Published as doi: 10.1096/fj.06-7052hyp.
(The FASEB Journal. 2007;21:995-1002.)
© 2007 FASEB
The relevance of salt bridges for the stability of the influenza virus hemagglutinin
P. Sivaramakrishna Rachakonda*,
Michael Veit
,
Thomas Korte*,
Kai Ludwig
,
Christoph Böttcher,
Qiang Huang
,
Michael F. G. Schmidt and
Andreas Herrmann*,1
* Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, Institut für Biologie/Biophysik, Berlin, Germany;
Institut für Immunologie und Molekularbiologie, Vet.-Med. Fakultät, Freie Universität Berlin, Berlin, Germany;
Forschungszentrum für Elektronenmikroskopie, Freie Universität Berlin, Berlin, Germany; and
State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China
1Correspondence: Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, Institut für Biologie/Biophysik, Invalidenstr. 43, D-10115 Berlin, Germany. E-mail: andreas.herrmann{at}rz.hu-berlin.de
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ABSTRACT
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Hemagglutinin (HA) of influenza virus undergoes an irreversible conformational change at acidic pH, mediating viral fusion with the host endosomal membrane. To unravel the molecular basis of the pH-dependent stability of HA, we demonstrate by mutagenesis of the prototype HA of virus strain X31 (H3 subtype) that salt bridges, especially a tetrad salt bridge within the monomers, are crucial for folding and stability of the trimeric ectodomain. This complex (tetrad) salt bridge is highly conserved among influenza virus subtypes. Introducing additional sites of electrostatic attraction between monomers in the distal region enhanced the stability of ectodomain at low pH mimicking the natural variant H2 subtype. We propose that distinct salt bridges in the distal domain may contribute to the enhanced stability of HA of natural virus variants. This hypothesis may provide clues to understanding adaptations of virus strains (for example, avian influenza viruses) in order to preserve stability of the protein in the host-specific environment.—Rachakonda, P. S., Veit, M., Korte, T., Ludwig, K., Böttcher, C., Huang, Q., Schmidt, M. F. G., Herrmann, A. The relevance of salt bridges for the stability of the influenza virus hemagglutinin.
Key Words: conformational change • fusion • ectodomain • influenza virus HA • stability
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INTRODUCTION
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BINDING OF THE INFLUENZA VIRUS to the host cell and fusion of the viral envelope with the endosomal membrane are mediated by the glycoprotein hemagglutinin (HA). HA is organized as a noncovalent-associated homotrimer. Each monomer is post-translationally cleaved into disulfide-linked HA1 and HA2. The 3-dimensional (3D) crystal structure of the whole HA ectodomain at neutral pH is known for influenza viruses of various subtypes at atomic resolution (1
2
3
4
5)
.
After endocytic uptake of the virus, the acidic pH in the endosomal lumen triggers an irreversible conformational change of HA acquiring a fusion-competent state (6
, 7)
. The hinge region connecting the two antiparallel oriented 
-helical segments of HA2 in the neutral pH conformation undergoes a loop-to--helix transition. As a result, an extended trimeric rod-like coiled-coil structure is formed (8
9
10)
and the first 20 amino acids of the N terminus of HA2, the fusion peptide is moved to the distal end of HA and exposed to the target membrane (11)
. Destabilization of the target membrane by the fusion peptide is thought to initiate membrane fusion (12
, 13)
. However, alternative models for the role of the fusion peptide suggest that the fusion peptide incorporates into the viral membrane. Kozlov and Chernomordik (14)
proposed that formation of the extended coiled-coil pulls the peptides, and thereby bends the viral membrane toward the bound target membrane. Bentz (15)
suggested that refolding of the central rod extracts the fusion peptide from the viral envelope, causing a hydrophobic void that is "healed" by recruiting lipids from the target membrane along the center of closely packed HAs.
The mechanism underlying the low pH-triggered destabilization of the HA ectodomain is not known. The structure of any protein and its stability are based on noncovalent interactions like hydrophobic forces, van der Waal interactions, hydrogen bonds, and ionic interactions. Although the contribution of each interaction to the stability varies among proteins, alteration of the pH essentially effects ionic interactions and salt bridges (16)
.
