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This study used X-ray diffraction to solve the crystal structures of ParB in the pre- and post-hydrolysis state at high resolution (1.7 and 1.9 Å, respectively). Data for the post-hydrolysis state were collected at beamline ID30-B. ParB was found to form a dimer composed of two domain- swapped NBDs followed by adjacent parS-binding domains (PBD) (Figures 45b and 45c). The nucleotides complexed with Mg2+ ions are sandwiched at the dimer interface. They establish multiple hydrogen-bonding interactions with neighbouring amino acid side chains and backbone groups that contribute to NBD self-dimerisation.
Importantly, the crystal structures revealed Q52 and E93 as potential catalytic residues (Figure 45c). Substitution of these residues by alanine drastically reduced or completely abolished CTP hydrolysis (Figure 45d). It was hypothesised that E93 could act as a catalytic base, promoting the nucleophilic attack of a polarised water molecule on the γ-phosphate group, whereas Q52 may stabilise the catalytic product and, thus, slow down the reverse reaction.
Further biochemical analyses showed that, in the absence of CTPase activity, ParB forms hyperstable clamps that remain associated longer with the DNA. In vivo, catalytically inactive ParB variants spread further on the chromosome compared to the wild type protein and lead to severe DNA segregation defects.
CTP hydrolysis shapes bacterial DNA-partition complexes
Stable inheritance of genetic information is essential for the survival and propagation of all cellular life forms. In bacteria, the segregation of chromosomes and low-copy number plasmids is often mediated by the ParABS system. The crystal structures of ParB in the pre- and post- hydrolysis state were solved, shedding light on the catalytic mechanism of CTP hydrolysis.
The bacterial ParABS DNA-segregation system comprises centromere-like DNA sequences (parS), a DNA-sliding clamp (ParB) and a P-loop ATPase (ParA) that interacts with ParB, mediating the active translocation of partition complexes during cell division . The loading of ParB clamps onto target DNA molecules requires the nucleotide cofactor CTP and the catalyst parS. Together, CTP- and parS-binding promote the self-association of the N-terminal nucleotide- binding domains (NBDs) of a ParB dimer, which results in the formation of a clamp-like structure entrapping DNA (Figure 45a). Upon clamp closure at a parS sequence, ParB leaves its initial loading site and spreads laterally along the DNA. As a result, parS becomes available for the loading of new ParB dimers [2-4]. Remarkably, ParB is able to hydrolyse CTP to CDP and Pi, but the role of CTP hydrolysis remained elusive.
Fig. 45: Role of CTP in ParB function. a) Scheme of a closed ParB clamp. b) Crystal structure of a nucleotide-bound ParB dimer, one monomer is shown in cartoon and the second monomer in surface representation. c) Catalytic pocket of ParB in the pre- and post-hydrolysis state. d) CTPase activities of different ParB variants. e) Model of partition complex assembly. ParB clamps are loaded at parS and then they spread laterally on the DNA. CTP hydrolysis triggers clamp opening, leading to ParB unloading. Released ParB clamps undergo nucleotide exchange and are loaded again at parS, thereby re-entering the cycle.