel (continuous lines) and perpendicular (shaded areas) polarizations of infrared light without (b, c, d) and with (e, f, g) bound POPC/CL vesicles. The initial pH was 7.4 (b, e), then changed to 5 (c, f) and readjusted to 7.4 (d, g). HA was purified and reconstituted in supported phospholipid bilayers as described (Hinterdorfer et al., 1994; Tatulian et al., 1995). The membranes were flushed with 2H2 O buffers of pH 7.4 or 5 (5 mM Hepes, 10 mM Mes, 135 mM NaCl, pH adjusted with NaOH without corrections for the isotope effect). The ATR dichroic ratio of HA was determined as the ratio of the heights of parallel and perpendicular polarized amide I bands: R ATR = A> /A_ . The helical order parameter was determined as SH = B/fH Sa (B − 3Ez2 ), where B = Ex2 − R ATREy2 + Ez2 (Ex , Ey , and Ez are the electric vector components of the infrared light at the germanium/membrane interface), fH is the fraction of residues in helices of similar orientation, Sa = (3 cos2a − 1)/2, and a is the angle between the transition dipole moment and the helical axis (39°). The angles of orientation of the major helices relative to the membrane normal (u) were evaluated from: SH = (3cos2u − 1)/2. All experiments were carried out at room temperature and the system was kept for 35 minutes at each experimental condition.
different orientations of individual secondary structure elements, the dichroic ratio varies within the amide I region (Figure 2). The dichroic spectra of Figure 2a demonstrate that acidification in the absence of vesicles decreases the amide I R ATR of
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Table 1. ATR dichroic ratios of the amide I bands of reconstituted influenza hemagglutinin under different experimental conditions R ATR pH 7.4 7.4 (flush) 5.0 7.4
No vesicles
POPC/POPG
POPC/CL
1.9020.04 1.9020.04 1.6820.04 1.8320.05
1.8220.04 1.8220.03 1.7020.03 1.7120.03
1.7920.05 1.7820.04 1.6620.06 1.6720.05
The experimental conditions were changed from top to bottom.
HA by 0.2 to 0.3 unit and reneutralization results in a restoration of R ATR to a large extent. The R ATR decreases upon the binding of target membranes at neutral pH and acidification with bound membranes further decreases R ATR, which remains at this low level after reneutralization (Figure 2b). The acid-induced tilting motion of HA trimers which are bound to both (viral and target) membranes by the transmembrane and fusion peptides may bring the membranes to a close contact and thus serve as a critical step in membrane fusion. The orientational change of HA described here is likely related to the pH-dependent conformational change of HA. An acid-induced loop-helix transition of residues 56 to 75 of the HA2 chain moves the fusion peptide to the top of the trimer (Bullough et al., 1994; Carr & Kim, 1993). This causes a transition to a loop of a segment of the longest a-helix (residues 106 to 112 at the base of HA2), whose helical structure at neutral pH is stabilized by extensive interactions with the fusion peptide, resulting in an inversion of the most C-terminal part of the long helix (Bullough et al., 1994). We
Figure 2. Infrared dichroic spectra of reconstituted HA in the amide I region in the absence (a) and presence (b) of bound POPC/CL vesicles (4:1, mol/mol). The pH initially was 7.4 (broken lines), then was shifted to 5 (dotted lines) and readjusted to 7.4 (continuous lines). Unilamellar vesicles of 100 nm diameter were prepared as described (Tatulian et al., 1995). The dichroic spectra were obtained by dividing the parallel polarized absorbance spectra by the perpendicular polarized ones.