We hypothesize that electrostatic forces—in particular, salt bridges between the two subunits of a monomer (intramonomer) and between monomers (intermonomer)—play an essential role for the pH-dependent stability of the ectodomain. Indeed, protonation of charged amino acids at acidic pH reduces the electrostatic attraction between the HA1 and HA2 subunits. Molecular modeling studies (17)
have shown a strong electrostatic attraction between the HA1 subunits (positively charged) and the HA2 subunits (negatively charged) at neutral pH. Thus, at neutral pH, the electrostatic repulsion between the three HA1 subunits on the one hand and that between the three HA2 subunits on the other is overcome by electrostatic attraction between the HA1 and HA2 domains, preserving a stable trimeric association of the monomers. Moreover, our hypothesis gains support from the fact that the 3D structure of HA (X-31 strain, H3 subtype, PDB 1HGD) protein at neutral pH is stabilized by an extensive network of 15 single and complex salt bridges. A statistical analysis of 3D structures of at least 94 oligomeric proteins in the PDB database revealed that HA of influenza virus X31 has the highest number of complex salt bridges (18)
. A single salt bridge is a pair of charged amino acid residues, whereas a complex salt bridge consists of more than two residues in single or adjacent protein chains (18)
.
The present study provides further evidence for the hypothesis of the vital role of salt bridges for the pH-dependent stability of the HA ectodomain (X31) either by eliminating salt bridges or by introducing additional sites for salt bridges by site-directed mutagenesis. First, the ectodomain of mutant HA lacking a highly conserved intramonomer salt bridge became less stable at acidic pH compared with wild-type HA (wt-HA). Second, the introduction of additional intra- or intermonomer sites for electrostatic attraction (e.g., in the distal HA1 domain of X31) caused significant stabilization of the ectodomain and shifted the threshold of the conformational change to a more acidic pH. The ensuing hypothesis discusses how this observation could increase an understanding of the enhanced stability of the ectodomain of other influenza viruses.
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Selection and characterization of mutants
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Identification of residues involved in salt bridges was based on the crystal structure of the HA ectodomain of X31 (PDB 1HGD) (1)
and on the criterion that the distance of interacting residues of a salt bridge was less than 4Å (19)
(Fig. 1
). Two intramonomer salt bridges (Fig. 1A
) are located at the HA1-HA2 interface of the hinge region that undergoes a loop-to--helix transition at low pH. This interface is formed by residues 85–90, 104–115, and 265–270 of the HA1 subunit and 64–72 of the HA2 subunit. One of the salt bridges corresponds to a tetrad arrangement formed by residues E89, R109, R269 of HA1, and E67 of HA2. Selected residues of these bridges were mutated either to prevent the formation of salt bridges or to generate an electrostatic repulsion between residues.
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Figure 1. Site-directed mutations in the HA ectodomain (X31; 1HGD). HA1 and HA2 subunits are in gray and black, respectively (A, B: side views, C: top view). A) Sites selected for disruption of native salt bridges. Colored residues form intramonomer salt bridges: the tetrad with E89/R109/R269-E67 and K299-E69. B) Y308-I89 were selected for insertion of an additional intramonomer salt bridge. C) T212-N216 were chosen to stabilize intermonomer interactions (HA1) by electrostatic attraction.
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The degree of conservation, and thereby their evolutionary significance, was assessed by aligning all the available homologous amino acid sequences in PDB database on the crystal structure of 1HGD using the software tool "consurf 3D" (20)
. We found that typically any substitution of residues belonging to those salt bridges strictly preserved the charge. Amino acids involved in the tetrad salt bridge R109 and E89 of HA1 and E67 of HA2 are highly conserved except for R269 of HA1. In particular, R109 is conserved to a very high degree among influenza A subtypes and is rarely substituted by Lys(K), preserving the positive charge. Likewise, E67 of HA2 is replaced in some strains by the negatively charged Asp(D). Conservation of the tetrad salt bridge in the interface region of HA1 and HA2 is confirmed by an analysis of the crystal structure of HA of other subtypes (not shown). Residues in the salt bridge K299-E69 are also highly conserved.
