Structural Insights on the Potential Significance of the Twin Asn-Residue Found at the Base of the Hemagglutinin 2 Stalk in All Influenza A H1N1 Strains: A Computational Study with Clinical Implications

Introduction

Influenza A virus is an RNA virus that initiates infection to the host cell by binding to sialic acids found on the host cell surface (Kongchanagul et al., 2008; Skehel and Wiley, 2000). Influenza virus binding to the host cell is primarily attributed to the hemagglutinin (HA) glycoprotein. HA is a homotrimeric glycoprotein that constitutes most of the virus surface (Cherry et al., 2009; Das et al., 2010) and consists of a fibrous globular stem and a globular head containing three sialic acid binding sites (Daniels et al., 2003; Das et al., 2010; Wang et al., 2009).

HA consists of two disulfide-linked polypeptide chains, HA1 and HA2, created by a proteolytic cleavage of the precursor protein (Wiley and Skehel, 1987; Wilson et al., 1981). The HA glycoprotein has multiple roles in the virus life cycle (Daniels et al., 2003) and, during interaction with host cells, causes a dramatic structural reorganization of the HA2 that can affect the 75 Å-long α-helix normally known as the HA2 stalk (Lorieau et al., 2010; Sui et al., 2009; Weber et al., 1994). The HA2 stalk has previously been shown to play a significant role in stabilizing the whole HA protein (DuBois et al., 2011b). The relationship between protein intrinsic disorder and influenza virulence was earlier reported and a high level of predicted disorder is found at the H1N1 HA2 stalk base (Goh et al., 2009), which predominantly contains asparaginyl (Asn) residues.

Asn is a polar amino acid reported to play an important role in maintaining protein structure among certain amino acid interactions found within a protein (Worth and Blundell, 2010). In addition, Asn was shown to form carbonyl–carbonyl interaction between the side-chain carbonyl and local backbone carbonyls within itself or with a neighboring Asn (Deane et al., 1999), allowing it to form a stacking conformation that is energetically more favorable (Deane et al., 1999; Tsai et al., 2005). Surprisingly, the significance of Asn residues at the H1N1 HA2 stalk base has not been elucidated. Here, we utilized a computational approach to determine the significance of the twin Asn-residue found at the H1N1 HA2 stalk base.

Materials and Methods

Influenza A H1N1 HA amino acid sequences of human, swine, and avian strains from 1918–2012 were obtained from the NCBI website. A total of 2830 amino acid sequences were analyzed. Similarly, H1N1 HA crystal structures were obtained from both NCBI and RCSB Protein Data Bank. In labeling the amino acid residues in the predicted H1N1 HA structures, we followed the numbering scheme as previously published (Wilson et al., 1981) and we designated subscript 1 and 2 to refer to HA1 and HA2, respectively. Predicted HA structures were made using Phyre 2 (Kelley and Sternberg, 2009). Briefly, the Phyre server scans user-submitted sequences against the nonredundant sequence database and, afterwards, a profile is constructed. The secondary structure is predicted by three independent secondary structure prediction programs and the confidence values of each program are averaged. All predicted HA structures were compared with available HA crystal structures for verification and consistency.

The twin Asn-residue (N145₂–N146₂) can be structurally traced to the salt bridge between the 110-helix and B-loop (Rachakonda et al., 2007). We determined the measurements of the salt bridge (R103₁ and E85₂) and the 110-helix:B-loop distance (D98₁ and L89₂) using all H1N1 strains studied. Moreover, we simulated an HA structure with either or both residues 145₂ and 146₂ substituted with Ala to determine how it affected both the salt bridge and 110-helix:B-loop distance measurements. All measurements were obtained by measuring the space between pre-identified amino acid residues in both the salt bridge and 110-helix:B-loop distance measurements using Jmol (Herraez, 2006). Briefly, Jmol is a free open source applet developed for the interactive display of three-dimensional chemical structures. Moreover, Jmol can also read script files and allow instructions or commands to be applied to the predicted model.

