The SARS-Unique Domain (SUD) of SARS Coronavirus Contains Two Macrodomains That Bind G-Quadruplexes

Tan, Jinzhi; Vonrhein, Clemens; Smart, Oliver S.; Bricogne, Gerard; Bollati, Michela; Kusov, Yuri; Hansen, Guido; Mesters, Jeroen R.; Schmidt, Christian L.; Hilgenfeld, Rolf

Embedded between the X-domain (Nsp3b) and the PL2 pro (Nsp3d), the SARS-unique domain (SUD; Nsp3c) fails to show sequence relationship to any other protein in the databases [1] . We have produced full-length SUD (residues 389 to 726 of Nsp3), and a more stable, shortened 264-residue version (residues 389 to 652; henceforth called SUD core ), by expression in Escherichia coli. This definition of the boundaries of the SUD is based on the structural results described here. We report crystallization of SUD core and its X-ray structure in two crystal forms, at 2.2 and 2.8 Ã… resolution, respectively. The structure turns out to consist of two further copies of the macrodomain, in spite of the complete absence of sequence similarity. In addition, we demonstrate that each of the subdomains binds G-quadruplexes, both in DNA and RNA fragments, and that selected mutations of lysine residues in the first subdomain, SUD-N, lead to reduced nucleic-acid binding, whereas those in the second subdomain, SUD-M, abolish it.