To corroborate the role of electrostatic interactions for the stability of the HA ectodomain, the interacting residues Y308-I89 were selected to generate mutants with additional electrostatic attraction between HA1 and HA2 within a monomer (Fig. 1B
). These sites were chosen because of the proximity of respective residues of HA1 and HA2, thus allowing the insertion of additional salt bridges. For Y308-I89, two different mutants were designed: Y308R–I89E and the single mutant I89R.
To explore to what extent electrostatic attractions between monomers can stabilize the neutral pH structure of the HA ectodomain, two mutations (T212E-N216R and T212E-N216E) were generated in the distal domain of HA1 (Fig. 1C
).
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Transient expression of the mutants
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Wild-type HA and mutants (R109E, R109G, R269E, R269G, K299E, K299G, T212E-N216R, T212E-N216E, I89R, and I89E-Y308R) were expressed in CV-1 cells. Cells were treated with trypsin (TPCK-trypsin) in order to assess surface expression of HA. Only HA expressed at the cell surface can be cleaved by trypsin into HA1 and HA2. Except for the mutant R109E (Fig. 2
, T212E-N216R and T212E-N216E, not shown), all mutants were found to be expressed on the surface of CV-1 cells.
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Figure 2. Surface expression of wt-HA and destabilizing mutants. Cleavage of HA to HA1 and HA2 was assessed with (+) and without (–) trypsin. Cells were lysed and immunoprecipitated using the HA trimer-specific N2 antibody (19)
. R109E was stuck intracellularly and resistant to trypsin treatment.
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An essential precondition for the validity of data was that the site-directed mutations did not affect the protein folding and structure of the ectodomain. To characterize and compare wild-type and mutant HA assays for trimerization, glycosylation and conformational changes (proteinase K assay) were made by immunoprecipitation using the N2 antibody, which specifically recognizes HA trimeric form at neutral pH (21)
. As shown for trimerization and the proteinase K assay (see below), immunoprecipitation revealed that wt-HA and mutant HAs are expressed to a similar extent.
Treatment of cells with the chemical cross-linker 3-3'-dithiobissuccinymidylpropionate (DSP) revealed that all mutants, including R109E, were organized as trimers to a degree similar to that of wt-HA (see supplemental information: cross-linking of HA monomers).
The glycosylation pattern of wt-HA and mutants was analyzed by treatment with glycosidases-endoglycosidase H (Endo H) and peptide-N-glycosidase F (PNGase F). Endo H removes simple, high-mannose N-linked oligosaccharides characteristic of ER localization; PNGase F digests all N-linked oligosaccharides regardless of their state of processing (22)
. Glycosylation of mutants was similar to that of wt-HA (data not shown) except for R109E. Whereas R109G and wt-HA were resistant to Endo H, R109E was sensitive to Endo H treatment, which indicated that this mutant was trapped in either the ER or cis-Golgi due to improper folding.
Thus, except for the mutant R109E (Fig. 2
, T212E-N216R and T212E-N216E; not shown), all mutants were found to have properties similar to those of wt-HA in terms of surface expression, trimerization, and glycosylation, indicating a proper folding of the mutants.
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Destabilizing mutations show higher pH threshold
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We measured the pH threshold at which the conformational change of the HA ectodomain was triggered. HA-expressing cells were incubated at various pH, then treated with proteinase K. On low pH-triggered conformational change, the ectodomain becomes sensitive to proteinase K (23)
. For wt-HA, the threshold is at pH 5.4. Mutants with deleted salt bridges were found to become sensitive to proteinase K treatment at a less acidic pH (Fig. 3
and supplemental information: proteinase K assay), with a shift between 0.2 and 0.6 pH units with respect to wt-HA. Mutations of the tetrad salt bridge (R109, R269) caused a larger pH shift than those of a "simple" salt bridge (K299-E69). Furthermore, a mutation in R109 caused a stronger destabilization of the tetrad salt bridge than of R269. In particular, the substitution of R109 by an amino acid of opposite charge (Glu) had a dramatic effect for the surface expression of HA. This indicates that the tetrad salt bridge is essential for folding and stability of the native ectodomain structure of HA. Moreover, R109 is a key residue of this salt bridge. Arg(R) has been found to be the predominant residue in most of the complex salt bridges in proteins (18)
and more often in the interface regions of oligomeric proteins (24)
. This also signifies the conservation of R109.
