Cargando…
Metal-Induced Stabilization and Activation of Plasmid Replication Initiator RepB
Initiation of plasmid rolling circle replication (RCR) is catalyzed by a plasmid-encoded Rep protein that performs a Tyr- and metal-dependent site-specific cleavage of one DNA strand within the double-strand origin (dso) of replication. The crystal structure of RepB, the initiator protein of the str...
Autores principales: | , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2016
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5030251/ https://www.ncbi.nlm.nih.gov/pubmed/27709114 http://dx.doi.org/10.3389/fmolb.2016.00056 |
_version_ | 1782454642127405056 |
---|---|
author | Ruiz-Masó, José A. Bordanaba-Ruiseco, Lorena Sanz, Marta Menéndez, Margarita del Solar, Gloria |
author_facet | Ruiz-Masó, José A. Bordanaba-Ruiseco, Lorena Sanz, Marta Menéndez, Margarita del Solar, Gloria |
author_sort | Ruiz-Masó, José A. |
collection | PubMed |
description | Initiation of plasmid rolling circle replication (RCR) is catalyzed by a plasmid-encoded Rep protein that performs a Tyr- and metal-dependent site-specific cleavage of one DNA strand within the double-strand origin (dso) of replication. The crystal structure of RepB, the initiator protein of the streptococcal plasmid pMV158, constitutes the first example of a Rep protein structure from RCR plasmids. It forms a toroidal homohexameric ring where each RepB protomer consists of two domains: the C-terminal domain involved in oligomerization and the N-terminal domain containing the DNA-binding and endonuclease activities. Binding of Mn(2+) to the active site is essential for the catalytic activity of RepB. In this work, we have studied the effects of metal binding on the structure and thermostability of full-length hexameric RepB and each of its separate domains by using different biophysical approaches. The analysis of the temperature-induced changes in RepB shows that the first thermal transition, which occurs at a range of temperatures physiologically relevant for the pMV158 pneumococcal host, represents an irreversible conformational change that affects the secondary and tertiary structure of the protein, which becomes prone to self-associate. This transition, which is also shown to result in loss of DNA binding capacity and catalytic activity of RepB, is confined to its N-terminal domain. Mn(2+) protects the protein from undergoing this detrimental conformational change and the observed protection correlates well with the high-affinity binding of the cation to the active site, as substituting one of the metal-ligands at this site impairs both the protein affinity for Mn(2+)and the Mn(2+)-driven thermostabilization effect. The level of catalytic activity of the protein, especially in the case of full-length RepB, cannot be explained based only on the high-affinity binding of Mn(2+) at the active site and suggests the existence of additional, lower-affinity metal binding site(s), missing in the separate catalytic domain, that must also be saturated for maximal activity. The molecular bases of the thermostabilizing effect of Mn(2+) on the N-terminal domain of the protein as well as the potential location of additional metal binding sites in the entire RepB are discussed. |
format | Online Article Text |
id | pubmed-5030251 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-50302512016-10-05 Metal-Induced Stabilization and Activation of Plasmid Replication Initiator RepB Ruiz-Masó, José A. Bordanaba-Ruiseco, Lorena Sanz, Marta Menéndez, Margarita del Solar, Gloria Front Mol Biosci Molecular Biosciences Initiation of plasmid rolling circle replication (RCR) is catalyzed by a plasmid-encoded Rep protein that performs a Tyr- and metal-dependent site-specific cleavage of one DNA strand within the double-strand origin (dso) of replication. The crystal structure of RepB, the initiator protein of the streptococcal plasmid pMV158, constitutes the first example of a Rep protein structure from RCR plasmids. It forms a toroidal homohexameric ring where each RepB protomer consists of two domains: the C-terminal domain involved in oligomerization and the N-terminal domain containing the DNA-binding and endonuclease activities. Binding of Mn(2+) to the active site is essential for the catalytic activity of RepB. In this work, we have studied the effects of metal binding on the structure and thermostability of full-length hexameric RepB and each of its separate domains by using different biophysical approaches. The analysis of the temperature-induced changes in RepB shows that the first thermal transition, which occurs at a range of temperatures physiologically relevant for the pMV158 pneumococcal host, represents an irreversible conformational change that affects the secondary and tertiary structure of the protein, which becomes prone to self-associate. This transition, which is also shown to result in loss of DNA binding capacity and catalytic activity of RepB, is confined to its N-terminal domain. Mn(2+) protects the protein from undergoing this detrimental conformational change and the observed protection correlates well with the high-affinity binding of the cation to the active site, as substituting one of the metal-ligands at this site impairs both the protein affinity for Mn(2+)and the Mn(2+)-driven thermostabilization effect. The level of catalytic activity of the protein, especially in the case of full-length RepB, cannot be explained based only on the high-affinity binding of Mn(2+) at the active site and suggests the existence of additional, lower-affinity metal binding site(s), missing in the separate catalytic domain, that must also be saturated for maximal activity. The molecular bases of the thermostabilizing effect of Mn(2+) on the N-terminal domain of the protein as well as the potential location of additional metal binding sites in the entire RepB are discussed. Frontiers Media S.A. 2016-09-21 /pmc/articles/PMC5030251/ /pubmed/27709114 http://dx.doi.org/10.3389/fmolb.2016.00056 Text en Copyright © 2016 Ruiz-Masó, Bordanaba-Ruiseco, Sanz, Menéndez and del Solar. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Molecular Biosciences Ruiz-Masó, José A. Bordanaba-Ruiseco, Lorena Sanz, Marta Menéndez, Margarita del Solar, Gloria Metal-Induced Stabilization and Activation of Plasmid Replication Initiator RepB |
title | Metal-Induced Stabilization and Activation of Plasmid Replication Initiator RepB |
title_full | Metal-Induced Stabilization and Activation of Plasmid Replication Initiator RepB |
title_fullStr | Metal-Induced Stabilization and Activation of Plasmid Replication Initiator RepB |
title_full_unstemmed | Metal-Induced Stabilization and Activation of Plasmid Replication Initiator RepB |
title_short | Metal-Induced Stabilization and Activation of Plasmid Replication Initiator RepB |
title_sort | metal-induced stabilization and activation of plasmid replication initiator repb |
topic | Molecular Biosciences |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5030251/ https://www.ncbi.nlm.nih.gov/pubmed/27709114 http://dx.doi.org/10.3389/fmolb.2016.00056 |
work_keys_str_mv | AT ruizmasojosea metalinducedstabilizationandactivationofplasmidreplicationinitiatorrepb AT bordanabaruisecolorena metalinducedstabilizationandactivationofplasmidreplicationinitiatorrepb AT sanzmarta metalinducedstabilizationandactivationofplasmidreplicationinitiatorrepb AT menendezmargarita metalinducedstabilizationandactivationofplasmidreplicationinitiatorrepb AT delsolargloria metalinducedstabilizationandactivationofplasmidreplicationinitiatorrepb |