Figure 3. Binding of phospholipid vesicles to reconstituted HA in planar supported membranes as measured by the integrated absorbance due to the stretching vibrations of the lipid acyl chain CH2 groups (2990 to 2820 cm−1 ). Three different sequential pH protocols were adopted: (a) vesicles were injected into reconstituted HA at pH 7.4, the cell was flushed with pH 7.4 buffer, the pH was decreased to 5 and readjusted to 7.4; (b) the pH was changed from 7.4 to 5, vesicles were added, and the cell was flushed with pH 5 and then with pH 7.4 buffers; (c) reconstituted HA was preincubated at pH 5, vesicles were added at pH 7.4, and the cell was flushed with pH 7.4 buffer. Squares, triangles, and circles correspond to 100% POPC, 60% POPC + 40% POPG, and 80% POPC + 20% CL vesicles, respectively. Vesicles were prepared in 2H2 O buffers at the pH of injection. All experiments were carried out at room temperature and the system was kept for 35 minutes at each experimental condition.
hypothesize that this segment may serve as a flexible hinge in the tilting motion of HA. A synthetic peptide corresponding to the loop56-75 and a fraction of the long helix of HA2 was shown to undergo a reversible acid-induced change from a monomeric irregular structure to a trimeric helical coiled coil (Yu et al., 1994). Therefore, one might speculate that the pH-dependent structural change of this stretch could be reversible even in the full-length HA, resulting in a partial sequestering of the fusion peptide and possibly other hydrophobic parts of HA upon reneutralization. Exposure and seclusion of the fusion peptide are expected to change the amphiphilic character and hence the lipid-binding capability of HA. When vesicles of POPC/CL, POPC/POPG, and pure POPC were bound to reconstituted HA at pH 7.4, the integrated absorbance due to the lipid acyl chain CH2 groups increased 5-, 3.5-, and 2.5-fold, respectively (Figure 3a). This binding is likely mediated by electrostatic interaction between the cationic globular head of HA and negatively charged membranes and van der Waals attraction. The difference between the binding of POPC/CL (4:1) and POPC/POPG (3:2) vesicles, which have similar surface charge density, indicates that additional (e.g. steric) factors are involved in vesicle-HA interactions. At pH 5, the capacity of reconstituted HA to bind POPC/CL vesicles was substantially increased (Figure 3b), evidently due to hydrophobic interactions between the exposed fusion peptide and the membranes (Harter et al.,
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1989) and additional protonation of HA. The amount of bound vesicles stays at the same level upon reneutralization, implying that the electrostatic effects play only a minor part in the acid-induced increase in vesicle binding to HA. Strikingly, when the vesicles were injected at pH 7.4 after HA was subjected to a pH cycle from 7.4 to 5 to 7.4, the vesicle binding was similar to that when they were injected at pH 7.4 without preincubation of HA at low pH (Figure 3c), indicating that the exposure of the fusion peptide and/or other amphiphilic structures at low pH in the absence of target membranes is reversed upon reneutralization. The data of Figure 3 are consistent with our results indicating that the acid-induced inclination of HA is reversible in the absence, but irreversible in the presence, of bound target membranes. The results of Figure 3 imply that after acid treatment for 35 minutes HA is not inactivated in terms of its lipid binding properties. Inactivation of influenza HA at low pH, in the absence of target membranes, is strongly dependent on temperature and on the virus strain. Treatment of X-47 virus at pH 5.2 for 15 minutes inactivated the virus above 30°C but had no effect at or below 20°C (Junankar & Cherry, 1986). The half-time of the inactivation of the strain PR8 virus at pH 5.2 is 30 seconds at 37°C and ten minutes at 0°C (Sato et al., 1983). After incubation of this virus for 35 minutes at room temperature, still 025% of the hemolytic activity is retained (Yewdell et al., 1983). On the other hand, the inactivation is believed to be caused by immobilization of HA due to its lateral aggregation, which requires