Metal-induced stabilization and activation of plasmid replication initiator RepuB

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.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (BFU2015-70052-R to MM; BFU2010-19597 to GdS, AGL2012-40084-C03 and AGL2015-71923-REDT to GdS). Additional funding to MM was provided by the CIBER de Enfermedades Respiratorias (CIBERES), an initiative of the Instituto de Salud Carlos III (ISCIII).Peer reviewe