Structure and kinase activity of bacterial cell cycle regulator CcrZ

Author summarySome bacteria enlist kinases to regulate cell cycle progression and developmental checkpoints. CcrZ is a recently identified cell cycle regulator and putative kinase showing similarity to choline kinases. We identified the ccrZ gene in Bacillus subtilis using a forward genetic approach with the ccrZ deletion conferring sensitivity to a broad range of DNA damage. We show that induced expression of CcrZ causes over-initiation of DNA replication, and we provide evidence for an interaction between CcrZ and replication initiation proteins DnaA and DnaB. We find that integrity of the kinase active site is required for function in vivo. To gain insight into kinase function, we solved the crystal structure of B. subtilis CcrZ bound to AMP-PNP. The structure demonstrates that CcrZ evolved from a choline kinase with a bilobed structure and a larger inter-lobe cleft relative to choline kinases. The larger inter-lobe cleft could help accommodate larger substrates than choline. As expected from the structure, CcrZ does not have activity on choline, but instead shows activity on ribose and 2 deoxy-ribose. With our results, we propose that CcrZ regulates replication initiation through phosphorylation of a small molecule and interaction with replication initiation proteins. CcrZ is a recently discovered cell cycle regulator that connects DNA replication initiation with cell division in pneumococci and may have a similar function in related bacteria. CcrZ is also annotated as a putative kinase, suggesting that CcrZ homologs could represent a novel family of bacterial kinase-dependent cell cycle regulators. Here, we investigate the CcrZ homolog in Bacillus subtilis and show that cells lacking ccrZ are sensitive to a broad range of DNA damage. We demonstrate that increased expression of ccrZ results in over-initiation of DNA replication. In addition, increased expression of CcrZ activates the DNA damage response. Using sensitivity to DNA damage as a proxy, we show that the negative regulator for replication initiation (yabA) and ccrZ function in the same pathway. We show that CcrZ interacts with replication initiation proteins DnaA and DnaB, further suggesting that CcrZ is important for replication timing. To understand how CcrZ functions, we solved the crystal structure bound to AMP-PNP to 2.6 angstrom resolution. The CcrZ structure most closely resembles choline kinases, consisting of a bilobal structure with a cleft between the two lobes for binding ATP and substrate. Inspection of the structure reveals a major restructuring of the substrate-binding site of CcrZ relative to the choline-binding pocket of choline kinases, consistent with our inability to detect activity with choline for this protein. Instead, CcrZ shows activity on D-ribose and 2-deoxy-D-ribose, indicating adaptation of the choline kinase fold in CcrZ to phosphorylate a novel substrate. We show that integrity of the kinase active site is required for ATPase activity in vitro and for function in vivo. This work provides structural, biochemical, and functional insight into a newly identified, and conserved group of bacterial kinases that regulate DNA replication initiation.