Construction of an extended form of GI.1 VP1 dimer based on the available cryo-EM data.

<p>(A) Fitting of GI.1 A-B dimer in the GII.10 cryo-EM Map (EMDB: 5374 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182056#pone.0182056.ref008" target="_blank">8</a>]). The connecting segments were generated with modeller 9.14. The termini were not modeled. Color code as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182056#pone.0182056.g001" target="_blank">Fig 1A</a>. (B) Definition of angle and height of S domains relative to the crystal structure's. The reference is the dimer of P, the height is of the center of mass of S along the P dimer axis (<i>O</i>Z) and the angle is between the S axes as defined in the methods section. In this panel, S of chain A is in orange and of chain B in yelllow.</p

Crystal structure and sequence diversity of the norovirus VP1 capsid protein.

<p>(A) Structure of the norovirus major capsid protein (VP1). The N-terminal S domain is in yellow and the C-terminal P domain in grey. They are separated by a connecting segment of approximately 10 amino acids (orange). The N-terminal arm of the S domain is colored in green. Boundaries of domains are indicated between the primary structure diagram. (B) Pairwise sequence identities between VP1 for GI.1 (crystal structure), GII.10 (Cryo-EM Map) and GIII.2 (TR-SAXS) and between their P and S domains. (C) Dendrogram of 29 representative VP1 sequences taken from Zheng et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182056#pone.0182056.ref006" target="_blank">6</a>]. The 29 genotypes' accession numbers are given in the "Materials and Methods" section. The genotypes are grouped in the five major norovirus genogroups GI to GV (colored circles). Capsids for which structural data are available (GI.1, GII.10 and GIII.2) are indicated by arrows.</p

Simulations of the extended GI.1 dimer.

<p>Top (A) and front (B) view for the extended model of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182056#pone.0182056.g004" target="_blank">Fig 4</a> and the endpoints of 5 simulations. S domains are red for chain A and blue for chain B. (C) Evolution of the angles and heights of S domains as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182056#pone.0182056.g004" target="_blank">Fig 4</a>. Values of 0 correspond to the S conformation of the GI.1 crystal structure.</p

Sequence conservation among noroviruses of the S-P interaction segments.

<p>The color code is the same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182056#pone.0182056.g006" target="_blank">Fig 6</a>. The conservation score was calculated with Jalview [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182056#pone.0182056.ref033" target="_blank">33</a>] on a scale from 0 to 11 based on one representative VP1 sequence for each of 29 genotypes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182056#pone.0182056.ref006" target="_blank">6</a>]. Averages for segments and domains are indicated in brackets.</p

Molecular dynamics simulations in different sets of protonation for the three acidic residues clustered in the GI.1 VP1.

<p>(A) Comparison of N-terminal arms in the GI.1 crystal structure (chain B) and after simulated annealing of our GIII.2 model. Color code as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182056#pone.0182056.g001" target="_blank">Fig 1A</a>. The side chains of the three acidic residues considered are displayed. (B-D) Evolution of the difference distances between D10, E22 and E152. A value of 0 corresponds to restoration of the initial distance in the GI.1 crystal structure while a negative value indicates a larger distance (see text for details). The illustrations represent the geometry of a representative frame of the largest cluster of each trajectory (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182056#pone.0182056.s002" target="_blank">S2 Fig</a>). The gray vertical line indicates the frame from which the picture originates.</p

Dynamics and asymmetry in the dimer of the norovirus major capsid protein

<div><p>Noroviruses are the major cause of non-bacterial acute gastroenteritis in humans and livestock worldwide, despite being physically among the simplest animal viruses. The icosahedral capsid encasing the norovirus RNA genome is made of 90 dimers of a single <i>ca</i> 60-kDa polypeptide chain, VP1, arranged with T = 3 icosahedral symmetry. Here we study the conformational dynamics of this main building block of the norovirus capsid. We use molecular modeling and all-atom molecular dynamics simulations of the VP1 dimer for two genogroups with 50% sequence identity. We focus on the two points of flexibility in VP1 known from the crystal structure of the genogroup I (GI, human) capsid and from subsequent cryo-electron microscopy work on the GII capsid (also human). First, with a homology model of the GIII (bovine) VP1 dimer subjected to simulated annealing then classical molecular dynamics simulations, we show that the N-terminal arm conformation seen in the GI crystal structure is also favored in GIII VP1 but depends on the protonation state of critical residues. Second, simulations of the GI dimer show that the VP1 spike domain will not keep the position found in the GII electron microscopy work. Our main finding is a consistent propensity of the VP1 dimer to assume prominently asymmetric conformations. In order to probe this result, we obtain new SAXS data on GI VP1 dimers. These data are not interpretable as a population of symmetric dimers, but readily modeled by a highly asymmetric dimer. We go on to discuss possible implications of spontaneously asymmetric conformations in the successive steps of norovirus capsid assembly. Our work brings new lights on the surprising conformational range encoded in the norovirus major capsid protein.</p></div

Radii of gyration and maximum dimensions of crystallographic dimer, cryo-EM based model, SAXS data and Dadimodo model.

<p>Radii of gyration and Dmax were calculated with Crysol for structural models, for which termini were included. P(R) analysis was used for experimental data.</p

Dynamic properties and conformations of the N-terminal arm in a homology model of the GIII.2 dimer.

<p>(A) RMSD analysis of the homology model simulation (100 ns). The protein core (black curve) is defined by residues 31 to 512 (without N-terminal and C-terminal arms). The N-terminal arm is analysed separately (green curve). (B) RMS fluctuation of the N-terminal arms (residues 1 to 30) in the homology model simulation. Heavy atoms only were considered for the RMSD and RMSF calculation. (C) RMS fluctuation of N-terminal arms in the 199 endpoints of simulated annealing. A representative position of each arm is also displayed on the protein picture. (D) Salt bridge network found for the arm of chain A in 84% of the simulated annealing endpoints. Interactions within the arm are represented in orange and interactions with S domain in blue. The color of the box is defined by the charge of the amino acid (negative in red, positive in green).</p

Solution structures of dissociated GI.1 dimer obtained by rigid body modeling of SAXS data.

<p>The fit of the curve computed from the model (in blue) to the experimental data (in red) and their associated reduced residuals (in gray) are displayed atop the models. (A) EOM results for 10000 symmetric models. The population of 3 selected models is displayed superposed on their P domains (in gray) with the 3 pairs of S domains in yellow, orange and magenta. (B) EOM results for 10000 asymmetric models. The population of 2 selected models is displayed superposed on their P domains (in gray) with the 2 pairs of S domains in yellow and magenta. (C) Fit of the single Dadimodo model. (D) Comparison of the GI.1 dimer before rigid body modeling (1IHM with arms modeled in, the fit to the experimental data is also shown) with the two main models from EOM (asymmetric) and Dadimodo. The best superposition of the <i>ab initio</i> envelope computed from the SAXS data is displayed for each model.</p

Simulated annealing clustering results.

<p>For each cluster the size is the number of endpoints and the spread the average RMSD between all endpoints composing this cluster. The spread can also be represented by the compactness of nodes in a dendrogram (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182056#pone.0182056.s001" target="_blank">S1 Fig</a>). a, the three members of this cluster are the first three endpoints.</p