The CTPase activity of ParB determines the size and dynamics of prokaryotic DNA partition complexes

Abstract

ParB-like CTPases mediate the segregation of bacterial chromosomes and low-copy number plasmids. They act as DNA-sliding clamps that are loaded at parS motifs in the centromere of target DNA molecules and spread laterally to form large nucleoprotein complexes serving as docking points for the DNA segregation machinery. Here, we solve crystal structures of ParB in the pre-and post-hydrolysis state and illuminate the catalytic mechanism of nucleotide hydrolysis. Moreover, we identify conformational changes that underlie the CTP-and parS-dependent closure of ParB clamps. The study of CTPase-deficient ParB variants reveals that CTP hydrolysis serves to limit the sliding time of ParB clamps and thus drives the establishment of a well-defined ParB diffusion gradient across the centromere whose dynamics are critical for DNA segregation. These findings clarify the role of the ParB CTPase cycle in partition complex assembly and function and thus advance our understanding of this prototypic CTP-dependent molecular switch.

The CTP-binding domain is disengaged from the DNA-binding domain in a co-crystal structure of Bacillus subtilis Noc-DNA complex

“…In previous structures of apo- or DNA-bound ParB/Noc, the equivalent helix α4 consistently folds back to pack with α5 from the same protein subunit (the folding-back conformation) (Figure 3B and Supplementary Figure 2). The swinging-out conformation of helices α4-5 is often associated with the nucleotide-bound state of ParB/Noc instead (Figure 3B and Supplementary Figure 2) 6,7,11,14 . It has been suggested that CTP-binding most likely facilitates the swinging-out conformation of ParB/Noc since nucleotides have been observed to make numerous contacts to both the equivalent α4 and the α4-α5 connecting loop in various ParB proteins 7,8,11,14 .…”

“…The swinging-out conformation of helices α4-5 is often associated with the nucleotide-bound state of ParB/Noc instead (Figure 3B and Supplementary Figure 2) 6,7,11,14 . It has been suggested that CTP-binding most likely facilitates the swinging-out conformation of ParB/Noc since nucleotides have been observed to make numerous contacts to both the equivalent α4 and the α4-α5 connecting loop in various ParB proteins 7,8,11,14 . The observation of a swingingout conformation in DNA-bound Noc is therefore surprising, given that CTP was not included in the crystallization drop and that CTP-binding is incompatible with high-affinity binding at the nucleation site NBS 6 .…”

“…CTP-binding switches nucleating ParB/Noc (bound at a high-affinity parS/NBS site) from an openclamp conformation (Figure 5A-B) to a closed-clamp conformation that can escape from parS/NBS to slide to neighboring DNA while still entrapping DNA (Figure 5C) 6,7,14,17,18 . The closed-clamp conformation is possible due to the new dimerization interface between the two adjacent N-terminal CTP-binding domains of ParB/Noc (the so-called NTD-NTD engagement, Figure 5C) 6,7,14,17 . Here, our NocΔCTD- NBS structure represents an openclamp conformation because there is no protein-protein contact between the majority of two adjacent NTDs of Noc, except for the swapping helices α4 and α4’ (Figure 1B and Figure 5B).…”

“…It has been observed that, without parS/NBS , CTP is unable to efficiently promote the NTD-NTD engagement to close the ParB/Noc clamp 6,7,14,17 . To rationalize this phenomenon, Antar et al (2021) noted that two ParB subunits would not be able to occupy a parS site if they were to adopt a conformation similar to apo-ParB (in which the NTD and the DBD of the same ParB subunit fold back on each other) because of a severe clash between opposing ParB subunits 15 .…”

Subcellular Dynamics of a Conserved Bacterial Polar Scaffold Protein

“…These inverted repeats are clustered around the oriC region [ 8 ]. ParB dimers close around the DNA in a CTP-dependent fashion and spread from parS sites across the DNA [ 13 , 14 ]. Upon CTP hydrolysis, the ParB proteins are released from the DNA and can be loaded again.…”