CTP-DEPENDENT MOLECULAR SWITCHES AS REGULATORS OF DNA SEGREGATION

The ParABS system ensures proper DNA segregation in the majority of bacterial species. In the Myxococcales, this system is complemented by PadC, a protein containing a ParB/Srx domain. The crystal structure of ParB/Srx-domain of PadC in the CTP-bound state suggests that CTP-binding is conserved among ParB-like proteins. CTP-dependent regulation might therefore be a more common theme in biological systems.

STRUCTURAL BIOLOGY

36 ESRF

The faithful inheritance of genetic information is critical to organisms from all domains of life and relies on a machinery that actively segregates sister DNA molecules into offspring cells. In the majority of bacterial species, chromosome segregation is mediated by the ParABS system [1], which consists of the ATPase ParA, the DNA-binding protein and CTPase ParB, and centromeric sequence motifs (parS), typically located near the chromosomal origin of replication [2]. Recognition of parS sites by ParB results in DNA condensation and formation of a large nucleoprotein complex. Once established, this so-called partition complex recruits ParA, which subsequently drives the directed movement of the ParB-DNA complex and thus mediates DNA segregation into the respective daughter cells [3].

ParB-like proteins have a conserved general architecture and can be subdivided into an N-terminal ParB/Srx-like domain, a central DNA-binding domain harbouring a helix-turn- helix (HTH) motif, and a C-terminal dimerisation domain (Figure 22a) [4]. In the Myxococcales, another ParB-like protein that complements the ParABS system is also present [5]. This protein, PadC, consists of a long, unstructured N-terminal domain followed by the ParB-homology domain and a C-terminal domain involved in interaction with the bactofilin cytoskeleton (Figure 22a). In this work, the crystal structure of the ParB- homology domain of PadC was solved to 1.7 Å resolution. Phase determination was achieved by single-wavelength anomalous dispersion (Se-SAD), using crystals of selenomethionine- labeled protein at beamline ID23-1. A high- resolution dataset was subsequently collected at ID23-2 and allowed complete model building of the C-terminal ParB/Srx-domain of PadC (Figure 22b). PadC forms a compact and strongly interconnected dimer highly reminiscent of ParB. Interestingly, additional electron density deeply buried in the PadC- dimer core was unambiguously identified as two molecules of CTP (Figures 22b,c). The CTP is tightly squeezed between helices H2 and H3 of one subunit and H4 of the opposing monomer (Figure 22c). A thorough analysis indicates that the CTP-bound ParB/Srx-domain is in a closed state, thus allowing the interaction of PadC with ParA. Sequence comparisons of CTP- coordinating residues revealed that CTP-binding is a conserved trait among ParB-like proteins. The ParB/Srx-domain of ParB also interacts with CTP, thereby transitioning to a dimeric state in a similar way as observed for PadC. Supported by other data [4], these findings suggest a model in which high-affinity binding of ParB to parS triggers dimerisation of the N-terminal ParB/Srx domains and formation of a ParB ring that embraces the bound DNA (Figure 22d). This structural transition reduces the affinity of ParB for parS, so that ParB slides away

Fig. 22: Structural insights into the CTP-binding of ParB- like proteins. a) Domain architecture of PadC and ParB. b) Crystal structure of the PadC-dimer bound to CTP. c) A close-up of the CTP-binding cleft reveals a tight interaction involving contributions from both monomers. d) Model of CTP-driven ParB-binding to parS.