The refined structure of Nudaurelia capensis ω Virus reveals control elements for a T = 4 capsid maturation

AbstractLarge-scale reorganization of protein interactions characterizes many biological processes, yet few systems are accessible to biophysical studies that display this property. The capsid protein of Nudaurelia capensis ω Virus (NωV) has previously been characterized in two dramatically different T = 4 quasi-equivalent assembly states when expressed as virus-like particles (VLPs) in a baculovirus system. The procapsid (pH 7), is round, porous, and approximately 450 Å in diameter. It converts, in vitro, to the capsid form at pH 5 and the capsid is sealed shut, shaped like an icosahedron, has a maximum diameter of 410 Å and undergoes an autocatalytic cleavage at residue 570. Residues 571–644, the γ peptide, remain associated with the particle and are partially ordered. The interconversion of these states has been previously studied by solution X-ray scattering, electron cryo microscopy (CryoEM), and site-directed mutagenesis. The particle structures appear equivalent in authentic virions and the low pH form of the expressed and assembled protein. Previously, and before the discovery of the multiple morphological forms of the VLPs, we reported the X-ray structure of authentic NωV at 2.8 Å resolution. These coordinates defined the fold of the protein but were not refined at the time because of technical issues associated with the approximately 2.5 million reflection data set. We now report the refined, authentic virus structure that has added 29 residues to the original model and allows the description of the chemistry of molecular switching for T = 4 capsid formation and the multiple morphological forms. The amino and carboxy termini are internal, predominantly helical, and disordered to different degrees in the four structurally independent subunits; however, the refined structure shows significantly more ordered residues in this region, particularly at the amino end of the B subunit that is now seen to invade space occupied by the A subunits. These additional residues revealed a previously unnoticed strong interaction between the pentameric, γ peptide helices of the A and B subunits that are largely proximal to the quasi-6-fold axes. One C-terminal helix is ordered in the C and D subunits and stabilizes a flat interaction in two interfaces between the protein monomers while the other, quasi-equivalent, interactions are bent. As this helix is arginine rich, the comparable, disordered region in the A and B subunits probably interacts with RNA. One of the subunit–subunit interfaces has an unusual arrangement of carboxylate side chains. Based on this observation, we propose a mechanism for the control of the pH-dependent transitions of the virus particle

Virus Capsid Dissolution Studied by Microsecond Molecular Dynamics Simulations

Dissolution of many plant viruses is thought to start with swelling of the capsid caused by calcium removal following infection, but no high-resolution structures of swollen capsids exist. Here we have used microsecond all-atom molecular simulations to describe the dynamics of the capsid of satellite tobacco necrosis virus with and without the 92 structural calcium ions. The capsid expanded 2.5% upon removal of the calcium, in good agreement with experimental estimates. The water permeability of the native capsid was similar to that of a phospholipid membrane, but the permeability increased 10-fold after removing the calcium, predominantly between the 2-fold and 3-fold related subunits. The two calcium binding sites close to the icosahedral 3-fold symmetry axis were pivotal in the expansion and capsid-opening process, while the binding site on the 5-fold axis changed little structurally. These findings suggest that the dissociation of the capsid is initiated at the 3-fold axis

Mutations to kirromycin resistance occur in the interface of domains I and III of EF-Tu·GTP

AbstractThe antibiotic kirromycin inhibits protein synthesis by binding to EF-Tu and preventing its release from the ribosome after GTP hydrolysis. We have isolated and sequenced a collection of kirromycin resistant tuf mutations and identified thirteen single amino acid substitutions at seven different sites in EF-Tu. These have been mapped onto the 3D structures of EF-Tu·GTP and EF-Tu·GDP. In the active GTP form of EF-Tu the mutations cluster on each side of the interface between domains I and III. We propose that this domain interface is the binding site for kirromycin

The three-dimensional structure of cocksfoot mottle virus at 2.7 å resolution

AbstractCocksfoot mottle virus is a plant virus that belongs to the genus Sobemovirus. The structure of the virus has been determined at 2.7 Å resolution. The icosahedral capsid has T = 3 quasisymmetry and 180 copies of the coat protein. Except for a couple of stacked bases, the viral RNA is not visible in the electron density map. The coat protein has a jelly-roll β-sandwich fold and its conformation is very similar to that of other sobemoviruses and tobacco necrosis virus. The N-terminal arm of one of the three quasiequivalent subunits is partly ordered and follows the same path in the capsid as the arm in rice yellow mottle virus, another sobemovirus. In other sobemoviruses, the ordered arm follows a different path, but in both cases the arms from three subunits meet and form a similar structure at a threefold axis. A comparison of the structures and sequences of viruses in this family shows that the only conserved parts of the protein–protein interfaces are those that form binding sites for calcium ions. Still, the relative orientations and position of the subunits are mantained

The crystal structure of bacteriophage Qβ at 3.5 å resolution

AbstractBackground: The capsid protein subunits of small RNA bacteriophages form a T=3 particle upon assembly and RNA encapsidation. Dimers of the capsid protein repress translation of the replicase gene product by binding to the ribosome binding site and this interaction is believed to initiate RNA encapsidation. We have determined the crystal structure of phage Qβ with the aim of clarifying which factors are the most important for particle assembly and RNA interaction in the small phages.Results The crystal structure of bacteriophage Qβ determined at 3.5 å resolution shows that the capsid is stabilized by disulfide bonds on each side of the flexible loops that are situated around the fivefold and quasi-sixfold axes. As in other small RNA phages, the protein capsid is constructed from subunits which associate into dimers. A contiguous ten-stranded antiparallel β sheet facing the RNA is formed in the dimer. The disulfide bonds lock the constituent dimers of the capsid covalently in the T=3 lattice.Conclusion The unusual stability of the Qβ particle is due to the tight dimer interactions and the disulfide bonds linking each dimer covalently to the rest of the capsid. A comparison with the structure of the related phage MS2 shows that although the fold of the Qβ coat protein is very similar, the details of the protein–protein interactions are completely different. The most conserved region of the protein is at the surface, which, in MS2, is involved in RNA binding

The 3-fold axis.

<p>Close-up of an icosahedral 3-fold symmetry axis from the final frame of the (<b>A, C</b>) and the (<b>B, D</b>) trajectories showing the main water permeation site. Calcium ion depicted as a cyan sphere and water molecules as blue sticks.</p

Structural dynamics of the capsid and water contacts.

<p>(<b>A</b>) The RMSd from the crystal structure (without over all rotation and translation of the entire capsid) for the of the shell domain (average over the last 0.1 and all 60 proteins). (<b>B</b>) As <b>A</b> but after aligning each protein individually to the crystal structure. (<b>C</b>) The RMSf for the of the shell domain (average over the last 0.1 ). (<b>D</b>) The number of contacts to water molecules crossing the capsid shell. The secondary structure elements of the crystal structure are shown as gray (-strand) or checkered (-helix) regions.</p

Structural changes of the capsid.

<p>Van der Waals surfaces of the capsid shell after 1 viewed along one of the 5-fold symmetry axes. Top row (<b>A, B</b>) shows and bottom row (<b>C, D</b>) shows . Right column (<b>B, D</b>) shows the interior structure when the 15 frontmost proteins has been removed. Each capsid inscribed in an icosahedron with 22 nm between opposing vertices. The N-terminal domains are represented as ribbons. Colors according to the RMSd from the crystal structure. The color scale goes from blue (0.0 nm) through cyan, green, lime-green, yellow and red (). Location of symmetry axes marked for one icosahedral face in (<b>A</b>). The outline of the proteins in one trimer traced in (<b>C</b>).</p