Author response: The structural basis for dynamic DNA binding and bridging interactions which condense the bacterial centromere (2017)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The ParB protein forms DNA bridging interactions around parS to condense DNA and earmark the bacterial chromosome for segregation. The molecular mechanism underlying the formation of these ParB networks is unclear. We show here that while the central DNA binding domain is essential for anchoring at parS, this interaction is not required for DNA condensation. Structural analysis of the C-terminal domain reveals a dimer with a lysine-rich surface that binds DNA non-specifically and is essential for DNA condensation in vitro. Mutation of either the dimerisation or the DNA binding interface eliminates ParB-GFP foci formation in vivo. Moreover, the free C-terminal domain can rapidly decondense ParB networks independently of its ability to bind DNA. Our work reveals a dual role for the C-terminal domain of ParB as both a DNA binding and bridging interface, and highlights the dynamic nature of ParB networks in Bacillus subtilis. https://doi.org/10.7554/eLife.28086.001 Introduction Bacterial chromosomes are actively segregated and condensed by the ParABS system and condensin (Wang et al., 2014; Song and Loparo, 2015). In B. subtilis, this machinery is physically targeted to the origin proximal region of the chromosome by eight palindromic DNA sequences called parS (consensus sequence 5′-TGTTNCACGTGAAACA-3′) to which the ParB (Spo0J) protein binds (Breier and Grossman, 2007; Lin and Grossman, 1998). These nucleoprotein complexes act as a positional marker of the origin and earmark this region for segregation in a manner somewhat analogous to eukaryotic centromeres and their binding partners. ParB is an unusual DNA binding protein. In addition to sequence-specific interactions with the parS sequence, the protein also spreads extensively around the site for about 18 kbp (Breier and Grossman, 2007; Murray et al., 2006; Lynch and Wang, 1995). The mechanistic basis for this behaviour is not well understood and a matter of active debate. Earlier models envisioned a lateral 1D spreading around parS to form a filament (Murray et al., 2006; Rodionov et al., 1999), principally because spreading can be inhibited in a polar manner by ‘roadblocks’ placed to the side of parS sequences. However, ParB foci appear to contain fewer proteins than are necessary to form a filament, and single molecule analyses using direct imaging (Graham et al., 2014) and magnetic tweezers (Taylor et al., 2015) have shown that binding of DNA by ParB is accompanied by condensation. These ‘networks’ were inferred to be dynamic and poorly-ordered, consisting of several DNA loops between distally bound ParB molecules. In cells, they are presumably anchored at parS sites by sequence-specific interactions but must also contain many interactions with non-specific DNA (nsDNA), as well as self-association interactions that bridge ParB protomers to form DNA loops. Modelling suggests that a combination of 1D spreading and 3D bridging interactions can explain the condensation activity and recapitulate the polar effect of roadblocks on ParB spreading (Broedersz et al., 2014). Recently, single-molecule imaging of the F-plasmid SopB led to a broadly similar model, defining ParB networks as fluid structures that localise around parS using a ‘nucleation and caging’ mechanism (Sanchez et al., 2015). Despite these recent experiments converging on DNA bridging models to explain the ParB spreading phenomenon, the mechanism underpinning this behaviour remains unresolved. In particular, the relationship between these dynamic nucleoprotein complexes and the molecular architecture of the ParB protein is unclear and is the subject of the work presented here. Genomically-encoded ParB proteins comprise three distinct domains (Figure 1A and Figure 1—figure supplement 1A,B and C). Our understanding of their structure is limited to the N-terminal domain (NTD) which binds ParA (Bouet and Funnell, 1999; Davey and Funnell, 1997; Davis et al., 1992; Radnedge et al., 1998) and the central DNA binding domain (CDBD) which binds parS and possibly also nsDNA (Leonard et al., 2004; Schumacher and Funnell, 2005). A structure of Thermus thermophilus ParB lacking the C-terminal domain (CTD) revealed a compact dimer in which the helix-turn-helix (HtH) motifs were symmetrically arranged in a manner suitable for binding to the palindromic parS sequence (Figure 1—figure supplement 1D) (Leonard et al., 2004). Analysis of the CTD by analytical ultracentrifugation suggested that it also formed a dimer, and it was argued that this interface might promote spreading interactions. Recently, a structure of Helicobacter pylori ParB, in which the protein was also truncated by removal of the CTD, showed a strikingly different conformation, where the NTD had moved away from the CDBD domain to form a tetrameric self-association interface (Figure 1—figure supplement 