A CTP-dependent gating mechanism enables ParB spreading on DNA

  1. Adam SB Jalal
  2. Ngat T Tran
  3. Clare EM Stevenson
  4. Afroze Chimthanawala
  5. Anjana Badrinarayanan
  6. David M Lawson
  7. Tung BK Le

  • Curated by eLife

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    Evaluation Summary:

    Bacterial ParB partition proteins have the novel property that they employ an unusual nucleotide cofactor for complex assembly at their specific DNA binding site, parS. The impact of this study is on our general understanding of this novel class of nucleotide-dependent processes, and the role that nucleotide-protein interactions play in DNA binding and bacterial physiology.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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Abstract

Proper chromosome segregation is essential in all living organisms. The ParA-ParB- parS system is widely employed for chromosome segregation in bacteria. Previously, we showed that Caulobacter crescentus ParB requires cytidine triphosphate to escape the nucleation site parS and spread by sliding to the neighboring DNA (Jalal et al., 2020). Here, we provide the structural basis for this transition from nucleation to spreading by solving co-crystal structures of a C-terminal domain truncated C. crescentus ParB with parS and with a CTP analog. Nucleating ParB is an open clamp, in which parS is captured at the DNA-binding domain (the DNA-gate). Upon binding CTP, the N-terminal domain (NTD) self-dimerizes to close the NTD-gate of the clamp. The DNA-gate also closes, thus driving parS into a compartment between the DNA-gate and the C-terminal domain. CTP hydrolysis and/or the release of hydrolytic products are likely associated with reopening of the gates to release DNA and recycle ParB. Overall, we suggest a CTP-operated gating mechanism that regulates ParB nucleation, spreading, and recycling.

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  1. Version published to 10.7554/elife.69676 on eLife
  2. Version published to 10.1101/816959v3 on bioRxiv
  3. eLife

    Evaluation Summary:

    Bacterial ParB partition proteins have the novel property that they employ an unusual nucleotide cofactor for complex assembly at their specific DNA binding site, parS. The impact of this study is on our general understanding of this novel class of nucleotide-dependent processes, and the role that nucleotide-protein interactions play in DNA binding and bacterial physiology.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    In a previous eLife paper, the authors showed CTP-dependent ParB spreading from a parS site in vitro on closed DNA substrates. But how ParB binds and spreads from the parS site to flanking DNA remained unclear. In this manuscript the authors provide rigorous structural and biochemical evidence showing how CTP-binding and -hydrolysis regulates (1) the loading of an open ParB dimer onto parS, (2) the sliding of the closed ParB clamp on flanking DNA, and (3) opening of the ParB clamp through CTP-hydrolysis and DNA release. The manuscript currently stands as one of the first papers with strong biochemical and structural support of a mechanism that explains how CTP-binding and CTPase activity can regulate a proteins transition from specific binding to sliding on chromosomal DNA.

    The authors provide a strong structural basis for the conformational transition in the N-terminal domain of ParB from the open clamp, where parS is captured by its DNA-binding domain, to the closed clamp upon CTP-binding, where parS is driven from the DNA-binding domain into a compartment of the ParB dimer that allows for sliding on flanking DNA. A series of clever in vitro cross-linking assays provide further biochemical support that ParB entraps DNA in a compartment between the DNA-binding domain and the C-terminal dimer domain after binding CTP. The authors first used CTPgammaS and cross-linking to trap the ParB dimer as a closed clamp. The authors go one step further and strategically mutated the CTP-binding pocket of ParB, allowing them to identify several mutant classes, such as CTP-binding defective and CTP-hydrolysis defective mutants, among others. The authors convincingly show through a suite of biochemical characterization (CTP-binding, CTPase, dimer cross-linking, and parS association assays) that ParB[E102A] is a CTP-trap mutant, capable of binding CTP but unable to hydrolyze CTP. The authors fairly conclude that [E102A] maintains a more stable closed-clamp conformation on closed DNA substrates containing a parS site.

    The authors take ParB[E102A] in vivo and performed ChIP-seq in Figure 8A. As the authors note, the mutant profile is significantly lower in height compared to WT. As a result, it is difficult to conclude whether ParB[E102A] has a more pronounced spreading activity from any of the parS sites on the chromosome. However, the authors do note a very interesting and dramatic ParB[E102A] signal amplified upstream of all parS sites. From this upstream "more extended" coverage, the authors propose ParB[E102A] can bind parS and then slide further away. As an interested reader, an explanation as to why this extension only occurs upstream of all parS sites, and particularly over the parAB operon, would have been appreciated.

