title: Conformational plasticity of RepB, the replication initiator protein of promiscuous streptococcal plasmid pMV158.
abstract: DNA replication initiation is a vital and tightly regulated step in all replicons and requires an initiator factor that specifically recognizes the DNA replication origin and starts replication. RepB from the promiscuous streptococcal plasmid pMV158 is a hexameric ring protein evolutionary related to viral initiators. Here we explore the conformational plasticity of the RepB hexamer by i) SAXS, ii) sedimentation experiments, iii) molecular simulations and iv) X-ray crystallography. Combining these techniques, we derive an estimate of the conformational ensemble in solution showing that the C-terminal oligomerisation domains of the protein form a rigid cylindrical scaffold to which the N-terminal DNA-binding/catalytic domains are attached as highly flexible appendages, featuring multiple orientations. In addition, we show that the hinge region connecting both domains plays a pivotal role in the observed plasticity. Sequence comparisons and a literature survey show that this hinge region could exists in other initiators, suggesting that it is a common, crucial structural element for DNA binding and manipulation.
authors.affiliation: Institute for Research in Biomedicine (IRB Barcelona), Barcelona, 08028 Spain.|Institut de Biologia Molecular de Barcelona (Consejo Superior de Investigaciones Científicas), Barcelona, 08028, Spain.
authors.forename: D Roeland
authors.initials: DR
authors.lastname: Boer
authors.affiliation: Centro de Investigaciones Biológicas (Consejo Superior de Investigaciones Científicas), Madrid, 28040, Spain.
authors.forename: José Angel
authors.initials: JA
authors.lastname: Ruiz-Masó
authors.affiliation: Institute for Research in Biomedicine (IRB Barcelona), Barcelona, 08028 Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: Rueda
authors.affiliation: European Molecular Biology Laboratory, Hamburg Unit, EMBL c/o DESY, Hamburg, 22607, Germany.
authors.forename: Maxim V
authors.initials: MV
authors.lastname: Petoukhov
authors.affiliation: Institute for Research in Biomedicine (IRB Barcelona), Barcelona, 08028 Spain.|Institut de Biologia Molecular de Barcelona (Consejo Superior de Investigaciones Científicas), Barcelona, 08028, Spain.
authors.forename: Cristina
authors.initials: C
authors.lastname: Machón
authors.affiliation: European Molecular Biology Laboratory, Hamburg Unit, EMBL c/o DESY, Hamburg, 22607, Germany.
authors.forename: Dmitri I
authors.initials: DI
authors.lastname: Svergun
authors.affiliation: Institute for Research in Biomedicine (IRB Barcelona), Barcelona, 08028 Spain.|Departament de Bioquímica, Facultat de Biologia, Universitat de Barcelona, Barcelona, 08028, Spain.
authors.forename: Modesto
authors.initials: M
authors.lastname: Orozco
authors.affiliation: Centro de Investigaciones Biológicas (Consejo Superior de Investigaciones Científicas), Madrid, 28040, Spain.
authors.forename: Gloria
authors.initials: G
authors.lastname: del Solar
authors.affiliation: Institute for Research in Biomedicine (IRB Barcelona), Barcelona, 08028 Spain.|Institut de Biologia Molecular de Barcelona (Consejo Superior de Investigaciones Científicas), Barcelona, 08028, Spain.
authors.forename: Miquel
authors.initials: M
authors.lastname: Coll
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D018360:Crystallography, X-Ray; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics; D004261:DNA Replication / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D056004:Molecular Dynamics Simulation; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D000072417:Protein Domains; D055503:Protein Multimerization; D018741:Replication Origin / Q000235:genetics; D013291:Streptococcus / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; C106846:RepB helicase; D004265:DNA Helicases