SARS Coronavirus-unique Domain (SUD): Three-domain Molecular Architecture in Solution and RNA Binding

Johnson, Margaret A.; Chatterjee, Amarnath; Neuman, Benjamin W.; Wüthrich, Kurt

The nonstructural protein 3 (nsp3) of the severe acute respiratory syndrome coronavirus (SARS-CoV) includes a “SARS-unique region” (SUD) consisting of three globular domains separated by short linker peptide segments. This paper reports NMR structure determinations of the C-terminal domain (SUD-C) and of a two-domain construct (SUD-MC) containing the middle domain (SUD-M) and the C-terminal domain, and NMR data on the conformational states of the N-terminal domain (SUD-N) and the SUD-NM two-domain construct. Both SUD-N and SUD-NM are monomeric and globular in solution, and in SUD-NM there is high mobility in the two-residue interdomain linking sequence, with no preferred relative orientation of the two domains. SUD-C adopts a frataxin-like fold and has structural similarity to DNA-binding domains of DNA-modifying enzymes. The structures of both SUD-M (previously determined) and SUD-C (from the present study) are maintained in SUD-MC, where the two domains are flexibly linked. Gel shift experiments showed that both SUD-C and SUD-MC bind to single-stranded RNA and recognize purine bases more strongly than pyrimidine bases, whereby SUD-MC binds to a more restricted set of purine-containing RNA sequences than SUD-M. NMR chemical shift perturbation experiments with observation of the (15)N-labeled proteins further resulted in the delineation of the RNA binding sites, i.e., in SUD-M a positively charged surface area with a pronounced cavity, and in SUD-C several residues of an antiparallel β-sheet. Overall, the present data provide evidence for molecular mechanisms involving concerted actions of SUD-M and SUD-C, which result in specific RNA-binding that might be unique to the SUD, and thus to the SARS-CoV.