Cargando…
Combined computational and experimental analysis of a complex of ribonuclease III and the regulatory macrodomain protein, YmdB
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 direct...
Autores principales: | , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BlackWell Publishing Ltd
2015
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4329070/ https://www.ncbi.nlm.nih.gov/pubmed/25546632 http://dx.doi.org/10.1002/prot.24751 |
_version_ | 1782357391655829504 |
---|---|
author | Paudyal, Samridhdi Alfonso-Prieto, Mercedes Carnevale, Vincenzo Redhu, Shiv K Klein, Michael L Nicholson, Allen W |
author_facet | Paudyal, Samridhdi Alfonso-Prieto, Mercedes Carnevale, Vincenzo Redhu, Shiv K Klein, Michael L Nicholson, Allen W |
author_sort | Paudyal, Samridhdi |
collection | PubMed |
description | 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 K(D) 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 K(D) (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. Proteins 2015; 83:459–472. © 2014 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. |
format | Online Article Text |
id | pubmed-4329070 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | BlackWell Publishing Ltd |
record_format | MEDLINE/PubMed |
spelling | pubmed-43290702015-03-19 Combined computational and experimental analysis of a complex of ribonuclease III and the regulatory macrodomain protein, YmdB Paudyal, Samridhdi Alfonso-Prieto, Mercedes Carnevale, Vincenzo Redhu, Shiv K Klein, Michael L Nicholson, Allen W Proteins Articles 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 K(D) 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 K(D) (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. Proteins 2015; 83:459–472. © 2014 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. BlackWell Publishing Ltd 2015-03 2015-01-21 /pmc/articles/PMC4329070/ /pubmed/25546632 http://dx.doi.org/10.1002/prot.24751 Text en © 2014 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. |
spellingShingle | Articles Paudyal, Samridhdi Alfonso-Prieto, Mercedes Carnevale, Vincenzo Redhu, Shiv K Klein, Michael L Nicholson, Allen W Combined computational and experimental analysis of a complex of ribonuclease III and the regulatory macrodomain protein, YmdB |
title | Combined computational and experimental analysis of a complex of ribonuclease III and the regulatory macrodomain protein, YmdB |
title_full | Combined computational and experimental analysis of a complex of ribonuclease III and the regulatory macrodomain protein, YmdB |
title_fullStr | Combined computational and experimental analysis of a complex of ribonuclease III and the regulatory macrodomain protein, YmdB |
title_full_unstemmed | Combined computational and experimental analysis of a complex of ribonuclease III and the regulatory macrodomain protein, YmdB |
title_short | Combined computational and experimental analysis of a complex of ribonuclease III and the regulatory macrodomain protein, YmdB |
title_sort | combined computational and experimental analysis of a complex of ribonuclease iii and the regulatory macrodomain protein, ymdb |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4329070/ https://www.ncbi.nlm.nih.gov/pubmed/25546632 http://dx.doi.org/10.1002/prot.24751 |
work_keys_str_mv | AT paudyalsamridhdi combinedcomputationalandexperimentalanalysisofacomplexofribonucleaseiiiandtheregulatorymacrodomainproteinymdb AT alfonsoprietomercedes combinedcomputationalandexperimentalanalysisofacomplexofribonucleaseiiiandtheregulatorymacrodomainproteinymdb AT carnevalevincenzo combinedcomputationalandexperimentalanalysisofacomplexofribonucleaseiiiandtheregulatorymacrodomainproteinymdb AT redhushivk combinedcomputationalandexperimentalanalysisofacomplexofribonucleaseiiiandtheregulatorymacrodomainproteinymdb AT kleinmichaell combinedcomputationalandexperimentalanalysisofacomplexofribonucleaseiiiandtheregulatorymacrodomainproteinymdb AT nicholsonallenw combinedcomputationalandexperimentalanalysisofacomplexofribonucleaseiiiandtheregulatorymacrodomainproteinymdb |