Structural and functional characterization of neuraminidase-like molecule N10 derived from bat influenza A virus

Abstract

The recent discovery of the unique genome of influenza virus H17N10 in bats raises considerable doubt about the origin and evolution of influenza A viruses. It also identifies a neuraminidase (NA)-like protein, N10, that is highly divergent from the nine other well-established serotypes of influenza A NA (N1-N9). The structural elucidation and functional characterization of influenza NAs have illustrated the complexity of NA structures, thus raising a key question as to whether N10 has a special structure and function. Here the crystal structure of N10, derived from influenza virus A/little yellow-shouldered bat/Guatemala/153/2009 (H17N10), was solved at a resolution of 2.20 Å. Overall, the structure of N10 was found to be similar to that of the other known influenza NA structures. In vitro enzymatic assays demonstrated that N10 lacks canonical NA activity. A detailed structural analysis revealed dramatic alterations of the conserved active site residues that are unfavorable for the binding and cleavage of terminally linked sialic acid receptors. Furthermore, an unusual 150-loop (residues 147-152) was observed to participate in the intermolecular polar interactions between adjacent N10 molecules of the N10 tetramer. Our study of influenza N10 provides insight into the structure and function of the sialidase superfamily and sheds light on the molecular mechanism of bat influenza virus infection.

Fig. 1.

Overall crystal structure of N10. (A) N10 adopts a typical box-shaped NA tetramer structure, even though the highest primary sequence identity of N10 to all other influenza NAs is only 29%. (B) Each NA monomer has six blades (blades 1–6), with blade 6 of N10 (green) containing three β strands and N2 (magenta) has four β strands. (C) Each N10 monomer contains three N-glycosylation sites: N146, N259, and N269 (presented as sticks in green). (D) Each N10 monomer contains the calcium-binding site conserved in other influenza NA structures. Coordination of the calcium ion (green) is shown by the dashed blue lines. (E) The pocket accommodating sialic acid in the N2-Neu5Ac complex contains a positively charged “edge” region and a negatively charged “platform” region. Here the N2 and N10 structures appear in surface representation with the electrostatic potential scaled from −5kT/e to 5KT/e. (F) The electrostatic potential of the uncomplexed N10 reveals no obvious charge in the canonical sialic acid carboxylate-binding site. Furthermore, the platform located at the bottom of the binding site is positively charged compared with the negatively charged platform in the N2-Neu5Ac complex.

Fig. 2.

N10 has no sialidase activity. The reaction velocity (μM/min) of NAs is shown based on substrate conversion. Fluorogenic MUNANA substrate was used at a final concentration of 0–500 μM. 09N1 (rhombus), N2 (black cycle), and N5 (triangle) display obvious enzymatic activity, whereas N10 (square) lacks sialidase activity. BSA (white cycle) was used as a negative control. Mean values were determined from at least three duplicates and are presented with SDs indicated by error bars.

Fig. 3.

Comparison of the catalytic site and framework residues in the N2-Neu5Ac complex with those in N10. (A) Sequence alignment shows that 5 of the 8 key catalytic site residues and 8 of the 11 framework residues are substituted in N10. (B) Superimposition of the N10 structure (green) with the N2-Neu5Ac complex structure (magenta) reveals the dramatic changes of the eight catalytic site residues. The natural ligand sialic acid is shown in stick presentation (yellow). (C) Structural comparison of the 11 framework residues in the N2-Neu5Ac complex with the corresponding residues of N10.

Fig. 4.

N10 has a unique 150-loop conformation. (A) Typical group 1 NA:VN04N1 (orange) has the 150-cavity in its active site. (B) Typical group 2 NA: N2 (blue) has no 150-cavity. (C) N10 (green) has a more open pseudoactive site without obvious bounds and lacks the 150-cavity. (D) The 150-loop of N10 (green) adopts an unusually extended conformation, and L144 and L433 in N10 form a stable hydrophobic interaction. (E) The N10 150-loop in one monomer forms strong contacts with the 100-loop of the adjacent monomer, which makes the two loops in N10 much closer compared with those of other influenza NAs. (F) Detailed presentation of the polar interactions between the N10 150-loop of one monomer and the 100-loop of the other monomer. S151 forms one hydrogen bond with P105, and N146 forms two hydrogen bonds with E109. S151 also forms hydrogen bonds with G107 that are mediated by two water molecules.