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New insights into the spring-loaded conformational change of influenza virus hemagglutinin.

ABSTRACT: Influenza virus hemagglutinin undergoes a conformational change in which a loop-to-helix "spring-loaded" conformational change forms a coiled coil that positions the fusion peptide for interaction with the target bilayer. Previous work has shown that two proline mutations designed to disrupt this change disrupt fusion but did not determine the basis for the fusion defect. In this work, we made six additional mutants with single proline substitutions in the region that undergoes the spring-loaded conformational change and two additional mutants with double proline substitutions in this region. All double mutants were fusion inactive. We analyzed one double mutant, F63P/F70P, as an example. We observed that F63P/F70P undergoes key low-pH-induced conformational changes and binds tightly to target membranes. However, limited proteolysis and electron microscopy observations showed that the mutant forms a coiled coil that is only approximately 50% the length of the wild type, suggesting that it is splayed in its N-terminal half. This work further supports the hypothesis that the spring-loaded conformational change is necessary for fusion. Our data also indicate that the spring-loaded conformational change has another role beyond presenting the fusion peptide to the target membrane.

SUBMITTER: Gruenke JA

PROVIDER: S-EPMC155089 | biostudies-literature | 2002 May

REPOSITORIES: biostudies-literature

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New insights into the spring-loaded conformational change of influenza virus hemagglutinin.

Gruenke Jennifer A JA Armstrong R Todd RT Newcomb William W WW Brown Jay C JC White Judith M JM

Journal of virology 20020501 9

Influenza virus hemagglutinin undergoes a conformational change in which a loop-to-helix "spring-loaded" conformational change forms a coiled coil that positions the fusion peptide for interaction with the target bilayer. Previous work has shown that two proline mutations designed to disrupt this change disrupt fusion but did not determine the basis for the fusion defect. In this work, we made six additional mutants with single proline substitutions in the region that undergoes the spring-loaded ...[more]

PMID: 11932412

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Project description:The conformational change of the influenza virus hemagglutinin (HA) protein mediating the fusion between the virus envelope and the endosomal membrane was hypothesized to be induced by protonation of specific histidine residues since their pKas match the pHs of late endosomes (pK(a) of ? 6.0). However, such critical key histidine residues remain to be identified. We investigated the highly conserved His184 at the HA1-HA1 interface and His110 at the HA1-HA2 interface of highly pathogenic H5N1 HA as potential pH sensors. By replacing both histidines with different amino acids and analyzing the effect of these mutations on conformational change and fusion, we found that His184, but not His110, plays an essential role in the pH dependence of the conformational change of HA. Computational modeling of the protonated His184 revealed that His184 is central in a conserved interaction network possibly regulating the pH dependence of conformational change via its pKa. As the propensity of histidine to get protonated largely depends on its local environment, mutation of residues in the vicinity of histidine may affect its pK(a). The HA of highly pathogenic H5N1 viruses carries a Glu-to-Arg mutation at position 216 close to His184. By mutation of residue 216 in the highly pathogenic as well as the low pathogenic H5 HA, we observed a significant influence on the pH dependence of conformational change and fusion. These results are in support of a pK(a)-modulating effect of neighboring residues.The main pathogenic determinant of influenza viruses, the hemagglutinin (HA) protein, triggers a key step of the infection process: the fusion of the virus envelope with the endosomal membrane releasing the viral genome. Whereas essential aspects of the fusion-inducing mechanism of HA at low pH are well understood, the molecular trigger of the pH-dependent conformational change inducing fusion has been unclear. We provide evidence that His184 regulates the pH dependence of the HA conformational change via its pK(a). Mutations of neighboring residues which may affect the pK(a) of His184 could play an important role in virus adaptation to a specific host. We suggest that mutation of neighboring residue 216, which is present in all highly pathogenic phenotypes of H5N1 influenza virus strains, contributed to the adaptation of these viruses to the human host via its effect on the pKa of His184.

Project description:Transcription factors must scan genomic DNA, recognize the cognate sequence of their control element(s), and bind tightly to them. The DNA recognition process is primarily carried out by their DNA binding domains (DBD), which interact with the cognate site with high affinity and more weakly with any other DNA sequence. DBDs are generally thought to bind to their cognate DNA without changing conformation (lock-and-key). Here, we used nuclear magnetic resonance and circular dichroism to investigate the interplay between DNA recognition and DBD conformation in the engrailed homeodomain (enHD), as a model case for the homeodomain family of eukaryotic DBDs. We found that the conformational ensemble of enHD is rather flexible and becomes gradually more disordered as ionic strength decreases following a Debye-Hückel's dependence. Our analysis indicates that enHD's response to ionic strength is mediated by a built-in electrostatic spring-loaded latch that operates as a conformational transducer. We also found that, at moderate ionic strengths, enHD changes conformation upon binding to cognate DNA. This change is of larger amplitude and somewhat orthogonal to the response to ionic strength. As a consequence, very high ionic strengths (e.g., 700 mM) block the electrostatic-spring-loaded latch and binding to cognate DNA becomes lock-and-key. However, the interplay between enHD conformation and cognate DNA binding is robust across a range of ionic strengths (i.e., 45 to 300 mM) that covers the physiologically-relevant conditions. Therefore, our results demonstrate the presence of a mechanism for the conformational control of cognate DNA recognition on a eukaryotic DBD. This mechanism can function as a signal transducer that locks the DBD in place upon encountering the cognate site during active DNA scanning. The electrostatic-spring-loaded latch of enHD can also enable the fine control of DNA recognition in response to transient changes in local ionic strength induced by variate physiological processes.

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New insights into the spring-loaded conformational change of influenza virus hemagglutinin.

Publications

New insights into the spring-loaded conformational change of influenza virus hemagglutinin.

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