1E) (Chen et al., 2015). The CDBD was bound to a parS half site, and it was argued that tetramerisation of the NTD could be responsible for bridging interactions between specific and nsDNA bound to the CDBD. Figure 1 with 1 supplement see all Download asset Open asset A hypothetical model for ParB-mediated condensation of the origin of replication region. (A) Domains and regions as identified in (Bartosik et al., 2004; Kusiak et al., 2011). (B) ParB is thought to be anchored at parS (grey) via the HtH motif found in the CDBD (red). ParB protomers self-associate via poorly defined interactions and also make non-specific contacts with DNA segments, leading to the formation of ParB networks. In this work we have investigated the potential role of the CTD (green) in mediating ParB oligomerisation and non-specific DNA binding. https://doi.org/10.7554/eLife.28086.002 In previous work, we hypothesised that ParB contains a second DNA binding locus for nsDNA that functions independently of the HtH motif (Figure 1B) (Taylor et al., 2015). This idea was attractive to us for several reasons. Firstly, in a single DNA binding locus model, it is not straightforward to reconcile the strict localisation of ParB networks to just a few parS sites (and their surroundings) with the limited discrimination between specific and nsDNA binding that is observed in vitro (a <10 fold apparent difference in affinity) (Taylor et al., 2015; Broedersz et al., 2014). Secondly, although binding to parS protects the CDBD region from proteolysis, binding to nsDNA affords no such protection, implying that it interacts elsewhere on the protein (Taylor et al., 2015). Thirdly, the distantly-related ParB protein from plasmid P1 provides a precedent for a second DNA binding locus in a Type I centromere binding protein (albeit an additional specific DNA binding site), and highlights the CTD as the putative candidate region (Schumacher et al., 2007). However, the lack of any structural information for the CTD of a genomically-encoded ParB prevents a rigorous comparison of the systems because the primary structure similarity in this region is negligible. In this work, we have probed the role of the CDBD and CTD of B. subtilis ParB using a combination of structural, biochemical, single molecule and in vivo approaches. We find that while the CDBD is responsible for specific recognition of parS, the CTD provides both a second nsDNA binding site and a self-association interface that is important for bridging interactions and DNA condensation. Results The R149 residue within the HtH motif is essential for specific binding to parS, but not required for non-specific binding and condensation Genetic and structural analyses have suggested that residue R149 may be critically important for specific binding to parS at the HtH locus (Graham et al., 2014; Chen et al., 2015; Autret et al., 2001; Gruber and Errington, 2009). To probe the role of the HtH motif using biochemical techniques, we compared binding of parS by wild type ParB and ParBR149G using electrophoretic mobility shift assays containing Mg2+ cations (TBM-EMSA). As reported previously, inclusion of divalent cations in both the gel composition and running buffer enables the clear differentiation of specific and nsDNA-binding activities of ParB (Taylor et al., 2015). As expected, binding of wild type ParB to parS-containing DNA produced a distinct band shift corresponding to the ParB2-parS complex, as well as poorly migrating species at high [ParB] (Figure 2A). These latter complexes are also formed on DNA that does not contain parS, and are therefore indicative of ParB bound to nsDNA flanking the central parS sequence. ParB, and mutants thereof, were purified to homogeneity (Figure 2—figure supplement 1A). EMSA experiments with ParBR149G fail to produce the specific ParB2-parS complex whereas the formation of nsDNA complexes is largely unaffected (Figure 2A). The retention of nsDNA binding activity in ParBR149G is further supported by data using gels lacking Mg2+ ions (TBE-EMSA) (Figure 2—figure supplement 1C), as well as a solution-based protein-induced fluorescence enhancement (PIFE) assay (Figure 2—figure supplement 1B), in which an increase in Cy3 intensity reports ParB binding. For wild type ParB, the data were fitted to the Hill equation yielding an apparent Kd of 361 ± 14 nM and Hill coefficient of 3.2 ± 0.3 in reasonable agreement with published data (Lin and Grossman, 1998; Taylor et al., 2015). ParBR149G produced a similar binding isotherm yielding a moderately weaker Kd of 493 ± 18 nM. This apparent Kd was not significantly altered when the Hill coefficient was not shared between datasets indicating the cooperativity of binding was not impaired in this ParB variant. Figure 2 with 1 supplement see all Download asset Open asset The R149 residue within the HtH motif is essential for specific binding to parS, but not required for non-specific binding and condensation. (A) Representative TBM-EMSAs for wild type ParB and ParBR149G