the HA molecules to be mobile in the viral membrane (Junankar & Cherry, 1986; Gutman et al., 1993). Earlier work in this laboratory, using fluorescence recovery after photobleaching, showed that HA is immobile and therefore unable to undergo lateral aggregation in supported bilayers (P. Hinterdorfer, unpublished results). We also demonstrated that the fusion activity of PR8 HA in supported bilayers, as measured by the spread of lipid dye, declines only slightly when it is exposed to pH 5 buffer at room temperature over a time period of 60 minutes (L. Tamm, unpublished results; Hinterdorfer et al., 1994). These considerations imply that under our experimental conditions, when HA is reconstituted in supported membranes at room temperature, it can largely escape inactivation at low pH. Reversible components of the conformational change of HA are more readily detected in supported membranes than in suspensions of the virus with or without target membranes because the HA molecules are confined to a single membrane and their interaction with other membranes is prevented. Our findings lead to a new understanding of the energetics of the structural change of HA. According to the irreversible kinetic mechanism, which is currently more widely accepted, the native HA is kinetically prevented from adopting the more stable low pH structure;
lowering the pH reduces the energy barrier between the two states and thereby triggers the structural change. The alternative thermodynamic concept suggests that changes in pH switch the energy levels of the two states, resulting in a reversible mechanism of the conformational change (Baker & Agard, 1994). Our results show that the conformational change of HA per se, i.e. in the absence of target or viral membranes, is reversible and therefore thermodynamically controlled. The apparent irreversibility of the structural change in the presence of membranes is evidently due to mechanical obstructions and hydrophobic interactions between HA molecules and bound membranes. It should be noted that we have not yet shown unequivocally that the transmembrane domain of HA properly spans the supported membrane in our experiments. Formally, it is possible that HA is inserted into the supported membrane with a C-terminal hairpin loop. If this is the case, the reversible components we are observing may apply to the reactions leading to hemi-fusion rather than to those resulting in the final fusion product (Kemble et al., 1994). Finally, our results may be of epidemiological and immunological significance because the reversibility in the absence of target membranes would allow the virions to survive under diverse environmental conditions, and the HA molecules are less likely digested by the proteases of the host immune system before fusion and transfer of the viral genome into the cytosol take place.
References Baker, D. & Agard, D. A. (1994). Influenza hemagglutinin: kinetic control of protein function. Structure, 2, 907–910. Bullough, P. A., Hughson, F. M., Skehel, J. J. & Wiley, D. C. (1994). Structure of influenza hemagglutinin at the pH of membrane fusion. Nature, 371, 37–43. Carr, C. M. & Kim, P. S. (1993). A spring-loaded mechanism for the conformational change of influenza hemagglutinin. Cell, 73, 823–832. Clague, M. J., Schoch, C., Zech, L. & Blumenthal, R. (1990). Gating kinetics of pH-activated membrane fusion of vesicular stomatitis virus with cells: stopped-flow measurements by dequenching of octadecylrhodamine fluorescence. Biochemistry, 29, 1303–1308. Doms, R. W. & Helenius, A. (1986). Quaternary structure of influenza virus hemagglutinin after acid treatment. J. Virol. 60, 833–839. Doms, R. W., Helenius, A. & White, J. (1985). Membrane fusion activity of the influenza virus hemagglutinin. The low pH-induced conformational change. J. Biol. Chem. 260, 2973–2981. Gaudin, Y., Ruigrok, R. W. H., Knossow, M. & Flamand, A. (1993). Low-pH conformational changes of rabies virus glycoprotein and their role in membrane fusion. J. Virol. 67, 1365–1372. Gething, M.-J., Doms, R. W., York, D. & White, J. (1986). Studies on the mechanism of membrane fusion: site-specific mutagenesis of the hemagglutinin of influenza virus. J. Cell Biol. 102, 11–23.
