Probing conformational variations at the ATPase site of the RNA helicase DbpA by high-field electron-nuclear double resonance spectroscopy

J Am Chem Soc. 2011 Oct 5;133(39):15514-23. doi: 10.1021/ja204291d. Epub 2011 Sep 9.


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 Mg(2+) cofactor in the ATPase active site for paramagnetic Mn(2+) 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 Mn(2+) interactions with the nucleotide phosphates through the (31)P hyperfine couplings and the coordination by protein residues through (13)C hyperfine coupling from (13)C-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 (13)C and the (31)P ENDOR spectra were found to be highly sensitive to changes in the local environment of the Mn(2+) ion induced by the hydrolysis. More specifically, ATPγS was efficiently hydrolyzed upon binding of RNA, similar to ATP. Importantly, the Mn(2+) 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 Mn(2+) ion. Finally, a competitive Mn(2+) binding site was found for single-stranded RNA construct.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / metabolism*
  • Base Sequence
  • Catalytic Domain*
  • Coenzymes / metabolism
  • DEAD-box RNA Helicases / chemistry*
  • DEAD-box RNA Helicases / metabolism*
  • Electron Spin Resonance Spectroscopy / methods*
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / metabolism*
  • Manganese / metabolism
  • Models, Molecular
  • RNA / genetics
  • RNA / metabolism
  • Ribonucleotides / genetics
  • Ribonucleotides / metabolism


  • Coenzymes
  • Escherichia coli Proteins
  • Ribonucleotides
  • Manganese
  • RNA
  • Adenosine Triphosphatases
  • dbpA protein, E coli
  • DEAD-box RNA Helicases