monitoring binding of parS-containing or non-specific 147 bp dsDNA. (B) Schematic of the magnetic tweezer assay used to monitor ParB-dependent DNA condensation. (C) Mean force-extension curves for parS-containing DNA molecules in the presence of wild type ParB and ParBR149G. Non-condensed DNA data is fitted to the worm-like chain model. Solid lines in condensed data are guides for the eye. Errors are the standard error of the mean of measurements on different molecules (N ~15–35 molecules). https://doi.org/10.7554/eLife.28086.004 We next investigated the ability of ParBR149G to condense DNA tethers using magnetic tweezers (Figure 2B). We previously showed that wild type ParB mediates progressive condensation of DNA substrates which is reversible by both force and protein unbinding (Taylor et al., 2015). The condensed state is not highly ordered and its formation is not dependent upon parS sequences, indicating that nsDNA binding is required for condensation. At a concentration sufficient for efficient condensation by wild type ParB (1 µM), ParBR149G did not fully condense DNA, although fluctuations of the DNA tether were consistent with minor condensation events that do not greatly affect the mean extension value measured (data not shown). However, at moderately elevated concentrations (3-fold), reversible condensation did occur and was qualitatively equivalent to wild type behaviour (Figure 2C and Figure 2—figure supplement 1D). Together, these data show that mutation of the HtH motif effectively eliminates the ability of ParB to interact specifically with its cognate parS site, while nsDNA binding and condensation is relatively unaffected. This is consistent either with the R149G mutation exclusively affecting nucleobase-specific contacts in the ParB-parS complex, and/or with the idea that nsDNA binding may occur at a second DNA binding locus. The structure of BsParB CTD reveals a dimer with a putative DNA binding interface We next used solution NMR to determine the structure of the CTD alone (see Figure 3—source data 1 for structure validation and statistics and Figure 3—figure supplement 1A for an assigned 1H-15N HSQC spectrum). The structure forms a well-defined dimer containing two α-helices and two β-strands per monomer in a α1-β1-β2-α2 arrangement (Figure 3A and B). The dimer interface is formed via an intermolecular β-sheet and two domain-swapped C-terminal helices. Although this protein fold is somewhat similar to that seen in the plasmid P1 and SopB ParB proteins (Figure 3—figure supplement 1B,C and D) (Schumacher and Funnell, 2005; Schumacher et al., 2007), there are also significant differences especially in the N-terminal region: the α1 helix in the chromosomal ParB structure is replaced by an additional β-strand in the CTDs of P1 ParB and SopB. Analytical ultracentrifugation, native mass spectrometry and circular dichroism (CD) thermal melt scans further confirmed that the CTD was primarily dimeric in solution and measured a Tm of 68°C (Figure 3—figure supplement 1E and F). NMR H-D exchange data revealed that the dimer exchanges slowly (which will be relevant to the interpretation of later experiments), with those most stable being the intermolecular H-bonds between the two β2 strands (data not shown; the half-lives range from 10 min to 4 hr). This secondary structure element is at the centre of the hydrophobic core which is made up of several Ile, Val and Phe residues in the β-sheet (Figure 3C). The α1 helix forms a leucine zipper with the α2 helix, where alternating Leu residues interdigitate (Figure 3D). A striking feature of the structure is a highly electropositive face of the dimer arising from several conserved Lys residues (Figure 3E and F) analogous to the plasmid-encoded SopB and P1 ParB proteins (Figure 3—figure supplement 1G,H and I). Figure 3 with 1 supplement see all Download asset Open asset Solution NMR structure of the dimeric ParB C-terminal domain. (A) Ensemble overlay of the 14 lowest-energy CTD structures. Red and blue depict separate monomers within the dimer. (B) Secondary structure elements are identified. α1 indicates the N-terminus of each monomer. (C) The hydrophobic core of Ile, Val, Leu and Phe residues. For clarity, portions of both monomers were removed. (D) Interdigitating Leu residues of both monomers form a leucine-zipper interaction. (E) Surface charge representation reveals a large electropositive region across the β-sheet face (orientation as in B, right hand side). Continuum electrostatics calculations used the PDB2PQR web server (Dolinsky et al., 2004) and the APBS plugin for PyMOL (Lerner and Carlson, 2006; Baker et al., 2001). (F) Evolutionary conservation surface profile of the CTD of ParB prepared using ConSurf (Goldenberg et al., 2009; Celniker et al., 2013) (orientation as in E). The chemical shifts, restraints and structural co-ordinates have been deposited with the BMRB (34122) and PDB (5NOC). https://doi.org/10.7554/eLife.28086.006 Figure 3—source data 1 NMR assignment, structure