Insights into the molecular mechanism of ParABS system in chromosome partition by HpParA and HpParB

Abstract The ParABS system, composed of ParA (an ATPase), ParB (a DNA binding protein), and parS (a centromere-like DNA), regulates bacterial chromosome partition. The ParB-parS partition complex interacts with the nucleoid-bound ParA to form the nucleoid-adaptor complex (NAC). In Helicobacter pylori, ParA and ParB homologs are encoded as HpSoj and HpSpo0J (HpParA and HpParB), respectively. We determined the crystal structures of the ATP hydrolysis deficient mutant, HpParAD41A, and the HpParAD41A-DNA complex. We assayed the CTPase activity of HpParB and identified two potential DNA binding modes of HpParB regulated by CTP, one is the specific DNA binding by the DNA binding domain and the other is the non-specific DNA binding through the C-terminal domain under the regulation of CTP. We observed an interaction between HpParAD41A and the N-terminus fragment of HpParB (residue 1–10, HpParBN10) and determined the crystal structure of the ternary complex, HpParAD41A-DNA-HpParBN10 complex which mimics the NAC formation. HpParBN10 binds near the HpParAD41A dimer interface and is clamped by flexible loops, L23 and L34, through a specific cation-π interaction between Arg9 of HpParBN10 and Phe52 of HpParAD41A. We propose a molecular mechanism model of the ParABS system providing insight into chromosome partition in bacteria.