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Figure 3. pH dependence of the conformational change of HA ectodomain with mutations. A) R109G; B) R269E and R299G; C) K299E and K299G; D) Y308R-I89E and I89R; E) T212E-N216R. For comparison, wt-HA was treated in parallel. Proteinase K assay was performed as described (23). Gel bands (HA1 band) were quantified by ScanPak. The percentage of nonproteolyzed HA is shown as a function of pH (average±SE of estimate of 3 experiments)
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To confirm the importance of the tetrad salt bridge, we screened natural variants of influenza virus subtypes (25
26
27
28
29)
selected for their resistance to antiviral drugs raising the endosomal pH (e.g., amantadine hydrochloride). Variants showed fusion at a higher pH than wt-HA. Analysis of X31 and Weybridge viruses (25)
revealed that 75% of the mutations involved charged residues. Typically, mutations were found either at the interface regions (HA1-HA2 or HA2-HA2) or in the N terminus of HA2. This supports the relevance of electrostatic interactions between subunits within a monomer and between the positively charged N terminus of HA2 with negatively charged residues of the HA1 cavity (26
, 30
, 31)
for the stability of HA. Except for R269, however, no substitutions changing the charge of residues were found in any of the natural mutants for those salt bridges considered in our study. This again points to the crucial role of the selected salt bridges.
For mutations of R269 and K299, destabilization was independent of whether the native residue was substituted by a neutral residue (Gly) or by a residue of opposite charge. Only a slight shift of the threshold to higher pH values was observed when Lys(K) residues were substituted for by Glu(E) (see Fig. 3B, C
; compare mutants R269G and R269E, K299G and K299E).
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Stabilizing mutations enhance resistance to pH changes
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The mutation (Y308R-I89E) designed to strengthen the intramonomer interaction between HA1 and HA2 showed a significant increase of HA resistance to proteinase K treatment upon incubation at low pH (Fig. 3)
. At pH 5.0, a significant fraction of mutant HA remained resistant to degradation by proteinase K whereas wt-HA was almost completely degraded. Even the single mutation I89R in HA2 increased the stability of the HA. We surmise that a cation-
interaction between Y308 of HA1 and I89R of HA2 is responsible for the enhanced stability. As shown recently, cation- interactions (32)
are typical for protein-protein interfaces and are most abundant between Arg(R) and Tyr(Y) residues. Our results suggest that insertion of just one site of attraction along the interface of HA1 and HA2 may be sufficient to enhance significantly the stability of the ectodomain. These mutants signify the role of electrostatic forces for the stability of the HA ectodomain.
Intermonomer-stabilizing mutant (T212E-N216R) also strongly inhibited the low pH-triggered conformational change (Fig. 3)
. Even at pH 4.7, a major fraction of HA was not destroyed proteolytically by proteinase K (Fig. 3)
. When residues of the same charge were introduced (T212E-N216E), a slight shift of the threshold to higher pH values with respect to wt-HA was noted (not shown). This mutant confirms previous observations that locking of the HA1 distal domains interferes with the conformational change of HA (33
, 34)
. In those studies domains were locked by disulfide bridges formed by T212C-N216C between HA1 subunits. A conformational change and fusion were observed only after breaking the disulfide linkages. Our approach shows that even noncovalent interaction via electrostatic attraction is sufficient to clamp the domains efficiently, and supports the notion that a rearrangement of the distal HA1 globular domains is required for formation of the fusion active conformation (33
34
35)
.
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Fusion assay with double-labeled RBCs
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Human RBCs were double labeled with the fluorescent dyes R18 and calcein. Membrane fusion (R18) and fusion pore formation (calcein) were monitored at neutral pH (7.4), low pH (5.0), and intermediate pH values 5.4 and 5.6 at 37°C. Binding of RBCs to the mutants was comparable to that of wild-type (mutant R109E was not investigated for fusion activity) (Fig. 4
), which indicates that all the mutants and wt-HA have similar surface expression. This agrees with the immunoprecipitation data showing that wt-HA and mutant HAs are expressed to a similar extent (see above).
