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

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• 作者：Ilia Kaminker ; Anastasiya Sushenko ; Alexey Potapov ; Shirley Daube ; Barak Akabayov ; Irit Sagi ; Daniella Goldfarb
• 刊名：Journal of the American Chemical Society
• 出版年：2011
• 出版时间：October 5, 2011
• 年：2011
• 卷：133
• 期：39
• 页码：15514-15523
• 全文大小：999K
• 年卷期：v.133,no.39(October 5, 2011)
• ISSN：1520-5126

文摘

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鈥搉uclear double resonance) spectroscopy to detect and analyze fine conformational changes in the protein鈥檚 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 ATP纬S 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, ATP纬S 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鈥檚 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.