Introduction
Accurately delivering the replicated chromosomal and plasmid DNA to each daughter cell, referred to as DNA segregation or partition, is crucial for the stable inheritance of genetic material ( 1 ).The ParAB S system (partitioning ( par )) is a highly-conserved machinery responsible for DNA segregation in bacteria ( 2 ,3 ).This system comprises three key components: ParA (partitioning protein A), an ATPase motor protein; ParB (partitioning protein B), a centromerebinding protein, and parS , a centromere-like DNA site ( 4 ).The ParA can be classified into two types based on their structure and size ( 5 ): One features both a non-specific DNA (nsDNA)-binding domain and a specific DNA-binding do-main, such as plasmid-encoded Esc heric hia coli P1 ParA ( 6 ); while the other one possesses a nsDNA-binding domain, such as plasmid-encoded Streptococcus pyogenes pSM19035 Delta ( δ, Sp ParA), Salmonella newport TP228 ParF (TP228 ParA), and chromosomal-encoded Helicobacter pylori ParA ( Hp ParA) (7)(8)(9).ParB comprises multiple domains, including a ParA-interacting peptide, an N -terminal CTPase domain involved in protein-protein interactions (NTD), a middle parSbinding domain (DBD), a C -terminal dimerization domain (CTD), and a flexible linker connecting the DBD and CTD domains ( 10 ).ParB is a dual-feature DNA-binding protein that can specifically bind to the parS site and non-specifically spread on the neighboring DNA (11)(12)(13).In TP228 parti-tion system, the ParG (TP228 ParB), instead of conventional ParB, consists of a flexible N -terminus responsible for TP228 ParA interaction and a C -terminal ribbon-helix-helix domain involving in sequence-specific DNA binding (14)(15)(16).
In bacterial chromosomes, the homolog proteins of ParA and ParB are Soj (sporulation protein J) and Spo0J (stage 0 sporulation protein J), respectively.ParA forms as a dimer in the ATP-bound state that can bind to nsDNA ( 17 ,18 ) through a continuous basic binding patch formed by arginine / lysine residues ( 19 ,20 ).ParB specifically binds with parS forming the partition complex to mediate the paired and higher-order complex formation ( 21 ,22 ).The partition complex interacts with the nucleoid-bound ParA forming the nucleoprotein ParA-ParB-DNA complex, known as the nucleoid-adaptor complex (NAC) (23)(24)(25)(26).Archaea, the third domain of life, have been characterized to be the ancestors of eukaryotes.The chromosomal segregation system, SegAB system, regulates the genome segregation of Sulfolobus solfataricus ( 27 ).The ParA-homolog SegA forms a novel non-sandwich dimer and exhibits two DNA binding sites.SegB specifically binds with S1 DNA and forms a higher-order partition complex.The N -terminal domain of SegB significantly stimulates SegA ATPase activity and architecturally regulates the segrosome (SegA-SegB-DNA) formation ( 28 ).
In bacterial ParAB S system, ParA is a weak ATPase and is activated by ParB and DNA in the process of DNA partitioning ( 7 , 17 , 18 , 29 ), such as observed in P1 ParA.P1 ParA undergoes a slow conformational change to [ParA-ATP]* upon ATP-binding, which enables its binding to non-specific DNA ( 30 ).The gradual formation of [ParA-ATP]* establishes a gradient of ParA on the nucleoid through interactions with ParB, thereby stimulating the ATPase activity of ParA.The complex comprising P1 ParA, ParB, and DNA (known as NAC) has been isolated, and the assembly and disassembly of NAC depend on the presence of ATP ( 25 ).The nsDNA binding ability of ParA, which relies on the formation of [ParA-ATP]*, is crucial for DNA partition ( 31 ,32 ).A key residue, aspartic acid (D41 in Hp ParA), coordinates the water nucleophile (W Nu ) and initiates ATP hydrolysis, playing a significant role in ParA A TP-hydrolysis. Tt SojD44A, an A TP-hydrolysis deficient mutant, binds to DNA efficiently, even more so than Tt Soj, which dissociates from DNA time-dependently due to ATP hydrolysis ( 18 ).Further investigation is required to understand the regulatory role of ATP hydrolysis in ATP cycling and NAC formation for ParA-mediated DNA partition.ParA binds ATP to form a ParA-ATP dimer, which undergoes conformational changes to become [ParA-ATP]* dimer, enabling its binding to nsDNA ( 30 ).The [ParA-ATP]* complexes localize on the nucleoid, where it interacts with ParB-parS complex, forming the NAC ( 25 ).The ATP hydrolysis activity of [ParA-ATP]* is stimulated by the ParB-parS complex, converting [ParA-ATP]* to ParA-ADP.Upon ATP hydrolysis, ParA dissociates from the ParB-parS complex and releases from the nucleoid.ParA continues to cycle ATP hydrolysis, assisting in the faithful segregation of replicated DNA to the cell poles ( 6 ).ParB has been characterized as exhibiting CTPase activity which is parS -dependent and this function is required for regulation partition complex formation ( 2 ,33-37 ).Two conserved motifs, GxxRxxA and EN(I / L)QRE(D / N / E)L motifs, located in the NTD of ParB are responsible for CTP hydrolysis ( 34 ,35 ).Upon CTP-binding, the NTDs of the two ParB monomers undergo domain-swapped dimerization resulting in a closed conformation known as a DNA-clamp ( 2 ,33-37 ).
The closed DNA-clamp state enables ParB dimer sliding alone the DNA and condensing the DNA efficiently ( 38 ,39 ).
The Hp ParA, Hp ParB and parS are three components of the ParAB S system of Helicobacter pylori ( 7 ).The crystal structure of the Ct-Hp ParB ( C -terminal truncated) and parS complex reveals an elongated structure, with a flexible N -terminal domain for protein-protein interaction and a conserved DNA-binding domain for parS binding ( 11 ).The crystal structures of the Hp ParA in complex with ATP and DNA were previously determined, revealing its non-specific DNA binding through a lysine-rich basic binding patch and a single DNA-binding site ( 19 ).Electron microscopy studies demonstrated the potential NAC complex formation involving Hp ParA, Hp ParB and DNA in H. pylori chromosome partitioning system ( 19 ).Although the model organisms of chromosome segregation typically are B. subtilis and C. crescentus , the ATP-hydrolysis deficient mutant in the two species cannot be obtained for further investigations of molecular mechanism of chromosome partition ( 17 ,20 ).In this study, we determined the crystal structures of ATP-hydrolysis deficient mutant Hp ParAD41A and its DNA complex, highlighting the crucial role of Asp41 and the essential water nucleophile in the ATP hydrolysis of Hp ParA.Furthermore, we investigated the specific and non-specific DNA binding modes of Hp ParB, which are regulated by CTP.Additionally, we observed interactions among Hp ParA, Hp ParB, and DNA leading to NAC formation.Finally, we determined the ternary complex structure of Hp ParAD41A-DNA-Hp ParBN10, mimicking a potential NAC complex.The Hp ParAD41A-DNA-Hp ParBN10 complex allows us to elucidate the molecular mechanism of ParAB S system in chromosome partition in bacteria.

Cloning, expression and purification of proteins
Cloning, expression and purification of H. pylori Soj / ParA (HP1139) and Spo0J / ParB (HP1138) have been described previously ( 11 ).Recombinant proteins were grown in Luria-Bertani medium and induced overnight at 20 • C by adding 1 mM isopropylβ-d -1-thiogalactopyranoside (IPTG).The Ni-NTA system (Cytiva) was used to purify proteins with elution buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 110 mM imidazole, 5 mM MgCl 2 and 10% glycerol).The eluted proteins were applied to a Superdex™ 200 increase 10 / 300 size exclusion chromatography column (Cytiva) pre-equilibrated with the elution buffer and run at 0.5 ml •min −1 .Molecular weight and purity of proteins were assessed by SDS-PAGE.Hp ParAD41A mutant was generated by site-directed mutagenesis method and verified by DNA sequencing and the protein purification was similar to that of Hp ParA ( 11 ).