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Figure 4. Fusion activity of wt-HA and mutant HAs. Membrane fusion and pore formation were measured by redistribution of R18 (left panel, red fluorescent) and calcein (right panel, green fluorescent), respectively. Wt-HA and mutant HA-expressing cells were bound to double-labeled RBC (top row). Images were taken 5 min after the pH trigger.
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For wt-HA as well as for HA with destabilizing mutations, extensive fusion was observed at pH 
5.4 (Fig. 4
, only shown for R109G, R269E, K299E), also indicating that mutant HAs were functional and thus properly folded. For destabilizing mutations, fusion events could be seen even at pH 5.6, which was not observed for wt-HA. This correlates with the shift of pH thresholds of the conformational change observed for these mutants. Gruenke et al. (36)
performed mutagenesis in the loop region of HA2 (55–75 residues) to prevent loop-to--helix transition and therefore the formation of a long extended helix at low pH. The fusion activity of these mutants was either restricted to hemifusion or completely abolished. In contrast, destabilizing mutations in the hinge region abolished neither membrane fusion nor pore formation, indicating that the formation of the long helix was not affected. Nevertheless, our results do not allow us to decide whether the shift of the pH dependence of fusion of HAs with destabilizing mutations is a direct consequence of the shift of pH thresholds of the conformational change or could be related to changes of the 3D structure distant of the mutation sites. Shental-Benchor et al. (37)
provided evidence that destabilizing mutations affect the structural stability as well as function of HA at sites distant from the mutation site. Specifically, they found that mutations in the fusion peptide affected the receptor binding site 
100 Å away.
Fusion activity of HA with stabilizing mutations was either shifted to lower pH values (I89R) or was completely inhibited. At pH 5.4, I89R showed only R18 transfer but not redistribution of calcein among cells, indicating hemifusion. A further decrease in the pH (5.0) caused full fusion of the mutant I89R as deduced from a full transfer of both R18 and calcein (Fig. 4)
. We observed neither membrane fusion nor pore formation for the two other stabilizing mutants, I89E-Y308R and T212E-N216R, in the entire pH range investigated (pH 7–pH 5.0). Efforts to trigger fusion at pH < 5.0 were hampered by cell damage.
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Influence of salt bridges on conformational flexibility of HA
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From simulation of the conformational flexibility of the HA ectodomain by an elastic network model, Isin et al. (38)
identified cooperative molecular motion modes of different frequency leading to global conformational changes. For example, the mode with the lowest frequency suggests a global torsion of the trimer about its longitudinal axes and is essential for the organization of trimers and genesis of the fusion pore. Two categories of sites critically to this mode have been found. Sites with minimum fluctuations are essential for the coordinated cooperative motions of the overall molecule. The regions involved in tetrad salt bridge (85–90, 104–115, 265–270 of HA1, and 64–72 of HA2), and even more, the residues E89, R109, R269 of HA1, and E67 of HA2, show minimum fluctuations that are typical for the hinge region (38)
. This suggests that these salt bridges are not only important for the stability of the ectodomain, but also for the coordination of cooperative motions essential for the fusion process. Sites with maximum fluctuations reflect regions of high flexibility. Regions of such sites are localized in the globular domain of HA1, including residues T212 and N216. It was suggested that this mode induces a partial relaxation in the globular heads, relieving the steric constraints that would resist the refolding of HA at low pH (38)
. The prediction would be that a reduction of the flexibility in the globular domain—for example, by salt bridges—would stabilize the conformation and thus interfere with low pH-triggered conformational change. This is exactly what we have seen for the mutant T212E-N216R.
