Plasticity of the 340-Loop in Influenza Neuraminidase Offers New Insight for Antiviral Drug Development

Abstract

The recently discovered 340-cavity in influenza neuraminidase (NA) N6 and N7 subtypes has introduced new possibilities for rational structure-based drug design. However, the plasticity of the 340-loop (residues 342-347) and the role of the 340-loop in NA activity and substrate binding have not been deeply exploited. Here, we investigate the mechanism of 340-cavity formation and demonstrate for the first time that seven of nine NA subtypes are able to adopt an open 340-cavity over 1.8 μs total molecular dynamics simulation time. The finding that the 340-loop plays a role in the sialic acid binding pathway suggests that the 340-cavity can function as a druggable pocket. Comparing the open and closed conformations of the 340-loop, the side chain orientation of residue 344 was found to govern the formation of the 340-cavity. Additionally, the conserved calcium ion was found to substantially influence the stability of the 340-loop. Our study provides dynamical evidence supporting the 340-cavity as a druggable hotspot at the atomic level and offers new structural insight in designing antiviral drugs.

Keywords: 340-cavity; influenza; molecular dynamics simulations; neuraminidase.

Figure 1

Potential of mean force (PMF) of nine neuraminidase (NA) subtypes based on 340-cavity volume calculation. Potential of mean force (PMF) based on the volume of the 340-cavity in N1 and N4 (A), N3, N5, and N8 (B), N2 and N9 (C), N6 and N7 (D).

Figure 2

Surface of the NA binding pocket in different subtypes. Surface of the binding pocket in the crystal structures of different NA subtypes. N6 and N7 have an open 340-cavity in the crystal structures.

Figure 3

Mechanism for 340-cavity formation. Cluster analysis based on the 340-loop root mean squared fluctuation was performed. Cluster centers representing the open conformation of the 340-cavity were superimposed on the crystal structures of type-1 and type-2 NAs. Panels (A–E) show a structural comparison between the cluster center and crystal structure of the 340-loop in type-1 NAs N1, N3, N4, N5, and N8. Panels (G,H) show a structural comparison between the cluster center and crystal structure of the 340-loop in type-2 NAs N2 and N9. The 340-loops of cluster centers and crystal structures are colored in blue and orange, respectively. Panels (F,I) illustrate the 340-cavity and binding pocket of type-3 NA N6 and N7 with surface representation.

Figure 4

Binding pathway of sialic acid (SA) and distribution of binding energies along the pathway. The two lowest binding energy pathways of sialic acid (SA), the climbing path and tunneling path, are illustrated in N1 (A), N2 (B), and N7 (C). The harbors of the climbing path, active site, and tunneling path were abbreviated as C, A, and T, respectively. Panels (D–F) represent binding energies in accordance with the harbor sites in N1, N2, and N7, respectively. Boxes represent binding energies between 25% and 75% of the cluster components, lines within the boxes represent mean values, and vertical bars represent binding energies within 1.5 interquartile range (IQR).

Figure 5

Interaction energy between the conserved calcium ion and interacted residues in all NA subtypes. Interaction energy, Lennard-Jones (LJ) and Coulomb (Coul), between the calcium ion and interacted residues was calculated based on the whole trajectory of each NA subtype. Panels (A) to (I) represent the LJ and Coul interactions of N1 to N9, respectively.

Figure 6

Free energy landscape of the 340-loop from dihedral principle component analysis. Free energy landscapes of the 340-loop in N1, N2, and N7 calcium-bound and calcium-free systems were obtained using dihedral principle component analysis (dPCA).