Hp ParB N -terminal peptide ( Hp ParBN10) preparation
The N -terminal peptide of Hp ParB (residue 1-10), Hp ParBN10, labeled with the 5-FAM-Ahx fluorophore, was synthesized and purchased from MDBio, inc.The excitation and emission wavelength of 5-FAM are 490 and 520 nm, respectively.The peptide was dissolved at a concentration of 3 mg / mL in a buffer containing 20 mM Tris-HCl (pH 8.0) and 100 mM NaCl, and it was stored at -20 • C until use.

Electrophoretic mobility shift assay
To investigate the DNA-binding mode of Hp ParB and the formation of NAC, electrophoretic mobility shift assay (EMSA) was conducted.For the DNA-binding mode of Hp ParB, reactions were performed in a 20 μl volume using a reaction buffer (20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 11 mM imidazole, 5 mM MgCl 2 , 10% glycerol).Hp ParB or Ct-Hp ParB was incubated with 30 pmol parS or nsDNA at different molar ratios of protein to DNA (5, 10, 20 and 40), with or without 2 mM CTP.The reactions were incubated at 37 • C for 15 minutes and then loaded onto a 7% acrylamide gel in Trisglycine buffer.Electrophoresis was conducted for 1 hour and 35 minutes at 60 V and 4 • C. For NAC formation, reactions were performed in a 20 μl volume in using the reaction buffer as described above.Cy3-parS (5 pmol) and Cy5-nsDNA (3.3 pmol) were used.Hp ParA and Hp ParB were mixed with Cy5-nsDNA and Cy3-parS , respectively, in a protein to DNA molar ratio of 30:1 and 20:1, with or without 2 mM CTP, and incubated at 37 • C for 15 min to form [ Hp ParA-nsDNA] and [ Hp ParB-parS ] complexes, separately.The pre-incubated complexes were then further incubated in a 1:1 molar ratio with or without 2 mM CTP at 37 • C for 30 min.The reactions were loaded onto a 7% acrylamide gel in Tris-glycine buffer and subjected to electrophoresis for 1 h and 35 min at 60 V and 4 • C and visualized by GelRed™ Nucleic Acid Gelstaining (Biotium).

CTPase assay
CTPase activity assays of Hp ParB were performed by the malachite green method with some modification ( 40 ). 4 μM ParB was incubated with or without 4 μM parS DNA in 4 mM CTP.The reaction was in buffer 20 mM Tris-HCl (pH 8.0), 175 mM NaCl, 5 mM MgCl 2 and 10% glycerol with the final volume of 200 μl at 37 • C for 2 h.The reaction was terminated with 200 μl 10% SDS, and followed by 200 μl of 1.25% ammonium molybdate in 6.5% H 2 SO 4 , and 200 μl of 9% ascorbic acid for coloring.The hydrolyzed phosphate product and molybdic acid form a complex that can be reduced and produces a deep blue color by ascorbic acid and is monitored at 660 nm.Three independent repeats for Hp ParB CTPase activity were conducted with error bars representing standard deviations.

Microscale thermophoresis
The protein-protein interaction between Hp ParA or Hp ParAD41A and Hp ParBN10 were measured using the microscale thermophoresis (MST) assay.Serial dilutions of unlabeled Hp ParA or Hp ParAD41A (300 μM) were prepared in a buffer (20 mM Tris-HCl (pH 8.0), 5% Glycerol, 200 mM NaCl, 50 mM Imidazole, 5 mM MgCl 2 ) containing 0.05% Tween20 over 16 tubes, each containing 10 μl protein solution.Aliquots were mixed with 10 μl of 5-FAM-Ahx labeled Hp ParBN10 (200 nM), which was diluted to an optimal fluorescence intensity of approximately 2000 counts).Subsequently, 4 μl of each reaction mixture was loaded into premium capillaries (NanoTemper Technologies).The thermophoresis was measured at 25 • C for 20 s with 40% LED power and 60% microscale thermophoresis power.The data obtained from three independent measurements were combined and analyzed using MO Affinity Analysis software (NanoT emper T echnologies) to fit a binding curve.

