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. 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 may subsequently re-open the gates. Overall, we suggest a CTP-operated gating mechanism that regulates ParB nucleation and spreading.
INTRODUCTION
Proper chromosome segregation is essential in all domains of life. In most bacterial species, faithful chromosome segregation is mediated by the tripartite ParA-ParB-parS system1–11. ParB, a CTPase and DNA-binding protein, nucleates on parS before spreading to adjacent non-specific DNA to form a higher-order nucleoprotein complex1,12–17. The ParB-DNA nucleoprotein complex stimulates the ATPase activity of ParA, driving the movement of the parS locus (and subsequently, the whole chromosome) to the opposite pole of the cell18– 23. ParB spreads by sliding along the DNA, in a manner that depends on the binding of a co-factor, cytidine triphosphate (CTP)24–26. A co-crystal structure of a C-terminal domain truncated Bacillus subtilis ParB (ParBΔCTD) together with CDP showed the nucleotide to be sandwiched between adjacent subunits, thus promoting their dimerization26. A similar arrangement was seen in the co-crystal structure of an N-terminal domain truncated version of the Myxococcus xanthus ParB homolog, PadC, bound to CTP25. Self-dimerization at the N-terminal domain (NTD) of B. subtilis ParB creates a clamp-like molecule that enables DNA entrapment26. Biochemical studies with M. xanthus and C. crescentus ParBs showed that CTP facilitates the dissociation of ParB from parS, thereby switching ParB from a nucleating mode to a sliding mode24,25. ParB can hydrolyze CTP to CDP and inorganic phosphate24–26, however hydrolysis is not required for spreading since ParB in complex with a non-hydrolyzable CTP analog (CTPγS) can still self-load and slide on DNA24,26. Furthermore, M. xanthus PadC does not possess noticeable CTPase activity25. As such, the role of CTP hydrolysis in bacterial chromosome segregation is not yet clear.
Here, we solve co-crystal structures of a C-terminal domain truncated C. crescentus ParB in complex with either parS or CTPγS to better understand the roles of CTP binding and hydrolysis. Consistent with the previous report26, the NTDs of C. crescentus ParB also self-dimerize upon binding to nucleotides, thus closing a molecular gate at this domain (the NTD-gate). Furthermore, the two opposite DNA-binding domains (DBD) move closer together to close a second molecular gate (the DNA-gate). We provide evidence that the CTP-induced closure of the DNA-gate drives parS DNA from the DBD into a 20-amino-acid long compartment between the DNA-gate and the C-terminal domain, thus explaining how CTP binding enables ParB to escape the high-affinity parS site to spread while still entrapping DNA. Lastly, we identify and characterize a ParB “clamp-locked” mutant that is defective in CTP hydrolysis but otherwise competent in gate closing, suggesting a role for CTP hydrolysis/release of hydrolytic products in the re-opening ParB gates. Collectively, we suggest a CTP-operated gating mechanism that regulates ParB nucleation and spreading in C. crescentus.
RESULTS
Co-crystal structure of a C. crescentus ParBΔCTD-parS complex reveals an open conformation at the NTD
We sought to solve a co-crystal structure of C. crescentus ParB nucleating at parS. After screening several constructs with different lengths of ParB and parS, we obtained crystals of a 50 amino acid C-terminally truncated ParB in complex with a 22-bp parS DNA (Figure 1). This protein variant lacks the CTD reponsible for ParB dimerization (Figure 1A)27. Diffraction data for the ParBΔCTD-parS co-crystal were collected to 2.9 Å resolution, and the structure was solved by molecular replacement (see Materials and Methods). The asymmetric unit contains four copies of ParBΔCTD and two copies of the parS DNA (Figure 1-figure supplement 1A-B).
Each ParBΔCTD subunit consists of an NTD (helices α1-α4 and sheets β1-β4) and a DBD (helices α5-α10) (Figure 1B). Each ParBΔCTD binds to a half parS site but there is no protein-protein contact between the two adjacent subunits (Figure 1B). We previously reported a 2.4 Å co-crystal structure of the DBD of C. crescentus ParB bound to parS28 and elucidated the molecular basis for specific parS recognition, hence we focus on the conformation of the NTD here instead. We observed that helices α3 and α4 are packed towards the DBD and are connected to the rest of the NTD via an α3-β4 loop (Figure 1B-C). While the DBD and helices α3-α4 are near identical between the two ParBΔCTD subunits (RMSD=0.19 Å, Figure 1C), the rest of the NTD, from α1 to β4, adopts notably different conformations in the two subunits (Figure 1C-D). Specifically, NTDs (α1-β4) from the two ParBΔCTD subunits are related by a rotation of approximately 80°, due to changes in a flexible loop in between α3 and β4 (Figure 1D). Furthermore, by superimposing the C. crescentus ParBΔCTD-parS structure onto that of Helicobacter pylori29, we observed that the NTDs of ParB from both species can adopt multiple alternative orientations (Figure1-figure supplement 2). Taken together, these observations suggest that the ability of the NTD to adopt multiple open conformations is likely a general feature of nucleating ParB.
Co-crystal structure of a C. crescentus ParBΔCTD-CTPγS complex reveals a closed conformation at the NTD
Next, to gain insight into the spreading state of ParB, we solved a 2.7 Å resolution structure of C. crescentus ParBΔCTD in complex with CTPγS (see Materials and Methods). At this resolution, it was not possible to assign the position of the ligand sulfur atom. Indeed, the placement of the sulfur atom relative to the terminal phosphorus atom may vary from one ligand to the next in the crystal leading to an averaging of the electron density. Hence, we modeled CTP, instead of CTPγS, into the electron density (Figure 2 and Figure 2-figure supplement 1). The asymmetric unit contains two copies of ParBΔCTD, each with a CTPγS molecule and a coordinated Mg2+ ion bound at the NTD (Figure 2A). In contrast to the open conformation of the ParBΔCTD-parS structure, nucleotide-bound NTDs from opposite subunits self-dimerize (with an interface area of 2111 Å2, as determined by PISA30), thus adopting a closed conformation (Figure 2A). Multiple CTPγS-contacting residues also directly contribute to the NTD self-dimerization interface (summarized in Figure 2-figure supplement 2), indicating a coupling between nucleotide binding and self-dimerization. Furthermore, the C. crescentus ParBΔCTD-CTPγS structure is similar to that of the CDP-bound B. subtilis ParBΔCTD (RMSD=1.48 Å) and the CTP-bound M. xanthus PadCΔNTD (RMSD=2.23 Å) (Figure 2-figure supplement 3A), suggesting that the closed conformation at the NTD is structurally conserved in nucleotide-bound ParB/ParB-like proteins.
