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

When we investigated possible interactions between SUD and nucleic acids by zone-interference gel electrophoresis, we found that the domain binds oligo(G) and oligo(dG) stretches with a K D of ,1 mM, but not oligo(dA), (dC), or (dT) [31] . Single-stranded nucleotides of random sequence are only bound if they are longer than ,15 nucleotides. Here we demonstrate that each of the two individual SUD subdomains also binds oligo(dG) ( Figure 4A ). With oligo(dH), where H stands for A, C, or T, but not G, only very small gel shifts, if at all, were observed. As oligo(G) stretches are known to form G-quadruplexes, i.e. four-stranded nucleic-acid structures formed by contiguous guanines [32] , we also examined the binding to the oligodeoxynucleotide 59-GGGCGCGGGAG-GAATTGGGCGGG-39, a G-rich sequence present in the bcl-2 promoter region. This oligonucleotide has been shown by NMR spectroscopy to form a G-quadruplex ( [33] ; PDB code 2F8U). We found that both full-length SUD and SUD core do indeed bind this oligodeoxynucleotide and that this process is enhanced by the addition of K + ions, which are known to stabilize G-quadruplex structures ( Figure 4B ). In agreement with the ability of SUD to non-specifically bind to oligonucleotides of .15 bases [31] , both SUD and SUD core were found to bind the reverse-complementary sequence, but with low affinity and, more importantly, independent of K + ions.