A new pentameric structure of rotavirus NSP4 revealed by molecular replacement

Abstract

The region spanning residues 95-146 of the rotavirus nonstructural protein NSP4 from the asymptomatic human strain ST3 has been purified and crystallized and diffraction data have been collected to a resolution of 2.6 Å. Several attempts to solve the structure by the molecular-replacement method using the available tetrameric structures of this domain were unsuccessful despite a sequence identity of 73% to the already known structures. A more systematic approach with a dimer as the search model led to an unexpected pentameric structure using the program Phaser. The various steps involved in arriving at this molecular-replacement solution, which unravelled a case of subtle variation between different oligomeric states unknown at the time of solving the structure, are presented in this paper.

Structural, spectroscopic, molecular docking, thermal and DFT studies on metal complexes of bidentate orthoquinone ligand

“…The TPMBQ ligand (guest) executed with various protein receptors (host) by molecular docking and it showed a strong interaction with all receptors (Figures ). This indicated to mutable capacity for the hydrogen bonding interaction by multi‐central groups internal docking complexes and all proteins have enter hydrogen bonding …”

Severe diffraction anisotropy, rotational pseudosymmetry and twinning complicate the refinement of a pentameric coiled-coil structure of NSP4 of rotavirus

“…The molecular-replacement solution was not straightforward and was obtained from data in space group P4 2 2 1 2 (Chacko et al, 2012) using Phaser (McCoy et al, 2007. Discussed below are the steps in identifying the inherent crystal pathology, which led to the determination of the true space group (P4 2 ) and ultimately resulted in successful refinement of the structure.…”

“…The selfrotation peaks were strongly masked by the crystallographic fourfold and twofold peaks and hence no information could be gained about the oligomeric state and the symmetry of the molecule. Since a clear molecular-replacement solution was obtained in space group P4 2 2 1 2, with a rotational Z-score of 5.6, a translational Z-score of 26.6 and an LLG of 1121 (Chacko et al, 2012), model building and refinement were carried out in this space group. However, the R factors could not be improved beyond an R work of 31.2 and an R free of 39.0%.…”

“…After overcoming the initial challenge with molecular replacement (Chacko et al, 2012), we encountered several pathologies associated with the data set that complicated the refinement process in the case of the pentameric structure. The manner in which the data pathologies were identified and overcome is described in this paper.…”

Understanding the penetrance of intrinsic protein disorder in rotavirus proteome

“…Amino acid substitution mutations P168A, Y166S, and M175I in the unstructured C-terminus of NSP4 lead to the complete loss of interaction of the protein with DLPs [113]. This multifunctional protein is pleiotropic in nature because of the oligomeric state combined with high conformational dynamics of the disordered cytoplasmic domain of the protein as evident from the calcium-lacking pentameric (PDB ID: 1G1J & 2O1K) [118,119] and the calcium-containing tetrameric (PDB ID: 2O1J) [120][121][122] coiled-coil structures of the diarrhea-inducing region from ST3, and SA11 and I321 strains of rotavirus, respectively. NSP4 has a cholesterol recognition amino acid consensus sequence (CRAC), which is predicted to be a completely disordered region and it interacts with cholesterol and caveolin, leading to its localization to the plasma membrane [123].…”