Each CTPγS molecule is sandwiched between helices α1, α2, α3 from one subunit and helix α3’ from the opposite subunit (Figure 2B). Ten amino acids form hydrogen-bonding contacts with three phosphate groups of CTPγS, either directly or via the coordinated Mg2+ ion (Figure 2C). These phosphate-contacting residues are referred to as P-motifs 1 to 3, respectively (P for phosphate motif, Figure 2C). Four amino acids at helix α1 and the α1-β2 intervening loop provide hydrogen-bonding interactions to the cytosine ring, hence are termed the C-motif (C for cytosine motif, Figure 2C). Lastly, six additional residues contact the ribose moiety and/or the pyrimidine moiety via hydrophobic interactions (Figure 2C). Nucleotide-contacting residues in C. crescentus ParB and their corresponding amino acids in ParB/ParB-like homologs are summarized in Figure 2-figure supplement 2 and Figure 2-figure supplement 3B. The C-motif forms a snug fit to the pyrimidine moiety, thus is incompatible with larger purine moieties such as those from ATP or GTP. Hydrogen-bonding contacts from the G79 main chain and the S74 side chain to the amino group at position 4 of the cytosine moiety further distinguish CTP from UTP (Figure 2C). Taken all together, our structural data are consistent with the known specificity of C. crescentus ParB for CTP24.
Conformational changes between the nucleating and the spreading state of C. crescentus ParB
A direct comparison of the C. crescentus ParBΔCTD-parS structure to the ParBΔCTD-CTPγS structure further revealed the conformational changes upon nucleotide binding. In the nucleating state, as represented by the ParBΔCTD-parS structure, helices α3 and α4 from each subunit bundle together (32° angle between α3 and α4, Figure 3). However, in the spreading state, as represented by the ParBΔCTD-CTPγS structure, α3 swings outwards by 101° to pack itself with α4’ from the opposing subunit (Figure 3). Nucleotide binding most likely facilitates this “swinging-out” conformation since both α3 and the α3-α4 loop i.e. P-motif 3 make numerous contacts with the bound CTPγS and the coordinated Mg2+ ion (Figure 2C). The reciprocal exchange of helices ensures that the packing in the α3-α4 protein core remains intact, while likely driving the conformational changes for the rest of the NTD as well as the DBD (Figure 4A). Indeed, residues 44-121 at the NTD rotate wholesale by 94° to dimerize with their counterpart from the opposing subunit (Figure 4A and Figure 4-figure supplement 1A). Also, residues 161-221 at the DBD rotate upward by 26° in a near rigid-body movement (Figure 4A and Figure 4-figure supplement 1A). As the result, the opposite DBDs are closer together in the spreading state (inter-domain distance = ∼27 Å) than in the nucleating state (inter-domain distance = ∼36 Å) (Figure 4-figure supplement 1B). By overlaying the CTPγS-bound structure onto the parS DNA complex, it is clear that the DBDs in the spreading state clash severely with DNA, hence are no longer compatible with parS DNA binding (Figure 4B). Our structural data are therefore consistent with the previous finding that CTP decreases C. crescentus ParB nucleation on parS or liberates pre-bound ParB from parS site24. Overall, we suggest that CTP binding stabilizes a conformation that is incompatible with DNA-binding and that this change might facilitate ParB escape from the high-affinity nucleation parS site.
C. crescentus ParB entraps parS DNA in a compartment between the DBD and the CTD in a CTP-dependent manner
To verify the CTP-dependent closed conformation of ParB, we performed site-specific crosslinking of purified proteins using a sulfhydryl-to-sulfhydryl crosslinker bismaleimidoethane (BMOE)26. Residues Q35, L224, and I304 at the NTD, DBD, and CTD, respectively (Figure 5A) were substituted individually to cysteine on an otherwise cysteine-less ParB (C297S) background24, to create ParB variants where symmetry-related cysteines become covalently linked if they are within 8 Å of each other (Figure 5B). We observed that the crosslinking of both ParB (Q35C) and ParB (L224C) were enhanced ∼2.5 to 3-fold in the presence of parS DNA and CTP (Figure 5B), consistent with CTP favoring a conformation when the NTD and the DBD are close together. In contrast, ParB (I304C) crosslinked independently of CTP or parS (Figure 5B), supporting the known role of the CTD as a primary dimerization domain27,31.
Previously, Soh et al (2019) showed that B. subtilis ParB-CTP forms a protein clamp that entraps DNA26, however the location of DNA within the clamp is not yet clear. To locate such DNA-entrapping compartment, we employed a double crosslinking assay26 while taking advantage of the availability of crosslinkable cysteine residues in all three domains of C. crescentus ParB (Figure 5A). A C. crescentus ParB variant with crosslinkable NTD and CTD interfaces (Q35C I304C) was first constructed and purified (Figure 5C). ParB (Q35C I304C) could form high molecular weight (HMW) species near the top of the polyacrylamide gel in the presence of CTP, a 3-kb parS plasmid, and the crosslinker BMOE (Lane 7, Figure 5C-Left panel). The HMW smear on the polyacrylamide gel contained both protein and DNA as apparent from a dual staining with Coomassie and Sybr Green (Figure 5C-Left panel). Slowly migrating DNA-stained bands were also observed when resolving on an agarose gel (Figure 5C-Right panel). The HMW smear most likely contained DNA-protein catenates between a circular parS plasmid and a denatured but otherwise circularly crosslinked ParB (Q35C I304C) polypeptide. Indeed, a post-crosslinking treatment with Benzonase, a non-specific DNA nuclease (Lane 8, Figure 5C-Left panel) or the use of a linear parS DNA (Lane 4, Figure 5C-Left panel) eliminated the HMW smear, presumably by unlinking the DNA-protein catenates. Lastly, the HMW smear was not observed when a plasmid containing a scrambled parS site was used (Lane 10, Figure 5C-Left panel) or when CTP was omitted from the crosslinking reaction (Lane 6, Figure 5C-Left panel), indicating that the DNA entrapment is dependent on parS and CTP. Collectively, these experiments demonstrate that as with the B. subtilis ParB homolog, C. crescentus ParB is also a CTP-dependent molecular clamp that can entrap parS DNA in between the NTD and the CTD.
