Conformation changes in E. coli Rho monitored by hydrogen/deuterium exchange and mass spectrometry: response to ligand binding.
Escherichia coli Rho is a doughnut-shaped homohexameric ATP-dependent RNA-DNA helicase that releases newly synthesized RNA molecules from transcription complexes. Rho binds 60-80 bases of RNA among six primary RNA binding sites around the inside of its N-terminal crown; the RNA then passes through the central hole of the hexamer. Here it triggers ATP hydrolysis and is moved with respect to the protein. We study protein conformation changes upon ligand binding using amide proton hydrogen/deuterium exchange and mass spectrometry. Global-exchange studies indicate net mass differences of about 15 Da after 1 h of exchange in the presence--versus in the absence--of the ligand MgATP or the RNA poly(C). Sites of ligand-dependent exchange differences were localized by mass determination of the peptic peptides of Rho. A peptide of the N-terminal domain near the known primary RNA sites (aa 56-63) was protected from amide proton exchange in the presence of poly(C), as was a novel N-terminal domain peptide that is not near RNA in the crystal structures or in NMR structures with RNA oligomers (aa 37-46). This result may further define the primary interaction site of RNA with Rho. The Q-loop-containing peptide in the central hole of the protein that interacts with RNA was also protected by RNA (aa 271-286). The exchange rate of one peptide near the ATPase active site (aa 206-218) slowed in the presence of MgATP and increased in the presence of RNA. Overall, the results show changes in a few protein segments rather than a different overall conformation.
Stitt
Barbara L
BL
Department of Biochemistry and Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140, USA. stitt@temple.edu
Xiao
Hui
H
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D003903:Deuterium / Q000032:analysis; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D006859:Hydrogen / Q000032:analysis; D013058:Mass Spectrometry / Q000379:methods; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D012329:RNA, Bacterial / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*
D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; C534627:Rho protein, E coli; D006859:Hydrogen; D000255:Adenosine Triphosphate; D003903:Deuterium