title: Combined computational and experimental analysis of a complex of ribonuclease III and the regulatory macrodomain protein, YmdB.
abstract: Ribonuclease 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 (ΔG) 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) (ΔΔG = +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 (ΔΔG = +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.
authors.affiliation: Department of Biology, Temple University, Philadelphia, Pennsylvania, 19122.
authors.forename: Samridhdi
authors.initials: S
authors.lastname: Paudyal
authors.forename: Mercedes
authors.initials: M
authors.lastname: Alfonso-Prieto
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Carnevale
authors.forename: Shiv K
authors.initials: SK
authors.lastname: Redhu
authors.forename: Michael L
authors.initials: ML
authors.lastname: Klein
authors.forename: Allen W
authors.initials: AW
authors.lastname: Nicholson
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D011485:Protein Binding; D043244:Ribonuclease III / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment; D013499:Surface Properties; D013816:Thermodynamics
chemical_list: D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C534806:YmdB protein, E coli; D043244:Ribonuclease III
keywords: ADP-ribose; RNA processing; RNase III; double-stranded RNA; macrodomain; post-transcriptional regulation