Employing the same strategy, we further narrowed down the DNA-entrapping compartment by constructing a ParB (L224C I304C) variant in which both the DBD and the CTD are crosslinkable (Figure 4D). We found that crosslinked ParB (L224C I304C) also entrapped circular plasmid efficiently in a parS- and CTP-dependent manner, as judged by the appearance of the HMW smear near the top of the gel (Lane7, Figure 5D-Left panel). By contrast, ParB (Q35C L224C) that has both the NTD and the DBD crosslinkable, was unable to entrap DNA in any tested condition (Figure 5-figure supplement 1). We therefore hypothesized that ParB clamps entrap DNA within a compartment created by a 20-amino-acid linker in between the DBD and the CTD. To investigate further, we constructed a ParB (L224C I304C)TEV variant, in which a TEV protease cleavage site was inserted within the DBD-CTD linker (Figure 5-figure supplement 2A). Again, ParB (L224C I304C)TEV entrapped a circular parS plasmid efficiently in the presence of CTP (the HMW smear on lane 7, Figure 5-figure supplement 2A). However, a post-crosslinking treatment with TEV protease eliminated such HMW smear, presumably by creating a break in the polypeptide through which a circular plasmid could escape (Lane 8, Figure 5-figure supplement 2A). Lastly, we extracted crosslinked ParB (L224C I304C) from gel slides that encompassed the HMW smear, and electrophoresed the eluted proteins again on a denaturing gel to find a single band that migrated similarly to a double-crosslinked protein (Lane 9, Figure 5-figure supplement 2B). Therefore, our results suggest that a ParB dimer, rather than ParB oligomers, is the major species that entraps DNA. Taken together, we suggest that C. crescentus ParB dimer functions as a molecular clamp that entraps parS-containing DNA within a DBD-CTD compartment upon CTP binding.
C. crescentus ParB (E102A) is a clamp-locked mutant that is defective in clamp re-opening
Next, we investigated the potential role(s) of CTP hydrolysis. Hydrolysis is unlikely to be required for DNA entrapment and translocation since ParB in complex with CTPγS can still self-load and slide on DNA24,26. M. xanthus ParB (N172A) and B. subtilis ParB (N112S) mutants, which bind but cannot hydrolyze CTP, failed to form higher-order protein-DNA complexes inside the cells25,26. However, these ParB variants are already impaired in NTD self-dimerization26, hence the mechanistic role of CTP hydrolysis is still unclear. We postulated that creation of a ParB variant defective in CTP hydrolysis but otherwise competent in NTD self-dimerization, would enable us to investigate the possible role of CTP hydrolysis. To this end, we performed alanine scanning mutagenesis on the CTP-binding pocket of C. crescentus ParB (Figure 2C). Eleven purified ParB variants were assayed for CTP binding by a membrane-spotting assay (DRaCALA) (Figure 6A), and for CTP hydrolysis by measuring the releasing rate of inorganic phosphate (Figure 6B). Moreover, their propensity for NTD self-dimerization was also analyzed by crosslinking with BMOE (Figure 6C and Figure 6-figure supplement 1). Lastly, their ability to nucleate, slide, and entrap a closed parS DNA substrate was investigated by a bio-layer interferometry (BLI) assay (Figure 6D and Figure 6-figure supplement 2A). Immobilizing a dual biotin-labeled DNA on a streptavidin-coated BLI surface created a closed DNA substrate that can be entrapped by ParB-CTP clamps (Figure 6-figure supplement 2A)24. The BLI assay monitors wavelength shifts resulting from changes in the optical thickness of the probe surface during the association/dissociation of ParB with a closed DNA substrate in real-time (Figure 6-figure supplement 2).
Overall, we identified several distinct classes of ParB mutants:
(i) Class I: ParB (R60A), (R103A), (R104A), (R139A), (N136A), (G79S), and (S74A) did not bind or bound radiolabeled CTP only weakly (Figure 6A), thus also showed weak to no CTP hydrolysis (Figure 6B) or clamp-closing activity (Figure 6C-D).
(ii) Class II: ParB (Q58A) and (E135A) that are competent in CTP-binding (Figure 6A), but defective in CTP hydrolysis (Figure 6B) and in entrapping a closed parS DNA substrate (Figure 6D). We noted that ParB (Q58A) and ParB (E135A) had an elevated crosslinking efficiency even in the absence of CTP (Figure 6C) but did not result in a wild-type level of DNA entrapment (Figure 6D).
(iii) Class III: ParB (E102A) did not hydrolyze CTP (Figure 6B) but nevertheless bound CTP efficiently (Figure 6A) to self-dimerize at the NTD and to entrap DNA to the same level as ParB (WT) at all CTP concentrations (Figure 6C-D).
Upon a closer inspection of the BLI sensorgrams (Figure 6-figure supplement 2B and Figure 7), we noted that the entrapped ParB (E102A) did not noticeably dissociate from a closed DNA substrate when the probe was returned to a buffer-only solution (Dissociation phase, koff = 8.0 × 10−4 ± 1.9 × 10− 4 s-1, Figure 6-figure supplement 2B and Figure 7). By contrast, entrapped ParB (WT) dissociated approx. 15-fold faster into buffer (koff = 1.2 × 10−2 ± 3.7 × 10−4 s-1). Further experiments showed that DNA-entrapment by ParB (E102A), unlike ParB (WT), is more tolerable to high-salt solution (up to 1 M NaCl, Figure 7A). Nevertheless, ParB (E102A)-CTP could not accumulate on a BamHI-restricted open DNA substrate (Figure 7B-C)24, suggesting that ParB (E102A)-CTP, similar to ParB (WT), also form a closed clamp that runs off an open DNA end. Collectively, our results suggest that parS DNA and CTP induced a stably closed clamp conformation of ParB (E102A) in vitro.
