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.

Metal-Induced Stabilization and Activation of Plasmid Replication Initiator RepB

“…In RepB, the Mn 2+ ion probably interacts with the oxygen atoms of the scissile DNA phosphate, polarizing the bond and favoring the nucleophilic attack by the catalytic Tyr99 (Boer et al, 2009 ). The presence of additional divalent cation binding sites at the interface of OBD and OD domains has been reported for the C2 crystal structure of RepB 6 (Boer et al, 2016 ).…”

“…To investigate whether the first thermal transition affects a particular protein domain, we analyzed the thermal stability of the separate RepB domains, purified from Escherichia coli as described (Boer et al, 2009 ). The N-terminal OBD, which has been shown by analytical ultracentrifugation to be in a monomeric state, contains the endonuclease and DNA binding activities, and retains these abilities when separated from the C-terminal OD, which maintains its hexameric structure (Boer et al, 2009 , 2016 ). Of note, the near-UV CD spectra of the separate domains correlate fairly well with that of RepB 6 (i.e., the RepB 6 spectrum approximately matches the curve obtained by addition of the spectra of the separate domains weighted by the fractional contribution of their amino acid number to the complete protein), evidencing the conservation of tertiary/quaternary structure in both domains (Figure 4 ).…”

“…The structure of the RepB hexamer reveals a high degree of conformational plasticity, allowing differences of up to 55° in the orientation of the OBDs relative to the ODs (Boer et al, 2009 , 2016 ). As a result, RepB 6 can exists at least in two distinct structural conformations (C2 and C3 structures).…”

“…The movement of the OBDs and their position relative to the ODs is mainly determined by the flexibility of the hinge region connecting both domains and by the distinct interactions created between the OBD and the hinge region of a protomer and the OD helix α5 of a neighboring protomer (Boer et al, 2016 ).Interestingly, a divalent cation can bind to this region through the backbone of the hinge region of a protomer and side chains of residues from the own OD and that of an adjacent protomer in an OBD conformation-dependent way. Indeed, null, half or full site occupancy by Mg 2+ or Ba 2+ has been observed, respectively, for the inward, intermediate and outward positions of the OBD domains in RepB 6 C2 structure (Boer et al, 2016 ). The role of this metal binding site in the orientation of the OBD domains or RepB 6 activity is presently unknown.…”

“…The conformational ensemble of RepB 6 is characterized by a rigid cylindrical scaffold, formed by the ODs, to which the OBDs are attached as highly flexible appendages. The intrinsic flexibility allows RepB to adopt multiple conformational states and might be involved in the specific recognition of the dso (Boer et al, 2016 ). The N-terminal 131-residue OBD domain retains the DNA-binding and nuclease functions of the protein (Boer et al, 2009 ).…”