Mesoscale All-Atom Influenza Virus Simulations Suggest New Substrate Binding Mechanism

Abstract

Influenza virus circulates in human, avian, and swine hosts, causing seasonal epidemic and occasional pandemic outbreaks. Influenza neuraminidase, a viral surface glycoprotein, has two sialic acid binding sites. The catalytic (primary) site, which also binds inhibitors such as oseltamivir carboxylate, is responsible for cleaving the sialic acid linkages that bind viral progeny to the host cell. In contrast, the functional annotation of the secondary site remains unclear. Here, we better characterize these two sites through the development of an all-atom, explicitly solvated, and experimentally based integrative model of the pandemic influenza A H1N1 2009 viral envelope, containing ∼160 million atoms and spanning ∼115 nm in diameter. Molecular dynamics simulations of this crowded subcellular environment, coupled with Markov state model theory, provide a novel framework for studying realistic molecular systems at the mesoscale and allow us to quantify the kinetics of the neuraminidase 150-loop transition between the open and closed states. An analysis of chloride ion occupancy along the neuraminidase surface implies a potential new role for the neuraminidase secondary site, wherein the terminal sialic acid residues of the linkages may bind before transfer to the primary site where enzymatic cleavage occurs. Altogether, our work breaks new ground for molecular simulation in terms of size, complexity, and methodological analyses of the components. It also provides fundamental insights into the understanding of substrate recognition processes for this vital influenza drug target, suggesting a new strategy for the development of anti-influenza therapeutics.

Figure 1

Mesoscale simulations enhance conformational sampling of the viral glycoproteins. (A) A fully intact all-atom model of the influenza A H1N1 2009 (pH1N1) viral envelope, containing over 160 million atoms, shown without explicit water molecules, was simulated with all-atom MD simulations. Hemagglutinin (HA) glycoproteins shown in royal (dark) blue, neuraminidase (NA) glycoproteins shown in ice (light) blue. (B) Top view of a single NA monomer in surface representation with the catalytic site (white), secondary site (yellow), 150-loop (green), and 430-loop (red) highlighted. (C–E) Principal component analysis (PCA) was performed by considering the motions of the C_α atoms of 19 1° pocket residues. PCA histograms were independently normalized so the bins containing the minimum and maximum number of points were blue and red, respectively. (C) PCA of the four monomers sampled during a single-NA-tetramer simulation (“single-glycoprotein”). (D) PCA of the 120 monomeric trajectories extracted during the last 8.33 ns of the viral envelope simulation (“terminal-envelope”). (E) PCA of all 120 monomeric trajectories extracted from the full simulation of the viral envelope (“complete-envelope”).

Figure 2

Volumetric and ligand binding “hot spot” analyses of the 1° catalytic site and adjacent regions. (A) NA is shown in ice blue ribbon, and the pocket volume is filled with semitransparent gel. The 1° active site, 430-loop, and 150-loop are visible. (B) NA is shown as a solid gradient, and ligand-binding hotspots are metallic. A portion of the surface-rendered protein was removed to facilitate visualization of internal cavities. This NA conformation has a notably open G351 pocket, which has a high propensity to bind ligands. (C) A histogram of the NA catalytic-site volumes sampled during the MD simulations. As reference, the volumes of the same active-site cavity from two crystal structures are indicated with black circled stars. The 3NSS(20) structure (pH1N1 with a closed 150-cavity) has a volume of 800 ų, and the 2HTY(12) structure (H5N1 with an open 150-cavity) has a pocket volume of 1088 ų. The simulated-pocket volumes range from ∼450 to 4440 ų (intervals 3500–4500 not shown), as reported in Table S1; the average pocket volume is 1536 ų.

Figure 3

A two-state MSM with representative structures from the viral envelope simulation. The equilibrium populations of the open and closed states are approximately equal in both the viral envelope and single-glycoprotein simulations. Correspondingly, the mean first-passage times between the states are approximately equal. The 150-loop and 430-loop are represented as green and red ribbons, respectively.

Figure 4

Chlorine anion distribution within the NA binding sites. The chlorine anion distribution (A) and the projection of the electrostatic potential onto the NA surface (B) show the pathway between the 1° and 2° sites. In panel A, NA is drawn as ice blue cartoon. Regions of high chloride occupancy are illustrated as dotted silver bubbles. Two sialic acids (PDB ID: 1MWE(24)) are superimposed in the catalytic (center) and 2° (upper right) sites for reference. In panel B the NA surface is colored with a palette varying from red (negative) to royal blue (positive), representing electrostatic potential values of −1 k_bT/e_c and +1 k_bT/e_c, respectively. The path connecting the 2° site with the catalytic site is shown as a dashed arrow between circles fading from yellow (2° site) to white (catalytic site).

Figure 5

The predicted sialic acid “bind and transfer” mechanism. Yellow stars represent a sialic-acid-containing glycan receptor. Ice blue half circles represent NA. The 1° catalytic site and 2° site are also labeled.