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Measuring pKa of activation and pKi of inactivation for influenza hemagglutinin from kinetics of membrane fusion of virions and of HA expressing cells
Biophysical Journal, Nov 2002 by Mittal, Aditya, Shangguan, Tong, Bentz, Joe
ABSTRACT The data for the pH dependence of lipid mixing between influenza virus (A/PR/8/34 strain) and fluorescently labeled liposomes containing gangliosides has been analyzed using a comprehensive mass action kinetic model for hemaglutinin (HA)-mediated fusion. Quantitative results obtained about the architecture of HA-mediated membrane fusion site from this analysis are in agreement with the previously reported results from analyses of data for HA-expressing cells fusing with various target membranes. Of the eight or more HAs forming a fusogenic aggregate, only two have to undergo the "essential" conformational change needed to initiate fusion. The mass action kinetic model has been extended to allow the analysis of the pKa for HA activation and pKi for HA inactivation. Inactivation and activation of HA following protonation were investigated for various experimental systems involving different strains of HA (A/PR/8/34, X:31, A/Japan). We find that the pKa for the final protonation site on each monomer of the trimer molecule is 5.6 to 5.7, irrespective of the strain. We also find that the pKi for the PR/8 strain is 4.8 to 4.9. The inactivation rate constants for HA, measured from experiments done with PR/8 virions fusing with liposomes and X:31 HA-expressing cells fusing with red blood cells, were both found to be of the order of 10^sup -4^ s^sup -1^. This number appears to be the minimal rate for HA's essential conformational change at low HA surface density. At high HA surface densities, we find evidence for cooperativity in the conformational change, as suggested by other studies.
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INTRODUCTION
Elucidation of influenza HA-mediated membrane fusion site architecture has been the focus of intense research since it was the first fusion protein whose crystal structure was solved (Wilson et al., 1981; Bullough et al., 1994) and its structure is related to other fusion proteins (Skehel and Wiley, 1998). Furthermore, it is the only membrane fusion system for which there is quantitative data that can be used to deduce how many fusion proteins are required at the fusion site (Bentz et al., 1990; Ellens et al., 1990; Melikyan et al., 1995; Danieli et al., 1996; Blumenthal et al., 1996; Bentz, 2000a; Mittal and Bentz, 2001). Thus, the architecture of its fusion site is being elucidated.
Recently, Bentz (2000a) began development of a comprehensive mass-action model for HA-mediated fusion to analyze the first fusion pore kinetics measured by Melikyan et al. (1995). The model was extended in Mittal and Bentz (2001) to extract consensus parameters for the data of Melikyan et al. (1995), Danieli et al. (1996), and Blumenthal et al. (1996) for HA-expressing cells fusing with various target membranes. The model includes a rigorous distinction between the minimum number of HA trimers aggregated at the nascent fusion site and how many of those trimers that must undergo a slow essential conformational change before the first conductivity can be measured across the fusing systems. This distinction allowed us to show that HAs bound to sialates on glycophorin could be members of the fusogenic aggregate but not undergo the essential conformational change needed to form the first fusion pore (Mittal and Bentz, 2001).
Assuming a nucleation model for HA aggregation, it was found that at least eight HAs must aggregate to form the fusogenic aggregate that results in initiating membrane fusion. This nucleation model is unlikely to accurately describe the true distribution of HA aggregates over the cell population, but it yields the minimum estimate for the number of HAs required to form the fusogenic aggregate. In other words, more realistic distributions would require that there are more than eight HAs in a fusogenic aggregate (Bentz, 2000a). Thus, the minimal aggregate size is co = 8, and of these, only two need to undergo the slow essential conformational change required to initiate fusion.
However, it remained to be shown the extent to which the results for HA-expressing cells were applicable to the virus fusing with target membranes. Further, the HA surface density on virions is reasonably constant (Ruigrok et al., 1984, 1985), so the noise associated with surface density heterogeneity in the data is negligible, as compared with the data of HA-expressing cells fusing with target membranes. Although the surface density of HA on virions cannot be reduced without causing surface density heterogeneity, we can homogeneously reduce the surface density of "active" HA by raising the pH. This approach was used by Doms et al. (1985) and Blumenthal (1988) to estimate the number of HAs required for fusion. Here we analyzed the kinetic data of the influenza virus fusing with the ganglioside GDla containing liposomes from Shangguan (1995) and Shangguan et al. (1996, 1998). We find that the approach can estimate the number of fusogenic aggregates in the area of contact with the target membrane.
The mass-action model used in Mittal and Bentz (2001) has been extended here to include activation and inactivation kinetics following protonation of HA. We have analyzed the inactivation data for the A/PR/8/34 strain of HA in virions (Shangguan et al., 1998), the X:31 strain of HA expressed in cells (Leikina et al., 2000), and the pH-dependent activation data for Japan strain expressed in cells (Mittal et al., 2002).
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