2024-03-29T10:05:03Zhttp://digital.csic.es/dspace-oai/requestoai:digital.csic.es:10261/1373022020-10-30T11:09:02Zcom_10261_34com_10261_5com_10261_86com_10261_1col_10261_287col_10261_339
2016-09-23T12:43:58Z
urn:hdl:10261/137302
Metal-induced stabilization and activation of plasmid replication initiator RepuB
Ruiz-Masó, José A.
Bordanaba-Ruiseco, Lorena
Sanz, Marta
Menéndez, Margarita
Solar, Gloria del
Ministerio de Economía y Competitividad (España)
Instituto de Salud Carlos III
HUH endonucleases
Plasmid-encoded Repproteins
Metal-dependent catalyticactivity,
RepB thermostability
Mn2+ affinity
15 p.-8 fig.
Initiation of plasmid rolling circle replication(RCR)is catalyzed by a plasmid-encoded Repprotein that performs aTyr-andmetal-dependentsite-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 Repprotein structure from RCR plasmids.It forms a toroidal homohexamericring where each RepBprotomer consists of two domains:theC-terminal domain involved in oligomerization and the N-terminal domain containing the DNA-binding and endonuclease activities.Binding of Mn2+ 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.Mn2+ protects the protein from undergoing this detrimental conformational hange and the ob served 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 Mn2+ and the Mn2+-driven thermostabilization effect.The level of catalytic activity of the protein,especially in the caseof full-length RepB,cannot be explained based only on the high-affinity binding of Mn2+ at the active site and suggests the existence of additional,lower-affinity metalbinding site(s),missing in these parate catalytic domain,that must also be saturated for maximal activity.The molecular bases of the thermostabilizing effect of Mn2+ on the N-terminal domain of the protein as well as the potential location of additional metalbinding sitesin the entire RepB are discussed.
2016-09-23T12:43:58Z
2016-09-23T12:43:58Z
2016-09-21
artículo
Frontiers in Molecular Biosciences 3:56 (2016)
http://hdl.handle.net/10261/137302
10.3389/fmolb.2016.00056
2296-889X
http://dx.doi.org/10.13039/501100003329
http://dx.doi.org/10.13039/501100004587
27709114
eng
Publisher's version
http://dx.doi.org/10.3389/fmolb.2016.00056
Sí
http://creativecommons.org/licenses/by/4.0/
openAccess
Frontiers Media