title: Nuclear magnetic resonance structure shows that the severe acute respiratory syndrome coronavirus-unique domain contains a macrodomain fold.
abstract: The nuclear magnetic resonance (NMR) structure of a central segment of the previously annotated severe acute respiratory syndrome (SARS)-unique domain (SUD-M, for "middle of the SARS-unique domain") in SARS coronavirus (SARS-CoV) nonstructural protein 3 (nsp3) has been determined. SUD-M(513-651) exhibits a macrodomain fold containing the nsp3 residues 528 to 648, and there is a flexibly extended N-terminal tail with the residues 513 to 527 and a C-terminal flexible tail of residues 649 to 651. As a follow-up to this initial result, we also solved the structure of a construct representing only the globular domain of residues 527 to 651 [SUD-M(527-651)]. NMR chemical shift perturbation experiments showed that SUD-M(527-651) binds single-stranded poly(A) and identified the contact area with this RNA on the protein surface, and electrophoretic mobility shift assays then confirmed that SUD-M has higher affinity for purine bases than for pyrimidine bases. In a further search for clues to the function, we found that SUD-M(527-651) has the closest three-dimensional structure homology with another domain of nsp3, the ADP-ribose-1"-phosphatase nsp3b, although the two proteins share only 5% sequence identity in the homologous sequence regions. SUD-M(527-651) also shows three-dimensional structure homology with several helicases and nucleoside triphosphate-binding proteins, but it does not contain the motifs of catalytic residues found in these structural homologues. The combined results from NMR screening of potential substrates and the structure-based homology studies now form a basis for more focused investigations on the role of the SARS-unique domain in viral infection.
authors.affiliation: Department of Molecular Biology, Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Amarnath
authors.initials: A
authors.lastname: Chatterjee
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Johnson
authors.forename: Pedro
authors.initials: P
authors.lastname: Serrano
authors.forename: Bill
authors.initials: B
authors.lastname: Pedrini
authors.forename: Jeremiah S
authors.initials: JS
authors.lastname: Joseph
authors.forename: Benjamin W
authors.initials: BW
authors.lastname: Neuman
authors.forename: Kumar
authors.initials: K
authors.lastname: Saikatendu
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Buchmeier
authors.forename: Peter
authors.initials: P
authors.lastname: Kuhn
authors.forename: Kurt
authors.initials: K
authors.lastname: Wüthrich
mesh_terms: D024202:Electrophoretic Mobility Shift Assay; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000737:chemistry*; D017361:Viral Nonstructural Proteins / Q000737:chemistry*
chemical_list: D016601:RNA-Binding Proteins; D017361:Viral Nonstructural Proteins; D012313:RNA; C510959:Nsp3 protein, SARS-CoV; D012324:RNA-Dependent RNA Polymerase