A CTP-dependent gating mechanism enables ParB spreading on DNA

Abstract

The tripartite ParA-ParB-parS complex ensures faithful chromosome segregation in the majority of bacterial species. ParB nucleates on a centromere-like parS site and spreads to neighboring DNA to form a network of protein-DNA complexes. This nucleoprotein network interacts with ParA to partition the parS locus, hence the chromosome to each daughter cell. Here, we determine the cocrystal structure of a C-terminal domain truncated ParB-parS complex from Caulobacter crescentus, and show that its N-terminal domain adopts alternate conformations. The multiple conformations of the N-terminal domain might facilitate the spreading of ParB on the chromosome. Next, using ChIPseq we show that ParBs from different bacterial species exhibit variation in their intrinsic capability for spreading, and that the N-terminal domain is a determinant of this variability. Finally, we show that the C-terminal domain of Caulobacter ParB possesses no or weak non-specific DNA-binding activity. Engineered ParB variants with enhanced non-specific DNA-binding activity condense DNA in vitro but do not spread further than wild-type in vivo. Taken all together, our results emphasize the role of the N-terminal domain in ParB spreading and faithful chromosome segregation in Caulobacter crescentus. Proper chromosome segregation is essential in all domains of life. In two-thirds of known bacterial species, faithful chromosome segregation is mediated by the conserved ParA-ParB-parS system (1-10). This tripartite complex consists of a Walker-box ATPase ParA, a centromere-binding protein ParB, and a centromere-like DNA sequence parS. The parS site is the first DNA locus to be segregated after chromosome replication (2,5,11,12). ParB, a DNA-binding protein, nucleates on parS before binding to adjacent non-specific DNA to form a network of protein-DNA complexes. This nucleoprotein network interacts with ParA to partition the chromosome to each daughter cell. In Caulobacter crescentus, ParA forms a protein gradient emanating from the opposite pole of the cell to the ParB-parS complex (13-15). The DNA-bound ParB complexes stimulate the ATPase activity of ParA, causing the ParA gradient to retract, bringing the ParB-DNA complex to the opposite pole of the cell in a retreating gradient of ParA (15)(16)(17)(18)(19). In Caulobacter crescentus, ParA and ParB are essential for chromosome segregation and cell viability (3,13). In other bacterial species, engineered strains lacking ParB are still viable but have elevated numbers of anucleate cells due to defects in chromosome segregation (2,7,9,12,16,(20)(21)(22).The binding of multiple ParB molecules onto non-specific DNA after nucleation at parS (i.e. spreading) is a crucial event; bacterial cells harboring a nucleation-competent but spreadingdefective parB allele are impaired in plasmid/chromosome segregation (23-25). Spreading was first discovered for the F-plasmid-encoded SopB protein and the P1 plasmid-encoded ParB protein (26,27), and was subsequently found to be a general feature of many plasmid and chromo...

Rules and Exceptions: The Role of Chromosomal ParB in DNA Segregation and Other Cellular Processes

“…The role of CTP binding and hydrolysis in ParB-driven partition complex formation was also shown for M. xanthus ParB [141]. Interestingly, in a recent study, Jalal and co-workers demonstrated that ParBs from various bacterial species show variation in their intrinsic capabilities for spreading and that the determinant of this variability maps to the N-terminal domain (NTD) [133]. The study used ChIP-seq to analyze ParB spreading in a heterologous host, so the results may not recapitulate all the determinants affecting the extent of spreading in its original host (like DNA supercoiling, involvement of other proteins).…”

“…All models postulate that multiple ParB-ParB interaction interfaces must be involved in the assembly of higher-order complexes. Structural studies have indeed demonstrated the flexibility of ParB molecules, the ability to bridge different parS sequences, as well as various cross-monomer interactions [115,116,122,133]. Notably, the majority of mutational analyses show that the conserved arginine patch residues are required for ParB spreading and DNA partition, thereby indicating the crucial role of this motif in ParB functions [68,114,127].…”

“…A combination of biochemical, structural, and computational approaches can shed light on the possible architecture of the ParB-parS nucleoprotein complex (reviewed in [118]). Nevertheless, no common assembly mechanism has been proposed, thus suggesting dynamic and heterogeneous interactions between ParB molecules within the complex (Figure 2b) [116,122,133,134]. It is widely acknowledged that ParB loading on parS is a prerequisite for the conformational changes that prime ParB for nucleation [135][136][137].…”

“…The motif containing amino acid residues responsible for CTP binding and ParB CTPase activity is one of the most conserved elements within the ParB protein sequences [122,141]. This suggests that CTP binding and hydrolysis may be crucial for proper ParB functioning, not only in the abovementioned B. subtilis [122], M. xanthus [141] and C. crescentus [133], but also in other bacteria encoding ParAB-parS systems.…”

Chromosome Segregation Proteins as Coordinators of Cell Cycle in Response to Environmental Conditions

“…This bridging is mediated by dimerization of the arginine-rich ParB box II and was recently shown to be modulated by CTP binding in this region (Osorio-Valeriano et al, 2019;Soh et al, 2019). Notably, while in all bacterial species that use the parABS system, ParB specifically binds parS sequences, the affinity and specificity of ParB toward parS sequences vary among bacteria, resulting in differences in ParB spreading (Jalal et al, 2019). These differences, as well as variations in the number and distribution of parS sites, are reflected in the species-specific architecture of the ParB complex.…”