To investigate the function of ParB (E102A) in vivo, we expressed a FLAG-tagged version of parB (E102A) from a vanillate-inducible promoter (Pvan) in a C. crescentus strain where the native parB was under the control of a xylose-inducible promoter (Pxyl) (Figure 8A). Cells were depleted of the native ParB by adding glucose for 4 hrs, subsequently vanillate was added for another hour before cells were fixed with formaldehyde for ChIP-seq. Consistent with the previous report11, the ChIP-seq profile of FLAG-ParB (WT) showed a ∼10-kb region of enrichment above background with clearly defined peaks that correspond to the positions of parS sites (Figure 8A). By contrast, the ChIP-seq profile of FLAG-ParB (E102A) is reduced in height but is notably more extended than the profile of FLAG-ParB (WT) (shaded area, Figure 8A). The instability of FLAG-ParB (E102A) in vivo, hence the reduced protein level (Figure 8-figure supplement 1), might explain the lower height of its ChIP-seq profile (Figure 8). However, the more extended profile of FLAG-ParB (E102A) is likely due to a stably closed clamp formation of ParB (E102) that enables this variant to persist, and thus sliding further away from the loading site parS in vivo.
Altogether, the “clamp-locked” phenotype of ParB (E102A) implies a possible role of CTP hydrolysis and/or the release of hydrolytic products in re-opening wild-type ParB clamp to discharge DNA. Lastly, we noted that the producing ParB (E102A) could not rescue cells with depleted ParB (WT) (Figure 8B). However, due to the caveat of a lower ParB (E102A) protein level (Figure 8-figure supplement 1), we could not reliably attribute its lethal phenotype to the defective CTP hydrolysis alone.
DISCUSSION
In this study, we provide structural insights into the nucleating and sliding states of C. crescentus ParB. Nucleating ParB is an open clamp in which parS DNA is held tightly (nM affinity)11 at the DBD. The NTDs of nucleating ParB can adopt multiple alternative conformations, and crucially there is no contact between opposing NTDs. We liken this conformation of the NTD to that of an open gate (NTD-gate), through which parS DNA might gain access to the DNA-binding domain (Figure 9). In the sliding state, CTP promotes the self-dimerization of the NTDs, thus closing the NTD-gate (Figure 9). Opposing DBDs also move approximately 10 Å closer together, bringing about a conformation that is DNA incompatible. Again, we liken this conformation of the DBDs to that of a closed gate (DNA-gate) (Figure 9). Overall, the DNA-gate closure explains how CTP binding might switch ParB from a nucleating to a sliding state.
Our data suggest that the closure of the two gates drives parS DNA into a compartment in between the DBD and the CTD. Previously, Soh et al. (2019) compared the B. subtilis ParBΔCTD-CDP co-crystal structure to that of a H. pylori ParBΔCTD-parS complex and proposed that DNA must be entrapped in the DBD-CTD compartment26. Here, the available structures of nucleating and sliding ParB from the same bacterial species enabled us to introduce a crosslinkable cysteine (L224C) at the DBD, and subsequently provided a direct evidence that the DBD-CTD compartment is the DNA-entrapping compartment. The linker that connects the DBD and the CTD together is not conserved in amino acid sequence among chromosomal ParB orthologs (Figure 2-figure supplement 2), however we noted that the linker is invariably ∼20 amino acid in length and positively charge lysines are over-represented (Figure 2-figure supplement 2). The biological significance of the linker length and its lysines, if any, is currently unknown. However, it is worth noting that a human PCNA clamp was proposed to recognize DNA via lysine-rich patches lining the clamp channel, and that these lysine residues help PCNA to slide by tracking the DNA backbone32. Investigating whether lysine residues in the DBD-CTD linker of ParB have a similar role is an important subject for the future.
If not already bound on DNA, the closed ParB clamp presumably cannot self-load onto parS owing to its inaccessible DBD. In this study, we showed that parS DNA promotes the CTP-dependent NTD-gate closure (Figure 5B), thus is likely a built-in mechanism to ensure gate closure results in a productive DNA entrapment. However, the molecular basis for the parS-enhanced gate closure remains unclear due to the lack of a crystal structure of C. crescentus apo-ParB, despite our extensive efforts.
CTP functions as a molecular latch that stabilizes the closure of the NTD-gate of ParB. Here, we provide evidence that CTP hydrolysis might contribute to re-opening the closed NTD-gate. A previous structure of a B. subtilis ParBΔCTD-CDP complex also has its NTD-gate closed (CTP was hydrolyzed to CDP during the crystallization)26, hence it is likely that both CTP hydrolysis and the subsequent release of hydrolytic products are necessary to re-open the gates. However, ParB has a weak to negligible affinity for CDP, hence the CDP-bound ParB species might be short-lived in solution and might not play a significant biological role. Once the clamp is re-opened, entrapped DNA might escape via the same route that it first enters. Other well-characterized DNA clamps, for example, type II topoisomerases open their CTD to release trapped DNA. However, the CTDs of ParB are stably dimerized independently of parS and CTP (Figure 5B), hence we speculate that the CTD of ParB is likely to be impassable to the entrapped DNA. The released ParB clamp might re-nucleate on parS and bind CTP to close the gate, hence restarting the nucleation and sliding cycle. Such a recycling mechanism might provide a biological advantage since a ParB clamp once closed could otherwise become stably trapped on DNA and thus eventually diffuse too far from the parS locus, as evidenced by the E102A variant that is defective in CTP hydrolysis (Figure 8A).
The CTP-bound structure of a M. xanthus ParB-like protein, PadC, was solved to a high resolution (1.7 Å), however, PadC does not possess noticeable CTPase activity25. A co-crystal structure of B. subtilis ParB with CDP was also solved to a high resolution (1.8 Å) but represents a post-hydrolysis state instead. Lastly, our CTPγS-bound C. crescentus ParB crystals diffracted to 2.7 Å, thus preventing water molecules, including a potential catalytic water, from being assigned with confidence. Therefore, the mechanism of CTP hydrolysis by a ParB CTPase remains unresolved. Nevertheless, based on our alanine scanning experiment (Figure 8), we speculate that Q58 (P-motif 1) and E102 (P-motif 2) might be involved in the catalytic mechanism of C. crescentus ParB. Supporting this view, we noted that an equivalent Q37 in B. subtilis ParB does not contact the hydrolytic product CDP, and this residue is not conserved in the catalytic-dead M. xanthus PadC (F308, which does not contact CTP, occupies this position in PadC instead) (Figure 2-figure supplement 3). E102 is also not conserved in M. xanthus PadC (F348 occupies this equivalent position) (Figure 2-figure supplement 3). Given that ParB is the founding member of a new CTPase protein family25,26, further studies are needed to fully understand the molecular mechanism of CTP hydrolysis so that the knowledge gained might be generalized to other CTPases.