Similarly, to determine the effects of substituting Ala in either or both residues 145₂ and 146₂, the empirical distribution of amino acid residues in the unaltered (N145_2--N146₂) and altered (A145₂–N146₂, N145₂–A146₂, A145₂–A146₂) HA structures were determined by establishing the Ramachandran plot using RAMPAGE (Lovell et al., 2003). Briefly, the RAMPAGE server utilizes density-dependent smoothing of amino acid residues that would show sharp boundaries at critical edges and would indicate a clear delineation between large empty areas and regions that are allowed but disfavored. This would allow us to establish any shift in the empirical distribution and, furthermore, emphasize the structural significance of the twin Asn-residue.

Results

Asn in residues 145₂ and 146₂ favor salt bridge formation between the 110-helix and B-loop

We compared predicted H1N1 HA structures generated from protein sequences obtained from 1918–2012 and found that the N145₂–N146₂ residue is structurally conserved. This was further confirmed using known H1N1 HA crystal structures. In both the HA predicted structures and HA crystal structures, we consistently found a conserved N145₂–N146₂ residue that can be structurally traced to the salt bridge between the 110-helix and B-loop (Fig. 1A), which would insinuate that the N145₂-N146₂ residue could influence the salt bridge.

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FIG. 1.

Conserved twin Asn-residue influences the salt bridge between the 110-helix and B-loop. (A) Representative influenza H1N1 hemagglutinin structure. Encircled region indicates the position of the conserved N1452–N1462 residue. Boxed region indicates the position of the salt bridge between the 110-helix and B-loop. (B–E) Salt bridge (R1031 and E852) and distance measurements (D981 and L892) corresponding to the (B) conserved N1452–N1462 residue and amino acid substitutions made in (C) both or (D,E) either residue. 110-helix (violet) and B-loop (blue) are indicated.

To establish the significance of the conserved twin Asn-residue for the salt bridge, amino acid substitutions were made in residues 145₂ and 146₂ with the salt bridge and 110-helix:B-loop distance subsequently measured. As seen in Figure 1B, among the unaltered HA structures (N145₂–N146₂), we found the ideal measurements for the salt bridge (2.13 Å) and 110-helix:B-loop distance (11.74 Å), which is consistent in both predicted and crystal HA structures. Interestingly, altered HA structures with either or both amino acids in residues 145₂ and 146₂ substituted with Ala showed varying salt bridge and 110-helix:B-loop distance measurements. As seen in Figure 1C–E, among the altered residues (A145₂–A146₂, A145₂–N146₂ and N145₂–A146₂), we found that the salt bridges were measured with 6.1 Å, 2.82 Å, and 5.47 Å, respectively, and the 110-helix:B-loop distances were measured with 11.79 Å, 11.57 Å, and 11.47 Å, respectively.

Amino acid substitutions in either or both residues 145₂ and 146₂ shift the empirical distribution of H1N1 HA amino acids

To determine the effects of amino acid substitution in residues 145₂ and 146₂ on the HA amino acid conformation, empirical distribution of HA amino acids using the Ramachandran plot was performed. As seen in Figure 2, we showed that the empirical distribution of HA amino acids vary when either or both amino acids in residues 145₂ and 146₂ were substituted with Ala. In particular, we observed a major shift in the H1N1 HA empirical distribution (Figure 2B–D) found in the D (α_R-helices, partially allowed) and E (α_L-helices, generally allowed) regions of the Ramachandran plot as previously defined (Deane et al., 1999).

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FIG. 2.

Amino acid substitution in either or both amino acids in residues 1452 and 1462 alters the empirical distribution of HA amino acids. Ramachandran plots of the (A) conserved twin Asn-residue and amino acid substitutions made in (B) both or (C,D) either residues 1452 and 1462. D region is indicated by dashed lines. E region is indicated by solid lines.

Both partially-allowed α_R-helix and generally-allowed α_L-helix conformations in HA were affected by the amino acid found in either or both residues 145₂ and 146₂. We suspect that Asn at both residues 145₂ and 146₂ favor α_R-helix conformation while producing less α_L-helix conformations in HA. In contrast, substituting Asn with Ala in either or both residues 145₂ and 146₂ decreases α_R-helix conformations while increasing α_L-helix conformations in HA.

