ja204291d_si_001.pdf (235.98 kB)
Download file

Probing Conformational Variations at the ATPase Site of the RNA Helicase DbpA by High-Field Electron–Nuclear Double Resonance Spectroscopy

Download (235.98 kB)
journal contribution
posted on 05.10.2011, 00:00 authored by Ilia Kaminker, Anastasiya Sushenko, Alexey Potapov, Shirley Daube, Barak Akabayov, Irit Sagi, Daniella Goldfarb
The RNA helicase DbpA promotes RNA remodeling coupled to ATP hydrolysis. It is unique because of its specificity to hairpin 92 of 23S rRNA (HP92). Although DbpA kinetic pathways leading to ATP hydrolysis and RNA unwinding have been recently elucidated, the molecular (atomic) basis for the coupling of ATP hydrolysis to RNA remodeling remains unclear. This is, in part, due to the lack of detailed structural information on the ATPase site in the presence and absence of RNA in solution. We used high-field pulse ENDOR (electron–nuclear double resonance) spectroscopy to detect and analyze fine conformational changes in the protein’s ATPase site in solution. Specifically, we substituted the essential Mg2+ cofactor in the ATPase active site for paramagnetic Mn2+ and determined its close environment with different nucleotides (ADP, ATP, and the ATP analogues ATPγS and AMPPnP) in complex with single- and double-stranded RNA. We monitored the Mn2+ interactions with the nucleotide phosphates through the 31P hyperfine couplings and the coordination by protein residues through 13C hyperfine coupling from 13C-enriched DbpA. We observed that the nucleotide binding site of DbpA adopts different conformational states upon binding of different nucleotides. The ENDOR spectra revealed a clear distinction between hydrolyzable and nonhydrolyzable nucleotides prior to RNA binding. Furthermore, both the 13C and the 31P ENDOR spectra were found to be highly sensitive to changes in the local environment of the Mn2+ ion induced by the hydrolysis. More specifically, ATPγS was efficiently hydrolyzed upon binding of RNA, similar to ATP. Importantly, the Mn2+ cofactor remains bound to a single protein side chain and to one or two nucleotide phosphates in all complexes, whereas the remaining metal coordination positions are occupied by water. The conformational changes in the protein’s ATPase active site associated with the different DbpA states occur in remote coordination shells of the Mn2+ ion. Finally, a competitive Mn2+ binding site was found for single-stranded RNA construct.