FINAL PERSPECTIVES
In this study, we provide a structural basis for a CTP-operated gating mechanism that regulate the opening and closing of the DNA-clamp ParB. CTP functions as a molecular switch that converts ParB from a nucleating to a sliding and DNA-entrapping state. Overall, CTP is crucial for the formation of the higher-order ParB-DNA complex in vivo, and ultimately for the faithful chromosome segregation in the majority of bacterial species. ATP and GTP switches are extensively used to control conformations and functions of proteins in a wide range of biological processes. However, CTP switches have rarely been found in biology so far. It is tempting to speculate that CTP switches may also be widespread in biology and await discovery. Moreover, it will be interesting to establish if evolution has also exploited this framework of CTP-induced conformational changes to regulate other diverse biological processes.
ACCESSION NUMBER
The accession number for the sequencing data reported in this paper is GSE168968. Atomic coordinates for protein crystal structures reported in this paper were deposited in the RCSB Protein Data Bank with the accession number 6T1F and 7BM8. All data are open to the public upon the deposition of this preprint.
MATERIALS AND METHODS
Strains, media and growth conditions
Escherichia coli and Caulobacter crescentus were grown in LB and PYE, respectively. When appropriate, media were supplemented with antibiotics at the following concentrations (liquid/solid media for C. crescentus; liquid/solid media for E. coli (μg/mL)): carbenicillin (E. coli only: 50/100), chloramphenicol (1/2; 20/30), kanamycin (5/25; 30/50), and oxytetracycline (1/2; 12/12).
Plasmids and strains construction
All strains, plasmids, and primers used in this study are listed in Supplementary File S1.
Construction of pET21b::parBΔCTD-(his)6
The coding sequence of a C-terminally truncated C. crescentus ParB (ParBΔCTD, lacking the last 50 amino acids) was amplified by PCR using primers NdeI-Ct-ParB-F and HindIII-Ct-ParB-R, and pET21b::parB-(his)618 as template. The pET21b plasmid backbone was generated via a double digestion of pET21b::parB-(his)6 with NdeI and HindIII. The resulting backbone was subsequently gel-purified and assembled with the PCR-amplified fragment of parBΔCTD using a 2x Gibson master mix (NEB). Gibson assembly was possible owing to a 23-bp sequence shared between the NdeI-HindIII-cut pET21b backbone and the PCR fragment. These 23-bp regions were incorporated during the synthesis of primers NdeI-Ct-ParB-F and HindIII-Ct-ParB-R. The resulting plasmids were sequence verified by Sanger sequencing (Eurofins, Germany).
Construction of pET21b::parB-(his)6 (WT and mutants)
DNA fragments containing mutated parB genes (parB*) were chemically synthesized (gBlocks, IDT). The NdeI-HindIII-cut pET21b plasmid backbone and parB* gBlocks fragments were assembled together using a 2x Gibson master mix (NEB). Gibson assembly was possible owing to a 23-bp sequence shared between the NdeI-HindIII-cut pET21b backbone and the gBlocks fragment. The resulting plasmids were sequenced verified by Sanger sequencing (Genewiz, UK).
pUC57::attL1-parB (WT/mutant)-attL2
The coding sequences of C. crescentus ParB (WT/mutants) were amplified by PCR and Gibson assembled into plasmid pUC57::attL1-parB (WT/mutants)-attL2 so that parB is flanked by phage attachment sites attL1 and attL2 i.e. Gateway cloning compatible. Correct mutations were verified by Sanger sequencing (Genewiz, UK).
pMT571-1xFLAG-DEST
Plasmid pMT57133 was first digested with NdeI and NheI. The plasmid backbone was gel-purified and eluted in 50 µL of water. The FLAG-attR1-ccdB-chloramphenicolR-attR2 cassette was amplified by PCR using primers P1952 and P1953, and pML477 as template. The resulting PCR fragment and the NdeI-NheI-cut pMT571 were assembled together using a 2xGibson master mix (NEB). Gibson assembly was possible owing to a 23 bp sequence shared between the two DNA fragments. These 23 bp regions were incorporated during the primer design to amplify the FLAG-attR1-ccdB-chloramphenicolR-attR2 cassette. The resulting plasmid was sequence verified by Sanger sequencing (Eurofins, Germany).
pMT571-1xFLAG::ParB (WT/mutants)
The parB (WT/mutant) genes were recombined into a Gateway-compatible destination vector pMT571-1xFLAG-DEST via LR recombination reaction (Invitrogen). For LR recombination reactions: 1 µL of purified pUC57::attL1-parB (WT/mutant)-attL2 was incubated with 1 µL of the destination vector pMT571-1xFLAG-DEST, 1 µL of LR Clonase II master mix, and 2 µL of water in a total volume of 5 µL. The reaction was incubated for an hour at room temperature before being introduced into E. coli DH5α cells by heat-shock transformation. Cells were then plated out on LB agar + tetracycline. Resulting colonies were restruck onto LB agar + carbenicillin and LB agar + tetracycline. Only colonies that survived on LB + tetracycline plates were subsequently used for culturing and plasmid extraction.
Strains MT148 + pMT571-1xFLAG::ParB (WT/mutants)
Electro-competent C. crescentus (NA1000) cells were electroporated with pMT571-1xFLAG::ParB (WT/mutants) plasmid to allow for a single integration at the vanA locus. The correct integration was verified by PCR, and ΦCr30 phage lysate was prepared from this strain. Subsequently, van::Pvan-1xflag-parB (WT/mutant), marked by a tetracyclineR cassette, was transduced by phage ΦCr30 into MT14834.