calculation and validation statistics. acommonly assigned groups (i.e. excluding OH, Asp/Glu side-chain carbonyl, Lys amide and Arg guanidinium groups as well as tertiary aromatic carbons); bresidues 229–282; cvalues reported by ARIA 2.3 (Rieping et al., 2007); dordered residues (230-254, 259-278) as calculated by PSVS 1.5 (Bhattacharya et al., 2007); evalues reported by Procheck (Laskowski et al., 1993); fvalue reported by PDB validation software; g residues in secondary structure (231-245, 249-254, 257-264, 267-277). The structural validation programs used were as follows: CING (Doreleijers et al., 2012), Verify3D (Lüthy et al., 1992), Prosa II (Sippl, 1993), Procheck (Laskowski et al., 1993), MolProbity (Lovell et al., 2003). https://doi.org/10.7554/eLife.28086.008 Download elife-28086-fig3-data1-v1.docx The CTD binds DNA non-specifically via a lysine-rich surface To test the idea that the lysine-rich surface we had observed might bind to DNA, we performed TBE-EMSAs with the isolated CTD. These showed that the CTD was indeed able to bind dsDNA (Figure 4Ai) resulting in the formation of a ‘ladder’ of bands of decreasing mobility. This is highly reminiscent of patterns formed by full length ParB under the same conditions (Figure 4Aii and (Taylor et al., 2015)) except for the presence of smaller gaps between the ‘rungs’ as would be expected for a protein of smaller size. The CTD was also shown to bind to hairpin oligonucleotides as short as 10 bp and to ssDNA (Figure 4—figure supplement 1A and data not shown). We do not see substantial differences in the affinity of ParB for DNA substrates with different sequences and so this binding activity appears to be non-specific (data not shown). Native mass spectrometry of complexes formed between the CTD and a 15 bp duplex DNA revealed a stoichiometry of 1 DNA per dimer (Figure 4D). This is in contrast to the P1 ParB system where the CTD operates in a different binding mode, and can bind two 16-mers (Schumacher et al., 2007). Figure 4 with 1 supplement see all Download asset Open asset The CTD binds DNA via a lysine-rich surface. (A–C) TBE-EMSAs for the titration of full length ParB and CTD against 147 bp DNA. Wild type and mutant proteins, K255A + K257A and K252A + K255A + K259A, are indicated. (D) Native mass spectrometry. Titrations of a 15 bp nsDNA hairpin against CTD were performed between ratios of 1.1:1 and 1.5:1 DNA:CTD2. Example spectra are shown with the DNA-CTD2 complex shaded in grey. (E) Deviations in the assigned 1H-15N HSQC spectra of CTD upon titration with a 10 bp hairpin DNA. (F) Chemical shift perturbations exceeding 0.08 ∆δ are highlighted on the structure in yellow. (G) Lys residues thought most likely to bind to DNA are shown as sticks. https://doi.org/10.7554/eLife.28086.009 To further probe the putative DNA binding surface, we performed a titration of the 10 bp hairpin DNA against the isotopically-labelled CTD dimer (Figure 4E, assigned 1H-15N HSQC spectra are shown in Figure 4—figure supplement 1B). Residues with large chemical shift perturbations (CSPs, Δδ > 0.08) are either directly involved in DNA-binding or undergo a conformational change as an indirect result of DNA-binding, and these were mapped onto the structure (Figure 4F). Two regions of interest were identified: D231-V233, and K252-K259, which are found on the intermolecular β-sheet face and proximal loop regions to form a large, concave and positively-charged interaction surface (Figure 4G). To confirm that this surface was responsible for DNA binding we substituted several Lys residues with Ala and monitored the effect on DNA binding using EMSA and PIFE assays. In the first instance, a dual K255A/K257A substitution was studied in the context of both the CTD-only construct (CTDKK) and the full length ParB protein a greatly affinity for DNA, but the binding was not (Figure thermal melt analysis confirmed that this was not to (Figure 4—figure supplement The full length showed a DNA binding isotherm in a PIFE indicating cooperativity as observed for wild type ParB but the apparent Kd was weaker (Figure 4—figure supplement 1D). In EMSA this showed no in specific binding to parS as would be expected (Figure 4—figure supplement these to have a effect on nsDNA binding when using the assays (Figure This may well the that when a nsDNA binding locus is physically to the wild type CDBD domain (which is fully to bind We next a with the of fully the charge across the surface of the CTD, than the EMSA analysis showed that DNA binding was in up to concentrations of (Figure and Figure 4—figure supplement thermal melt analysis showed that was to wild type CTD at but with a Tm indicating a effect of the (Figure 4—figure supplement analysis of full length showed a clear and consistent in all nsDNA binding assays gels showed that ParB-parS complexes were although it be that their was to wild type (Figure 4—figure supplement gels showed a of the mobility bands which from nsDNA binding (Figure Moreover, nsDNA binding was using the