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Relevance of distal salt bridges for HA stability: a hypothesis
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We wonder whether such a stabilization of the HA ectodomain could be a natural strategy to prevent inactivation of HA. Typically, low pH pretreatment in the absence of the target membrane (particularly at pH 5.0 and 37°C) causes a rapid loss of fusion activity of HA as observed for X31 (39
, 40)
. This is accompanied by a fuzzy morphology of the ectodomain, indicating the dissociation of the monomers (40)
. In contrast, although the pKa of HA activiation is the same as that found for X31 (41)
, the HA ectodomain of A/JPN/305/57 (H2 subtype) was stable at low pH and inactivation of A/JPN/305/57 was slow (39
, 40
, 42)
. Indeed, cryo-electron microscopy has shown that the monomers remain closely associated upon acidification, preserving typical overall features of the trimeric ectodomain at neutral pH (43)
. The molecular basis of the enhanced stability of the HA ectodomain of A/JPN/305/57 is not yet clear. Based on our study, a reasonable hypothesis could be the presence of additional sites of electrostatic attractions, particularly sites in the distal domain of HA1, as demonstrated by the T212E-N216R mutant. Sequence alignment revealed that R208 and E212 of A/JPN/305/57 aligned with T212 and N216 of X31 (Fig. 5
), which may indicate the presence of a salt bridge in the distal region of A/JPN/305/57. The same residues, R208 and E212, were found for the avian influenza virus A/Duck/Singapore/3/97 belonging to subtype H5 (Fig. 5
and Fig. 6
). Notably, as deduced from sequence alignment and a comparison of 3D structures, the ectodomains of H2 and H5 are quite similar (see supplemental information: sequence alignment and comparison of 3D structure). Taking into account that avian influenza viruses replicate in the gastrointestinal tract of birds and are excreted via feces into lake water, those viruses are often exposed to the relatively low pH prevailing in lake water and therefore have to survive acidic environments. As has been shown, viruses with noncleaved HA are able to survive those conditions without inactivation of HA (44)
. We put forward the hypothesis that stabilization of the HA ectodomain by electrostatic attraction could be a natural strategy to adapt to such specific environments. Further studies will clarify whether residues such as R208 and E212 are essential for the observed stability of the H2 strain A/JPN/305/57 and for the stability of HA of viruses from other subtypes as H5. The residue Glu212 of H5 (A/Duck/Singapore/3/97) has been found to be substituted for by residues of opposite charge (Lys or Arg) in the highly human pathogenic H5 strains (45)
from outbreaks in 2004 (Fig. 5B
). This mutation may reduce the stability of the ectodomain at low pH enhancing the infectivity of the H5N1 virus.
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Figure 5. Comparison of the distal HA1 sequence between influenza viruses. A) Sequence alignment of the ectodomains (part of the alignment is shown) of HA from X31 (H3) with those from A/Japan/305/57 (H2) and A/Duck/Singapore/3/97 (H5). Arrows indicate charged amino acid residues (see text). Alignment was done using software clustalw (Jalview). B) A comparison of the distal HA1 sequence between influenza viruses of H5 subtypes. Glu 212 of avian H5 virus A/Duck/Singapore/3/97 is substituted by a residue of opposite charge (Lys or Arg) in the highly human pathogenic H5N1 viruses (A/chick/Macheng/2004; A/chicken/Viet Nam/VL-008/2004; A/goose/China/F3/2004; A/Thailand/3(SP-83)/2004; A/Thailand/5(KK-494)/2004; A/Thailand/2(SP-33)/2004; A/Thailand/1(KAN-1)/2004; A/Thailand/4(SP-528)/2004) identified from outbreaks in 2004 (for details and accession numbers, see ref. 45
). Arrows indicate charged amino acid residues contributing to association of monomers by electrostatic interaction. Alignment was achieved using software clustalw.
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These residues are also conserved and arranged similarly in the HA1 ectodomain of the H1 subtype, e.g., A/PR 8/34 (Arg 212, Glu 216, see Figs. 5
and 6
). Accordingly, one would predict that the ectodomain is stabilized against low pH-triggered inactivation comparable to that of A/JPN/305/57. However, the unfavorable interaction between Glu 219 and Glu 246 due to their close proximity is likely to weaken the stability in the distal region of A/PR8/34 (Fig. 6)
. This would agree with the fast inactivation of this influenza virus strain at low pH observed previously (40
, 46
, 47)
. For the H1 subtype "1918" strain, position 219 corresponds to Ala (Fig. 6)
and should not interfere with ectodomain stability. Unfortunately, the inactivation behavior of this pandemic strain is not known.
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ACKNOWLEDGMENTS
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We are indebted to Dr. Judy White (Department of Cell Biology, University of Virginia Health Systems, Charlottesville, VA, USA) for providing us the pTM1 vector for HA and N2 antibody against HA (X31 strain).
Received for publication August 17, 2006.
Accepted for publication November 9, 2006.
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