Protein overexpression and purification
Plasmid pET21b::parBΔCTD-(his)6 was introduced into E. coli Rosetta pRARE competent cells (Novagen) by heat-shock transformation. Forty mL overnight culture was used to inoculate 4 L of LB medium + carbenicillin + chloramphenicol. Cells were grown at 37°C with shaking at 250 rpm to an OD600 of ∼0.4. The culture was then left in the cold room to cool to 28°C before isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 0.5 mM. The culture was shaken for an additional 3 hours at 30°C before cells were pelleted by centrifugation. Pelleted cells were resuspended in a buffer containing 100 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM Imidazole, 5% (v/v) glycerol, 1 µL of Benzonase nuclease (Merck), 10 mg of lysozyme (Merck), and an EDTA-free protease inhibitor tablet (Merck). Cells were further lyzed by sonification (10 cycles of 15 s with 10 s resting on ice in between each cycle). The cell debris was removed through centrifugation at 28,000 g for 30 minutes and the supernatant was filtered through a 0.45 µm sterile filter (Sartorius). The protein was then loaded into a 1-mL HiTrap column (GE Healthcare) that had been pre-equilibrated with buffer A [100 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM Imidazole, and 5% (v/v) glycerol]. Protein was eluted from the column using an increasing (10 mM to 500 mM) Imidazole gradient in the same buffer. ParBΔCTD-containing fractions were pooled and diluted to a conductivity of 16 mS/cm before being loaded onto a 1-mL Heparin HP column (GE Healthcare) that had been pre-equilibrated with 100 mM Tris-HCl pH 8.0, 25 mM NaCl, and 5% (v/v) glycerol. Protein was eluted from the Heparin column using an increasing (25 mM to 1 M NaCl) salt gradient in the same buffer. ParBΔCTD fractions were pooled and analyzed for purity by SDS-PAGE. Glycerol was then added to ParBΔCTD fractions to a final volume of 10% (v/v), followed by 10 mM EDTA and 1 mM DTT. The purified ParBΔCTD was subsequently aliquoted, snap frozen in liquid nitrogen, and stored at -80°C. ParBΔCTD that was used for X-ray crystallography was further polished via a gel-filtration column. To do so, purified ParBΔCTD was concentrated by centrifugation in an Amicon Ultra-15 3-kDa cut-off spin filters (Merck) before being loaded into a Superdex-200 gel filtration column (GE Healthcare). The gel filtration column was pre-equilibrated with buffer containing 10 mM Tris-HCl pH 8.0 and 250 mM NaCl. ParBΔCTD fractions were then pooled and analyzed for purity by SDS-PAGE.
Other C-terminally His-tagged ParB mutants were purified using HIS-Select® Cobalt gravity flow columns as described previously28. Purified proteins were desalted using a PD-10 column (Merck), concentrated using an Amicon Ultra-4 10 kDa cut-off spin column (Merck), and stored at -80°C in a storage buffer [100 mM Tris-HCl pH 8.0, 300 mM NaCl, and 10% (v/v) glycerol]. Purified ParB mutants that were used in BMOE crosslinking experiments were buffer-exchanged and stored in a storage buffer supplemented with TCEP instead [100 mM Tris-HCl pH 7.4, 300 mM NaCl, 10% (v/v) glycerol, and 1 mM TCEP].
DNA preparation for crystallization, EnzCheck phosphate release assay, and differential radical capillary action of ligand assay (DRaCALA)
A 22-bp palindromic single-stranded DNA fragment (parS: GGATGTTTCACGTGAAACA TCC) [100 µM in 1 mM Tris-HCl pH 8.0, 5 mM NaCl buffer] was heated at 98°C for 5 minutes before being left to cool down to room temperature overnight to form 50 µM double-stranded parS DNA. The core sequence of parS is underlined.
Protein crystallization, structure determination, and refinement
Crystallization screens for the C. crescentus ParBΔCTD-parS complex were set up in sitting-drop vapour diffusion format in MRC2 96-well crystallization plates with drops comprised of 0.3 µL precipitant solution and 0.3 µL of protein-DNA complex, and incubated at 293 K. His-tagged ParBΔCTD (approx. 10 mg/mL) was mixed with a 22-bp parS duplex DNA at a molar ratio of 2:1.2 (protein monomer:DNA) in buffer containing 10 mM Tris-HCl pH 8.0 and 250 mM NaCl. The ParBΔCTD-parS crystals grew in a solution containing 20.5% (w/v) PEG 3350, 260 mM magnesium formate, and 10% (v/v) glycerol. After optimization of an initial hit, suitable crystals were cryoprotected with 20% (v/v) glycerol and mounted in Litholoops (Molecular Dimensions) before flash-cooling by plunging into liquid nitrogen. X-ray data were recorded on beamline I04-1 at the Diamond Light Source (Oxfordshire, UK) using a Pilatus 6M-F hybrid photon counting detector (Dectris), with crystals maintained at 100 K by a Cryojet cryocooler (Oxford Instruments). Diffraction data were integrated and scaled using XDS35 via the XIA2 expert system36 then merged using AIMLESS37. Data collection statistics are summarized in Table 1. The majority of the downstream analysis was performed through the CCP4i2 graphical user interface38.
The ParBΔCTD-parS complex crystallized in space group P21 with cell parameters of a = 54.3, b = 172.9, c = 72.9 Å and β = 90.5° (Table 1). Analysis of the likely composition of the asymmetric unit (ASU) suggested that it contains four copies of the ParBΔCTD monomer and two copies of the 22-bp parS DNA duplex, giving an estimated solvent content of ∼47%.
Interrogation of the Protein Data Bank with the sequence of the C. crescentus ParBΔCTD revealed two suitable template structures for molecular replacement: apo-ParBΔCTD from Thermus thermophilus39 (PDB accession code: 1VZ0; 46% identity over 82% of the sequence) and Helicobacter pylori ParBΔCTD bound to parS DNA29 (PDB accession code: 4UMK; 42% identity over 75% of the sequence). First, single subunits taken from these two entries were trimmed using SCULPTOR40 to retain the parts of the structure that aligned with the C. crescentus ParBΔCTD sequence, and then all side chains were truncated to Cβ atoms using CHAINSAW41. Comparison of these templates revealed a completely different relationship between the N-terminal domain and the DNA-binding domain. Thus, we prepared search templates based on the individual domains rather than the subunits. The pairs of templates for each domain were then aligned and used as ensemble search models in PHASER42. For the DNA component, an ideal B-form DNA duplex was generated in COOT43 from a 22-bp palindromic sequence of parS. A variety of protocols were attempted in PHASER42, the best result was obtained by searching for the two DNA duplexes first, followed by four copies of the DNA-binding domain, giving a TFZ score of 10.5 at 4.5 Å resolution. We found that the placement of the DNA-binding domains with respect to the DNA duplexes was analogous to that seen in the H. pylori ParBΔCTD-parS complex. After several iterations of rebuilding in COOT and refining the model in REFMAC544, it was possible to manually dock one copy of the N-terminal domain template (from 1VZ0) into weak and fragmented electron density such that it could be joined to one of the DNA-binding domains. A superposition of this more complete subunit onto the other three copies revealed that in only one of these did the N-terminal domain agree with the electron density. Inspection of the remaining unfilled electron density showed evidence for the last two missing N-terminal domains, which were also added by manual docking of the domain template (from 1VZ0). For the final stages, TLS refinement was used with a single TLS domain defined for each protein chain and for each DNA strand. The statistics of the final refined model, including validation output from MolProbity45, are summarized in Table 1.