PIFE analysis (Figure 4—figure supplement EMSA analysis showed that networks do form as mobility species that at high ParB that the protein a HtH this might well a role for the CDBD in binding to compared to the parS sequence. This highlights the potential of nsDNA binding in ParB which might binding by both the CDBD and CTD DNA binding by the CTD is essential for DNA condensation and bridging in vitro We next and mutant ParB proteins to test the role of the DNA-binding activity with the CTD in condensed ParB networks. These networks have been extensively previously for wild type ParB using magnetic tweezers with single DNA substrates (Taylor et al., 2015) and also in (Graham et al., 2014). full length ParB, the CTD was not of DNA tethers under any up to concentrations and under as as (Figure 1—figure supplement This is consistent with the expected for and/or to promote DNA and condensation. of full length with single DNA tethers in DNA condensation compared to wild type ParB (Figure it was condensation was than as for wild type and full condensation required the force to be to an value (Figure supplement The DNA molecules also showed large when by that was in interactions between isolated DNA regions (data not shown). of with single DNA tethers under standard conditions in no condensation under as as and at elevated concentrations (Figure and Figure supplement The work by ParB compared to the proteins these condensation events was from the difference between the of the force-extension in the presence of the protein and that of DNA This provides a to the condensation between mutants (Figure supplement 1B). We also performed experiments (Figure In this a single DNA molecule is at a 4 force by ParB is full buffer all are monitoring DNA of DNA extension from that expected of DNA is indicative of regions being by could interactions between isolated DNA regions but this was by large in the DNA tether extension increase which is the behaviour of wild type (Figure and Figure supplement was to structures that it bridge DNA in (Figure and Figure supplement 1D). Figure with 1 supplement see all Download asset Open asset DNA binding by the CTD is required for efficient DNA condensation in vitro. (A) Mean force-extension curves of DNA molecules with ParB at the Non-condensed DNA data is fitted to the worm-like chain model. Solid lines in condensed data are guides for the eye. Errors are the standard error of the mean of measurements on different molecules (N molecules). (B) Schematic of A single DNA molecule was at 4 force by This the tether length to the formation of in the DNA. is and all are monitoring DNA for ParB-dependent is by in the extension as a of as the is removed. (C) assay DNA, wild type ParB and The protein supported DNA bridging and large were observed in the (see for (D) assay wild type ParB and activity was for the protein. The CTD can both the formation and networks in vitro The CTD as both an oligomerisation interface and also a site of we hypothesised that the CTD might have a effect on full length ParB by for the DNA and protein that the formation of ParB networks in the magnetic tweezers CTD inhibited the formation of the condensed state with wild type ParB and DNA under the high force (Figure and Figure supplement 1A). We also the of free CTD to tethers was able to networks. ParB networks were stable in a of free ParB on the of these and the DNA tethers were also able to (Figure However, the inclusion of free CTD rapidly ParB with of observed in of all the molecules (Figure Moreover, those molecules which did not decondense could be by but were to when were This ability of the CTD to decondense ParB networks that the and/or that the condensed state under a force are dynamic (i.e. they are while the structure of the is Figure with 2 see all Download asset Open asset The CTD of ParB both and ParB-dependent DNA condensation. (A) Mean force-extension curves for DNA molecules with 1 in the presence or of Non-condensed DNA data is fitted to the worm-like chain model. Solid lines in condensed data are guides for the eye. (B) networks are stable in magnetic tweezers in the presence of 1 complexes decondense the of 1 and free complexes decondense the of 1 and (C) Mean force-extension curves for DNA molecules with 1 in the presence or of mutant Errors are the mean of measurements on different molecules (N > (D) (see Materials and methods for for DNA condensation events the addition of CTD to networks. We have shown that the CTD binds to its ability to condensation and might for the nsDNA that exchange of we have shown previously that free DNA is a of in the (Taylor et al., 2015). To test the idea that the CTD dimerisation interface is also important for the condensed we experiments with the and which are and to bind mutant proteins were as as wild type in condensation (Figure and Figure supplement 1B), and both were able to in of all molecules (Figure and This suggests that ParB is primarily by for the CTD dimerisation interface and further