NMR study of a single-stranded DNA binding to the C-terminal domain of retroviral protease


V. Motáčková, M. Nálezková, M. Švec, M. Kožíšek, L. Žídek, J. Konvalinka, and V. Sklenář


National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kotlarska 2, Brno, 611 37, Czech Republic
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo n. 2, Praha 6, 166 10, Czech Republic


Proteases play a crucial role in the retroviral viral infection but the mechanism of their regulation remains unclear. The C-terminal domain of the retroviral proteases, rich in glycines, has been proposed to bind RNA. Biochemical tests showed that the C-terminal domain binds single-stranded oligonucleotides (both RNA and DNA) without inhibiting the proteolytic activity. The N-15 labeled C-terminal domain of mouse intracisternal A-type particles endogenous retrovirus (CT) was investigated using NMR. CT was titrated with a single-stranded DNA 20-mer. The titration was monitored in 2D HN-HSQC and 1D proton spectra. 3D TOCSY-HSQC, NOESY-HSQC, and HNHB spectra were recorded on the over-titrated sample. The protocols of the overexpression on minimal media have been optimized and C-13, N-15 labeled C-terminal domain and C-13, N-15, H-2 labeled full-length protease samples were prepared. A set of triple resonance experiments have been measured. The assignment of the C-terminal region is presented.