    Overall the biochemical conclusions and mechanism of action proposed in Figure 9 are well justified by the data presented. The findings have major implications in our understanding of the most common DNA segregation system across the bacterial world.

    Going forward it is important to place these findings and mechanism in the context of how a ParB dimer oligomerizes with other ParB dimers as shown by many others in the field, as well as how these CTP-dependent activities regulate interactions with the ParA ATPase on the nucleoid that drives the chromosome segregation reaction.

  5. eLife

    Reviewer #2 (Public Review):

    Bacterial ParB partition proteins have the novel property that they employ a CTP nucleotide cofactor for complex assembly at their specific DNA binding site, parS. Here the authors present structural and biochemical data using Caulobacter crescentus ParB, and examine how CTP binding promotes ParB clamp formation around the DNA substrate which in turn results in parS release so the clamp can move away from parS along the DNA ("spreading"). In addition, they examine the role of CTP hydrolysis via isolation of mutant ParBs altered in interactions with CTP. Their data support the proposal that CTP hydrolysis opens the clamps to unload ParB from DNA and limit DNA spreading.

    The authors solve the crystal structure of ParB lacking the C-dimer domain in two forms, one with parS DNA and one with CTPgS, representing the prehydrolysis state of ParB. The latter is a new addition to ParB-CTP structures as the original B. subtilis structure was with CDP. The CTPgS structure is "closed" and shows how this closure creates clashes with DNA binding. The CTPgS structure provides a more detailed description of the CTP binding site, and allowed the authors to target a number of residues for mutagenesis to further probe the role of CTP hydrolysis. They use crosslinking of full length ParB to show that closure at the N-term/DBD region and at the C-dimer domain is essential for the clamp. The crosslinking at the DBD further implies that DNA has moved into the region between the DBD and dimer domain, since it can be released when this region is cleaved at an engineered TEV protease site. Overall the data are convincing and they provide important new information about this new class of CTP-dependent DNA binding proteins. The combination of structures, crosslinking and mutagenesis provides a relatively comprehensive analysis and supports the model they propose (Fig 7).

  6. eLife

    Reviewer #3 (Public Review):

    Jalal et al. investigated the mechanism of gating that enables ParB to clamp onto DNA in a reversible manner, using a combination of X-ray structures and in vitro assays. They find that a truncated version (delta CTD) of ParB from Caulobacter crescentus adopt two distinct conformations when bound to parS, revealing an open conformation. By contrast, the structure in the presence of the non-hydrolysable CTPgS nucleotide display a closed conformation of the NTD, similar to other ParB-CTP structures (B. subtilis and M. xanthus) indicating that this closed conformation is a conserved feature. By comparing the ParB-deltaCTD structures formed in the presence of parS or of CTPgS, they unravel the conformational changes upon CTP binding that convert ParB from the open to the closed conformation. They also characterize the clash with DNA in the closed conformation thus providing a molecular explanation for the escape of ParB dimer from parS site upon CTP binding. By performing a well-designed and carefully controlled double cross-linking assay, they fully demonstrate that the DNA is entrapped between the DBD and the CTD, in both a parS- and a CTP-dependent manner. These data clearly demonstrate that the ParB dimer functions as a molecular clamp that entraps parS-containing DNA within the 20 amino acids-long DBD-CTD compartment upon CTP binding. To investigate the role of CTP-hydrolysis in this mechanism, they perform an alanine scanning mutagenesis to uncover and characterize a ParB variant (E102A) defective in CTP hydrolysis but still able to self-dimerize. Comparison of ChIP-sequencing data performed with ParB WT and E102A display enrichment differences, both in intensity and extend. The authors suggest that the clamped state of the ParB variant is more stable explaining the extended profile compare to WT, and thus that CTP hydrolysis might be involved in opening the closed conformation.

    The manuscript is clearly written and well presented, and all the experiments are thoroughly controlled. This study thus provides novel structural and molecular insights of the two ParB dimer states - open and closed conformations - that are controlled by ParS and CTP binding. The conclusions of this paper are well supported by data, but some aspects of the ChIP-sequencing data analysis need to be clarified and discussed.

  7. Version published to 10.1101/816959v2 on bioRxiv
  8. Version published to 10.1101/816959v1 on bioRxiv