PDBsum entry 2r7c

RNA binding protein

PDB id: 2r7c

Contents

Protein chain

308 a.a.

Ligands

PO4

Waters ×15

References listed in PDB file

Key reference

Title

Crystallographic and biochemical analysis of rotavirus nsp2 with nucleotides reveals a nucleoside diphosphate kinase-Like activity.

Authors

M.Kumar, H.Jayaram, R.Vasquez-Del carpio, X.Jiang, Z.F.Taraporewala, R.H.Jacobson, J.T.Patton, B.V.Prasad.

Ref.

J Virol, 2007, 81, 12272-12284.

PubMed id

17804496

Abstract

Rotavirus, the major pathogen of infantile gastroenteritis, carries a nonstructural protein, NSP2, essential for viroplasm formation and genome replication/packaging. In addition to RNA-binding and helix-destabilizing properties, NSP2 exhibits nucleoside triphosphatase activity. A conserved histidine (H225) functions as the catalytic residue for this enzymatic activity, and mutation of this residue abrogates genomic double-stranded RNA synthesis without affecting viroplasm formation. To understand the structural basis of the phosphatase activity of NSP2, we performed crystallographic analyses of native NSP2 and a functionally defective H225A mutant in the presence of nucleotides. These studies showed that nucleotides bind inside a cleft between the two domains of NSP2 in a region that exhibits structural similarity to ubiquitous cellular HIT (histidine triad) proteins. Only minor conformational alterations were observed in the cleft upon nucleotide binding and hydrolysis. This hydrolysis involved the formation of a stable phosphohistidine intermediate. These observations, reminiscent of cellular nucleoside diphosphate (NDP) kinases, prompted us to investigate whether NSP2 exhibits phosphoryl-transfer activity. Bioluminometric assay showed that NSP2 exhibits an NDP kinase-like activity that transfers the bound phosphate to NDPs. However, NSP2 is distinct from the highly conserved cellular NDP kinases in both its structure and catalytic mechanism, thus making NSP2 a potential target for antiviral drug design. With structural similarities to HIT proteins, which are not known to exhibit NDP kinase activity, NSP2 represents a unique example among structure-activity relationships. The newly observed phosphoryl-transfer activity of NSP2 may be utilized for homeostasis of nucleotide pools in viroplasms during genome replication.

Secondary reference #1

Title

Rotavirus protein involved in genome replication and packaging exhibits a hit-Like fold.

Authors

H.Jayaram, Z.Taraporewala, J.T.Patton, B.V.Prasad.

Ref.

Nature, 2002, 417, 311-315. [DOI no: 10.1038/417311a]

PubMed id

12015608

Figure 1.
Figure 1: The X-ray structure of the monomeric subunit of NSP2. a, A stereo view of the ribbon representation of the structure. The secondary structural elements in the monomer are coloured: -helices in red, -strands in green and loops in blue. The locations of the major -helices are indicated following the same numbering scheme as in c. The basic loop between the subdomains in the N-terminal domain is shown by an arrow. b, Another view of the monomer showing the cleft (arrow) and the domain organization. The N- and C-terminal domains are shown in green and red respectively. The location of the N and the C termini are denoted in both a and b. c, The structure -sequence relationship in NSP2. The secondary structural elements along with the sequence are shown following the same colour scheme as in a. -helices and -strands are numbered sequentially. The strictly conserved residues are highlighted in orange. The residues that participate in the 4- and 2-fold contacts of the octamer are indicated by boxes below the sequence in blue and purple respectively. The HIT-homology (see Fig. 2) region is underlined in light blue.

Figure 3.
Figure 3: Octameric structure. a, Views of the crystallographic octamer along the two distinct 2-fold axes, separated by a 45° rotation about the 4-fold axis. The left side shows a ribbon representation of the octamer along one of these two 2-fold axes. One of the subunits, with N- and C-terminal ends marked, is shown using the same colouring scheme as in Fig. 1a. The rest of the subunits are shown in grey. A deep groove lined by the basic residues from the N-terminal loop, shown by an arrow, runs diagonally across this 2-fold axis. The right side shows the octamer as viewed along the other 2-fold axis of the 4-2-2 symmetry. This 2-fold axis is associated with protein -protein interactions that link the two tetrameric layers. The two subunits that participate in these 2-fold interactions are highlighted using the colour scheme in Fig. 1b. The N-terminal domains (in green) participate in the 4-fold related inter-subunit interactions, whereas the C-terminal domains (in red) participate in inter-subunit interactions across this 2-fold axis. b, Stereo view of the electrostatic potential surface of the octamer viewed along the 4-fold axis showing the central hole. c, Stereo view of the octamer, along the 2-fold axis, as in a (left) showing the grooves (black arrows). An open arrow shows the location of the cleft in one of the subunits. The surface is coloured deep blue in the most electropositive regions (20 k[B]T) and red in the most electronegative regions (-20 k[B]T), where k[B] is the Boltzmann constant, and T is temperature in kelvin.

The above figures are reproduced from the cited reference with permission from Macmillan Publishers Ltd