Crystallization screens for the C. crescentus ParBΔCTD-CTPγS complex crystal were also set up in sitting-drop vapour diffusion format in MRC2 96-well crystallization plates with drops comprised of 0.3 µL precipitant solution and 0.3 µL of protein solution (approx. 10 mg/mL) supplemented with 1 mM CTPγS (Jena Biosciences) and 1 mM MgCl2, and incubated at 293 K. The ParBΔCTD-CTPγS crystals grew in a solution containing 15% (w/v) PEG 3350, 0.26 M calcium acetate, 10% (v/v) glycerol, 1 mM CTPγS, and 1 mM MgCl2. Suitable crystals were cryoprotected with 20% (v/v) glycerol and mounted in Litholoops (Molecular Dimensions) before flash-cooling by plunging into liquid nitrogen. X-ray data were recorded on beamline I03 at the Diamond Light Source (Oxfordshire, UK) using an Eiger2 XE 16M hybrid photon counting detector (Dectris), with crystals maintained at 100 K by a Cryojet cryocooler (Oxford Instruments). Diffraction data were integrated and scaled using DIALS46 via the XIA2 expert system36 then merged using AIMLESS37. Data collection statistics are summarized in Table 1. The majority of the downstream analysis was performed through the CCP4i2 graphical user interface38.
The ParBΔCTD-CTPγS complex crystallized in space group P21 with cell parameters of a = 69.5, b = 56.1, c = 71.4 Å and β = 98.4° (Table 1). Analysis of the likely composition of the asymmetric unit (ASU) suggested that it contains two copies of the ParBΔCTD monomer giving an estimated solvent content of ∼50%. Molecular replacement templates were generated from the ParBΔCTD-parS complex solved above. Attempts to solve the structure in PHASER using individual subunits taken from the latter in both conformations did not yield any convincing solutions, suggesting that the subunits had adopted new conformations. Given that the two subunit conformations observed in the previous structure differed largely in the relative dispositions of DBD and NTDs, we reasoned that a better outcome might be achieved by searching for the DNA-binding domains and N-terminal domains separately. This time PHASER successfully placed two copies of each domain in the ASU such that they could be reconnected to give two subunits in a new conformation. The result was subjected to 100 cycles of jelly-body refinement in REFMAC5 before rebuilding with BUCCANEER47 to give a model in which 77% of the expected residues had been fitted into two chains and sequenced. The model was completed after further iterations of model editing in COOT and refinement with REFMAC5. In this case, TLS refinement was not used as this gave poorer validation results. The statistics of the final refined model, including validation output from MolProbity45, are summarized in Table 1.
Measurement of protein-DNA interaction by bio-layer interferometry (BLI) assay
Bio-layer interferometry experiments were conducted using a BLItz system equipped with High Precision Streptavidin 2.0 (SAX2) Biosensors (Molecular Devices). BLItz monitors wavelength shifts (nm) resulting from changes in the optical thickness of the sensor surface during association or dissociation of the analyte. All BLI experiments were performed at 22°C. The streptavidin biosensor was hydrated in a low-salt binding buffer [100 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM MgCl2, and 0.005% (v/v) Tween 20] for at least 10 minutes before each experiment. Biotinylated double-stranded DNA (dsDNA) was immobilized onto the surface of the SA biosensor through a cycle of Baseline (30 sec), Association (120 sec), and Dissociation (120 sec). Briefly, the tip of the biosensor was dipped into a binding buffer for 30 sec to establish the baseline, then to 1 μM biotinylated dsDNA for 120 sec, and finally to a low salt binding buffer for 120 sec to allow for dissociation.
After the immobilization of DNA on the sensor, association reactions were monitored at 1 μM dimer concentration of ParB with an increasing concentration of CTP (0, 1, 5, 10, 50, 100, 500, 1000 µM) for 120 sec. At the end of each binding step, the sensor was transferred into a protein-free binding buffer to follow the dissociation kinetics for 120 sec. The sensor can be recycled by dipping in a high-salt buffer [100 mM Tris-HCl pH 8.0, 1000 mM NaCl, 10 mM EDTA, and 0.005% (v/v) Tween 20] for 5 minutes to remove bound ParB.
For the dissociation step in the BLI experiments in Figure 7A, the probe was returned to either a low-salt binding buffer [100 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM MgCl2, and 0.005% (v/v) Tween 20] for 30 sec or a high-salt buffer [100 mM Tris-HCl pH 8.0, 1 M NaCl, 1 mM MgCl2, and 0.005% (v/v) Tween 20] for 30 sec.
For experiments in Figure 7C, DNA-coated tips were dipped into 300 µL of restriction solution [266 µL of water, 30 µL of 10x buffer 3.1 (NEB), and 3 µL of BamHI restriction enzyme (20,000 units/mL)] for 2 hours at 37°C. As a result, closed DNA on the BLI surface was cleaved to generate a free DNA end.
All sensorgrams recorded during BLI experiments were analyzed using the BLItz analysis software (BLItz Pro version 1.2, Molecular Devices) and replotted in R for presentation. Each experiment was triplicated, standard deviations were calculated in Excel, and a representative sensorgram was presented in Figure 6-figure supplement 2B and Figure 7.
Differential radical capillary action of ligand assay (DRaCALA) or membrane-spotting assay
Purified C. crescentus ParB-His6 (WT and mutants, at final concentrations of 0.7, 1.5, 3.1,, and 12.5 µM) were incubated with 5 nM radiolabeled P32-α-CTP (Perkin Elmer), 30 µM of unlabeled CTP (ThermoFisher), and 1.5 μM of 22-bp parS DNA duplex in the binding buffer [100 mM Tris pH 8.0, 100 mM NaCl, and 10 mM CaCl2] for 5 minutes at room temperature. Four μL of samples were spotted slowly onto a nitrocellulose membrane and air-dried. The nitrocellulose membrane was wrapped in cling film before being exposed to a phosphor screen (GE Healthcare) for two minutes. Each DRaCALA assay was triplicated, and a representative autoradiograph was shown. Data were quantified using Multi-Gauge software 3.0 (Fujifilm), the bound fraction were quantified as described previously48. Error bars represent standard deviations from triplicated experiments.