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Gutman, O., Danieli, T., White, J. M. & Henis, Y. I. (1993) Effects of exposure to low pH on the lateral mobility of influenza hemagglutinin expressed at the cell surface: correlation between mobility inhibition and inactivation. Biochemistry, 32, 101–106. Harter, C., James, P., Ba¨chi, T., Semezna, G. & Brunner, J. (1989). Hydrophobic binding of the ectodomain of influenza hemagglutinin to membranes accurs through the ‘‘fusion peptide’’. J. Biol. Chem. 264, 6459–6464. Hinterdorfer, P., Baber, G. & Tamm, L. K. (1994). Reconstitution of membrane fusion sites. A total internal reflection fluorescence microscopy study of influenza hemagglutinin-mediated membrane fusion. J. Biol. Chem. 269, 20360–20368. Junankar, P. R. & Cherry, R. J. (1986). Temperature and pH dependence of the haemolytic activity of influenza virus and of the rotational mobility of the spike glycoproteins. Biochim. Biophys. Acta, 854, 198–206. Kemble, G. W., Danieli, T. & White, J. M. (1994) Lipid-anchored influenza hemagglutinin promotes hemifusion, not complete fusion. Cell, 76, 383–391. Krumbiegel, M., Herrmann, A. & Blumenthal, R. (1994). Kinetics of the low pH-induced conformational changes and fusogenic activity of influenza hemagglutinin. Biophys. J. 67, 2355–2360. Reisdorf, W. C., Jr & Krimm, S. (1995). Infrared dichroism of amide I and amide II modes of aI- and aII-helix segments in membrane proteins. Biophys. J. 69, 271–273. Romalho-Santos, J., Nir, S., Du¨zgu¨nes, N., Carvalho, A. P. & Lima, M. P. C. (1993). A common mechanism for influenza virus fusion activity and inactivation. Biochemistry, 32, 2771–2779. Sato, S. B., Kawasaki, K. & Ohnishi, S. (1983). Hemolytic activity of influenza virus hemagglutinin glycoproteins activated in mildly acidic environments. Proc. Natl Acad. Sci. USA, 80, 3153–3157. Siegel, D. P. (1993). Energetics of intermediates in membrane fusion: comparison of stalk and inverted micellar intermediate mechanisms. Biophys. J. 65, 2124–2140. Stegmann, T. & Helenius, A. (1993). Influenza virus fusion: from models towards a mechanism. In Viral Fusion Mechanisms (Bentz, J., ed.), pp. 89–111, CRC Press, Boca Raton, FL.
Stegmann, T., Booy, F. P. & Wilschut, J. (1987). Effect of low pH on influenza virus. Activation and inactivation of the membrane fusion capacity of the hemagglutinin. J. Biol. Chem. 262, 17744– 17749. Stegmann, T., White, J. M. & Helenius, A. (1990). Intermediates in influenza induced membrane fusion. EMBO J. 9, 4231–4241. Stegmann, T., Delfino, J. M., Richards, F. M. & Helenius, A. (1991). The HA2 subunit of influenza hemagglutinin inserts into the target membrane prior to fusion. J. Biol. Chem. 266, 18404–18410. Tatulian, S. A., Hinterdorfer, P., Baber, G. & Tamm, L. K. (1995). Influenza hemagglutinin assumes a tilted conformation during memebrane fusion as detected by attenuated total reflection FTIR spectroscopy. EMBO J. 14, 5514–5523. Tsurudome, M., Glu¨ck, R., Graf, R., Falchetto, R., Schaller, U. & Brunner, J. (1992). Lipid interactions of the hemagglutinin HA2 NH2-terminal segment during influenza virus-induced membrane fusion. J. Biol. Chem. 267, 20225–20232. White, J. M. (1994). Fusion of influenza virus in endosomes: role of the hemagglutinin. In Cellular Receptors for Animal Viruses (Wimmer, E., ed.), pp. 281–301, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. White, J. M. & Wilson, I. A. (1987). Anti-peptide antibodies detect steps in a protein conformational change: low-pH activation of the influenza virus hemagglutinin. J. Cell Biol. 105, 2887–2896. Wiley, D. C. & Skehel, J. J. (1987) The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. Annu. Rev. Biochem. 56, 365–394. Wilson, I. A., Skehel, J. J. & Wiley, D. C. (1981). Structure of the hemagglutinin membrane glycoprotein of ˚ resolution. Nature, 289, influenza virus at 3 A 366–373. Yewdell, J. W., Gerhard, W. & Ba¨chi, T. (1983). Monoclonal anti-hemagglutinin antibodies detect irreversible antigenic alterations that coincide with the acid activation of influenza A/PR/834-mediated hemolysis. J. Virol. 48, 239–248. Yu, Y. G., King, D. S. & Shin, Y.-K. (1994). Insertion of a coiled-coil peptide from influenza virus hemagglutinin into membranes. Science, 266, 274–276.
Edited by R. Huber (Received 22 November 1995; received in revised form 25 April 1996; accepted 1 May 1996)