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Paudyal S , Alfonso-Prieto M , Carnevale V , Redhu SK , Klein ML , Nicholson AW
Combined computational and experimental analysis of a complex of ribonuclease III and the regulatory macrodomain protein, YmdB
Proteins. 2015 Mar;83(3) :459-72
PMID: 25546632 PMCID: PMC4329070
AbstractRibonuclease III is a conserved bacterial endonuclease that cleaves double-stranded(ds) structures in diverse coding and noncoding RNAs. RNase III is subject to multiple levels of control that in turn confer global post-transcriptional regulation. The Escherichia coli macrodomain protein YmdB directly interacts with RNase III, and an increase in YmdB amount in vivo correlates with a reduction in RNase III activity. Here, a computational-based structural analysis was performed to identify atomic-level features of the YmdB-RNase III interaction. The docking of monomeric E. coli YmdB with a homology model of the E. coli RNase III homodimer yields a complex that exhibits an interaction of the conserved YmdB residue R40 with specific RNase III residues at the subunit interface. Surface Plasmon Resonance (SPR) analysis provided a KD of 61 nM for the complex, corresponding to a binding free energy (DeltaG) of -9.9 kcal/mol. YmdB R40 and RNase III D128 were identified by in silico alanine mutagenesis as thermodynamically important interacting partners. Consistent with the prediction, the YmdB R40A mutation causes a 16-fold increase in K(D) (DeltaDeltaG = +1.8 kcal/mol), as measured by SPR, and the D128A mutation in both RNase III subunits (D128A/D128'A) causes an 83-fold increase in KD (DeltaDeltaG = +2.7 kcal/mol). The greater effect of the D128A/D128'A mutation may reflect an altered RNase III secondary structure, as revealed by CD spectroscopy, which also may explain the significant reduction in catalytic activity in vitro. The features of the modeled complex relevant to potential RNase III regulatory mechanisms are discussed.
NotesPaudyal, Samridhdi Alfonso-Prieto, Mercedes Carnevale, Vincenzo Redhu, Shiv K Klein, Michael L Nicholson, Allen W GM56772/GM/NIGMS NIH HHS/United States GM56772-08S1/GM/NIGMS NIH HHS/United States R01 GM056772/GM/NIGMS NIH HHS/United States Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. United States Proteins. 2015 Mar;83(3):459-72. doi: 10.1002/prot.24751. Epub 2015 Jan 21.