Measurement of CTPase activity by EnzCheck phosphate release assay
CTP hydrolysis was monitored using an EnzCheck Phosphate Assay Kit (ThermoFisher). Samples (100 µL) containing a reaction buffer supplemented with an increasing concentration of CTP (0, 1, 5, 10, 50, 100, 500, and 1000 µM), 0.5 µM of 22-bp parS DNA, and 1 µM ParB (WT or mutants) were assayed in a Biotek EON plate reader at 25°C for 8 hours with readings every minute. The reaction buffer (1 mL) typically contained: 740 μL Ultrapure water, 50 μL 20x reaction buffer [100 mM Tris pH 8.0, 2 M NaCl, and 20 mM MgCl2], 200 μL MESG substrate solution, and 10 μL purine nucleoside phosphorylase enzyme (1 unit). Reactions with buffer only or buffer + CTP + 22-bp parS DNA only were also included as controls. The plates were shaken at 280 rpm continuously for 8 hours at 25°C. The inorganic phosphate standard curve was also constructed according to the manual. The results were analyzed using Excel and the CTPase rates were calculated using a linear regression fitting in Excel. Error bars represent standard deviations from triplicated experiments.
In vitro crosslinking assay using a sulfhydryl-to-sulfhydryl crosslinker bismaleimidoethane (BMOE)
A 50 µL mixture of 8 µM ParB mutants (with residues at specific positions in the NTD, DBD, or CTD substituted to cysteine) ± CTP (0 to 1000 µM) ± 0.5 µM DNA (a 22-bp linear DNA or a 3-kb circular parS/scrambled parS plasmid) was assembled in a reaction buffer [10 mM Tris-HCl pH 7.4, 100 mM NaCl, and 1 mM MgCl2] and incubated for 5 minutes at room temperature. BMOE (1 mM final concentration from a 20 mM stock solution) was then added, and the reaction was quickly mixed by three pulses of vortexing. SDS-PAGE sample buffer containing 23 mM β-mercaptoethanol was then added immediately to quench the crosslinking reaction. Samples were heated to 50°C for 5 minutes before being loaded on 12% Novex WedgeWell Tris-Glycine gels (ThermoFisher). Protein bands were stained with an InstantBlue Coomassie solution (Abcam) and band intensity was quantified using Image Studio Lite version 5.2 (LI-COR Biosciences). The crosslinked fractions were averaged, and their standard deviations from triplicated experiments were calculated in Excel.
For the experiment described in Lane 8 of Figure 5C-D and Figure 5-figure supplement 1, crosslinking reactions were performed as described above, however the reaction were quenched using a quenching buffer [10 mM Tris-HCl pH 7.4, 100 mM NaCl, 1 mM MgCl2, and 2.3 mM β-mercaptoethanol] instead. Subsequently, 1 µL of a non-specific DNA nuclease (Benzonase, 250 units/ µL, Merck) was added, the mixture was incubated at room temperature for a further 10 minutes before SDS-PAGE sample buffer was added. Samples were heated to 50°C for 5 minutes before being loaded on 4-12% Novex WedgeWell Tris-Glycine gels (ThermoFisher).
For the experiments described in Lane 8 of Figure 5_S1-figure supplement 2A, crosslinking and quenching reactions were performed as described above before 1 µL of TEV protease (10 units/µL, ThermoFisher) was added. The mixture was incubated at room temperature for a further 30 minutes before SDS-PAGE sample buffer was added. Samples were heated to 50°C for 5 minutes before being loaded on 4-12% Novex WedgeWell Tris-Glycine gels.
For experiments described in Lane 9 of Figure 5-figure supplement 2B, proteins were released from gel slices by a “crush & soak” method. Briefly, ten gel slices were cut out from unstained SDS-PAGE gels and transferred to a 2-mL Eppendorf tube. Gel slices were frozen in liquid nitrogen and were crushed using a plastic pestle. The resulting paste was soaked in 500 µL of soaking buffer [10 mM Tris-HCl pH 8, 100 mM NaCl, 1 mM MgCl2, and1 µL of Benzonase (250 units/µL)], and the tube was incubated with rotation in a rotating wheel overnight. On the next day, the tube was centrifuged at 13,000 rpm for 5 minutes and the supernatant was transferred to a new 1.5 mL Eppendorf tube. The sample volume was reduced to approx. 50 µL using a SpeedVac vacuum concentrator before SDS-PAGE sample buffer was added in. The entire sample was loaded onto a single well of a 4-12% WedgeWell Tris-Glycine gel.
Gels were submerged in an InstantBlue Coomassie solution (Abcam) to stain for protein, or in a SYBR Green solution (ThermoFisher) to stain for DNA. Denatured samples were also loaded on 1% TAE agarose gels and electrophoresed at 120V for 40 minutes at room temperature. Afterwards, agarose gels were submerged in a SYBR green solution to stain for DNA.
Chromatin immunoprecipitation with deep sequencing (ChIP-seq)
ChIP-seq experiments and subsequent data analysis were performed exactly as reported previously11. Each ChIP-seq experiment was duplicated using biological replicates. For the list of ChIP-seq experiments and their replicates in this study, see Supplementary File S2.
Immunoblot analysis
For Western blot analysis, C. crescentus cells were pelleted and resuspended directly in 1xSDS sample buffer, then heated to 95°C for 5 min before loading. Total protein was run on 12% Novex WedgeWell gels (ThermoFisher) at 150 V for separation. Resolved proteins were transferred to PVDF membranes using the Trans-Blot Turbo Transfer System (BioRad) and probed with 1:10,000 dilution of α-FLAG HRP-conjugated antibodies (Sigma-Aldrich) antibody. Blots were imaged using an Amersham Imager 600 (GE Healthcare).
ACKNOWLEDGEMENTS
This study was funded by the Royal Society University Research Fellowship (UF140053), BBSRC grant (BB/P018165/1 and BBS/E/J/000PR9791), and Royal Society Research Grant (RG150448) (to T.B.K.L) and a DST-SERB CRG grant 2019/003321 (to A.B). We thank Diamond Light Source for access to beamlines I04-1 and I03 under proposals MX13467 and MX18565 with support from the European Community’s Seventh Framework Program (FP7/2007–2013) under Grant Agreement 283570 (BioStruct-X).
Footnotes
This version includes extensive work on C. crescentus ParB-CTP interactions. Only the structure of C. crescentus ParB-DNA from the previous version was included here.