title: Metal-Induced Stabilization and Activation of Plasmid Replication Initiator RepB.
abstract: 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 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 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 Mn2+and the Mn2+-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 Mn2+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 Mn2+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.
authors.affiliation: Molecular Biology of Gram-Positive Bacteria, Molecular Microbiology and Infection Biology, Centro de Investigaciones Biológicas (Consejo Superior de Investigaciones Científicas) Madrid, Spain.
authors.forename: José A
authors.initials: JA
authors.lastname: Ruiz-Masó
authors.affiliation: Molecular Biology of Gram-Positive Bacteria, Molecular Microbiology and Infection Biology, Centro de Investigaciones Biológicas (Consejo Superior de Investigaciones Científicas) Madrid, Spain.
authors.forename: Lorena
authors.initials: L
authors.lastname: Bordanaba-Ruiseco
authors.affiliation: Molecular Biology of Gram-Positive Bacteria, Molecular Microbiology and Infection Biology, Centro de Investigaciones Biológicas (Consejo Superior de Investigaciones Científicas) Madrid, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Sanz
authors.affiliation: Biological Physical Chemistry, Protein Structure and Thermodynamics, Instituto de Química-Física Rocasolano (Consejo Superior de Investigaciones Científicas)Madrid, Spain; CIBER of Respiratory DiseasesMadrid, Spain.
authors.forename: Margarita
authors.initials: M
authors.lastname: Menéndez
authors.affiliation: Molecular Biology of Gram-Positive Bacteria, Molecular Microbiology and Infection Biology, Centro de Investigaciones Biológicas (Consejo Superior de Investigaciones Científicas) Madrid, Spain.
authors.forename: Gloria
authors.initials: G
authors.lastname: Del Solar
keywords: HUH endonucleases; Mn2+ affinity; RepB thermostability; metal-dependent catalytic activity; plasmid-encoded Rep proteins