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. 2016 Aug 25;120(33):8590-9.
doi: 10.1021/acs.jpcb.6b02655. Epub 2016 May 12.

Microsecond Molecular Dynamics Simulations of Influenza Neuraminidase Suggest a Mechanism for the Increased Virulence of Stalk-Deletion Mutants

Jacob D Durrant  1 , Robin M Bush  2 , Rommie E Amaro  1
Affiliations

Microsecond Molecular Dynamics Simulations of Influenza Neuraminidase Suggest a Mechanism for the Increased Virulence of Stalk-Deletion Mutants

Jacob D Durrant et al. J Phys Chem B. .

Abstract

Deletions in the stalk of the influenza neuraminidase (NA) surface protein are associated with increased virulence, but the mechanisms responsible for this enhanced virulence are unclear. Here we use microsecond molecular dynamics simulations to explore the effect of stalk deletion on enzymatic activity, contrasting NA proteins from the A/swine/Shandong/N1/2009 strain both with and without a stalk deletion. By modeling and simulating neuraminidase apo glycoproteins embedded in complex-mixture lipid bilayers, we show that the geometry and dynamics of the neuraminidase enzymatic pocket may differ depending on stalk length, with possible repercussions on the binding of the endogenous sialylated-oligosaccharide receptors. We also use these simulations to predict previously unrecognized druggable "hotspots" on the neuraminidase surface that may prove useful for future efforts aimed at structure-based drug design.

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Conflict of interest statement

The authors declare the following competing financial interest(s): R.E.A. is a co-founder of Actavalon, Inc.

Figures

Figure 1
Figure 1
Equilibrated neuraminidase models used in the current study: (A) wild-type (long-stalk) NAwt and (B) stalk-deletion NAdel. The four domains of the glycoprotein are indicated.
Figure 2
Figure 2
RMSDs of all trajectory monomers to their respective reference structures. In all cases, only the Cα of the head domain (i.e., the Cα of all residues homologous to the 388 residues present in the 3NSS crystal structure) were considered. (A) NAwt (5 tetrameric trajectories × 4 monomers per tetramer = 20 runs); (B) NAdel (5 tetrameric trajectories × 4 monomers per tetramer = 20 runs); and (C) NAhead (1 tetrameric trajectory × 4 monomers per tetramer = 4 runs).
Figure 3
Figure 3
Dynamics of the sialic-acid-binding pocket. (A) Simulated apo binding-pocket conformations are compared to the crystallographic (2HU4) holo conformation. Geometric similarity was judged by calculating the RMSDs between simulated and crystallographic pocket residues. The NAwt pocket assumes two distinct conformational states. (B) Dynamics of the arginine triad. Geometric similarity was judged by calculating the RMSDs between simulated and crystallographic triad conformations. Note that the NAwt simulation again assumes two conformational states. (C) Representative NAwt arginine-triad conformations sampled from the 2.4 Å state (in yellow) and the 3.4 Å state (in red). The crystallographic position of the triad residues is shown in thick, white licorice. An oseltamivir molecule has been positioned within the binding pocket for reference (in orange), though the simulations did not include any ligand. (D) The simulated conformations of NAwt and NAdel active-site heavy atoms, in black and red, respectively, projected onto the first two principal components of the NAwt simulation. The NAwt site assumes a greater variety of conformations, in harmony with the RMSD analysis.
Figure 4
Figure 4
NA active-site residues colored by ΔRMSF = RMSFwt – RMSFdel, from −0.2 to 0.2. Residues in red and blue were more flexible in the NAwt and NAdel simulations, respectively. The backbones of key binding-pocket loops are subtly outlined. A crystallographic oseltamivir molecule is shown for reference, though no ligand was included in the simulations.
Figure 5
Figure 5
Volumetric analysis. (A) The smallest and (B) largest binding-pocket conformations sampled by the NAwt simulation. The smallest and largest conformations from the NAdel and NAhead simulations were similar (data not shown). Sialic acid molecules (in licorice) have been placed in each pocket for reference, though the simulations included no ligand. () Histograms of the volumes sampled over the course of the NAwt and NAdel simulations, in black and gray, respectively. The NAwt pocket tended to be larger (p = 0.0).
Figure 6
Figure 6
Unique druggable hotspots (outlined). The 371, 430, and 150 loops are shown in mauve, pink, and green, respectively. (A) A druggable hotspot was identified near the 430 loop of the NAwt pocket. (B) A second druggable hotspot was identified beneath the 371 loop of both the NAwt and NAdel pockets. (C) A large druggable hotspot was identified near the base of the NAwt and NAdel heads.
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