Discussion

Asparaginyl residues are found to occur frequently in irregular regions (Srinivasan et al., 1994) and, among H1N1 strains, this would seem to be a common feature at the HA2 stalk base (Goh et al., 2009). Throughout this study, we were able to identify a conserved twin Asn-residue found at the H1N1 HA2 stalk base and that amino acid substitutions made in either or both residues 145₂ and 146₂ altered the measurements in both the salt bridge and 110-helix:B-loop distance. This highlights the structural significance of having Asn in both residues 145₂ and 146₂, that is, to maintain the salt bridge between the 110-helix and B-loop.

HA stability is shown to be regulated by salt bridges across the receptor binding domain (RBD) interfaces and that any alteration in HA stability is suggested to influence viral infectivity (DuBois et al., 2011a; Rachakonda et al., 2007; Xu and Wilson, 2011), emphasizing the importance of the salt bridge between the 110-helix and B-loop in stabilizing the RBD (DuBois et al., 2011b). A salt bridge is a combination of two noncovalent interactions (hydrogen bonding and electrostatic interactions) and the most common salt bridges occur from the anionic carboxylate of either aspartic acid or glutamic acid and the cationic ammonium from lysine or the guanidinium of arginine (Kumar and Nussinov, 2002). Salt bridges between the influenza HA 110-helix and B-loop have been correlated to both the stability of the ectodomain and coordination of cooperative motions essential for the fusion process (Rachakonda et al., 2007). This emphasizes the importance of the N145₂–N146₂ residue and, likewise, its role in maintaining the salt bridge between the 110-helix and B-loop.

Similarly, we were able to observe that the empirical distribution of HA amino acids shifted when Asn was substituted with Ala in either or both residues 145₂ and 146₂ favoring an increase in α_L-helix conformations and a decrease in partially-allowed α_R-helix conformations. Asn residues are known to adopt to structural conformations that favor an α_L-helix conformation and other partially-allowed conformations found in the Ramachandran plot more readily than any other nonglycyl amino acids (Srinivasan et al., 1994) ascribable to the carbonyl–carbonyl interaction occurring in their own backbone or with that of the previous residue (Deane et al., 1999). Consequentially, carbonyl–carbonyl interaction produces stabilization energy comparable with hydrogen bonds (Choudhary et al., 2009; Deane et al., 1999). The decrease in the number of partially-allowed α_R-helix conformation of HA amino acids would insinuate that there is less stabilization energy available. Consequentially, this may explain the increase in α_L-helix conformations of HA amino acids since this may partially compensate for the energy requirement needed for protein stability (Deane et al., 1999). However, we emphasize that even though α_L-helix conformations increased, it was not enough to maintain the salt bridge. We hypothesize that Asn in both residues 145₂ and 146₂ creates an energy-favorable HA conformation essential for salt bridge formation between the 110-helix and B-loop which, in turn, contributes to HA stability.

At the moment, there are several proposed anti-influenza therapies based on varying strategies (Palese and Garcia-Sastre 2002; Saladino et al., 2010). Based on our results, we propose that designing influenza vaccines and antiviral strategies that target the twin Asn-residue could have the potential to be another anti-influenza strategy.

Conclusion

In conclusion, we found that from 1918–2012 the N145₂–N146₂ residue is conserved in all strains of the influenza A H1N1 subtype. We were able to trace the conserved N145₂–N146₂ residue to the salt bridge between the 110-helix and B-loop and, in addition, show that amino acid substitution in either or both residues 145₂ and 146₂ could alter the measurements in the salt bridge and 110-helix:B-loop distance. Furthermore, simulated amino acid substitutions in either or both residues 145₂ and 146₂ were also shown to change the empirical distribution of HA amino acids, resulting in a structural conformation that is less energetically favorable. Thus, we believe that both the N145₂ and N146₂ residues found in the conserved twin Asn-residue located at the H1N1 HA2 stalk base provide the necessary structural requirement needed to maintain and allow salt bridge formation between the 110-helix and B-loop. This, in turn, is necessary to stabilize the overall HA protein. Furthermore, this would insinuate that the twin-Asn-residue could be an ideal target for future anti-influenza therapies.