Fluorescence polarization binding isotherms
The equilibrium DNA binding assays with Hp ParA, Hp ParAD41A and Hp ParB were done by fluorescence polarization (FP) binding isotherms.The DNA substrates were fluorescently (Cy3 and Cy5) labeled on the 5' end, which allows to measure the increase in FP of the protein-DNA complex relative to the value obtained from the proteinunbound DNA.Twenty micromolar proteins with a 2-fold serial dilution of proteins were made in 20 mM Tris-HCl (pH 8.0), 175 mM NaCl, 5 mM MgCl 2 , 10% glycerol and 1 mM ATP or CTP before being incubated with 5 nM fluorophorelabeled DNA at room temperature.DNA binding by proteins were determined by measuring the changes in fluorescence polarization using a Paradigm plate reader (Molecular Devices).The FP signal was read at 595 nm at an excitation of 535 nm and calculated by determining the concentration of protein required to bind 50% of the fluorophore-labeled DNA.The unbound state is represented by the fluorescence anisotropy of the fluorophore-labeled DNA in the presence of buffer alone.The average of three independent experiments is shown, with error bars representing standard deviations.

Data collection and structure determination
X-ray diffraction data for all the crystals used in this study were collected from beamlines TLS 15A1 and TPS 05A, National Synchrotron Radiation Research Center (NSRRC), Taiwan.All datasets were processed using HKL-2000 software ( 41 ).The structural phase was determined by molecular replacement (MR) with Phaser-MR ( 42 ), using Hp ParA-ATP-DNA structure (PDB ID: 6IUC) ( 19) as a search model.Structural refinements were performed in PHENIX ( 43 ), and structural model adjustment was carried out in COOT ( 44 ).All structural figures shown in this report were generated using PyMOL ( http:// pymol.org/).X-ray diffraction data and structural refinements are summarized in Table 1 .

Hp ParAD41A, an ATP hydrolysis-deficient mutant
We have observed the formation of the nucleoid adaptor complex (NAC) by the Hp ParA-DNA and Hp ParB-parS complexes ( 19 ).Upon ATP hydrolysis, the Hp ParA-ATP dimer dissociates into Hp ParA monomers, resulting in the disruption of the interaction between Hp ParA and Hp ParB within the NAC.In the ParA superfamily, a conserved functional aspartic acid residue plays a critical role in ATP hydrolysis ( Supplementary Figure S1 A); for example, Asp44 of Tt Soj is known to have this function ( 18 ), and its corresponding residue in Hp ParA is Asp41.The side chain of Hp ParA Asp41 coordinates with the nucleophilic water (W Nu ), which is crucial for initiating ATP hydrolysis and has been observed in the Hp ParA-ATP complex (PDB ID: 6IUB, ( 19)).We have utilized the ATP hydrolysis deficient mutant, Hp ParAD41A, to calculate the ATP hydrolysis rate, which was determined to be 0.4 ± 0.2 mol Pi released / mol Hp ParAD41A •hour −1 that was 50% lower than that of Hp ParA ( 19 ).We assessed the DNA-binding ability of Hp ParA and Hp ParAD41A by EMSA using a 24-bp non-specific DNA (nsDNA) ( Supplementary Figure S2 ).We observed that both Hp ParA and Hp ParAD41A can shift the nsDNA in a concentration-dependent manner to a maximal shift at which the nsDNA was saturated with proteins.Since the mobility of the shifted bands between Hp ParA and Hp ParAD41A was different ( Supplementary Figure S2 ), the nature of the Hp ParAD41A-nsDNA complex may differ from that of Hp ParA-nsDNA complex.We suggested that the DNA-bound competent state (ParA * 2 -ATP 2 ) formation of Hp ParAD41A may be different from that of Hp ParA.Additionally, we determined the dissociation constant ( K d ) for DNA binding of Hp ParA and Hp ParAD41A as 117.3 ± 15.2 and 148.8 ± 18.1 nM, respectively, using fluorescence polarization (Figure 1 ).

Regulation of specific and non-specific DNA binding modes of Hp ParB by CTP
ParB has been reported as a CTPase that binds CTP at its flexible N -terminal domain and catalyzes CTP hydrolysis ( 34 ,35 ).Two conserved motifs, GxxRxxA and ENLQRE, have been identified as participating in CTP binding and hydrolysis in Bs ParB ( 35 ).The corresponding CTP binding motifs, 87 GERRLRA 93 and 121 ENIQRE 126 , have also been observed in N -terminal domain of Hp ParB ( Supplementary Figure S1 C).We assayed the CTPase activity of Hp ParB by the malachite green method ( 40 ).Indeed, Hp ParB achieved CTP hydrolysis of 3.9 ± 0.2 μM CTP / μM Hp ParB •hour −1 (Figure 2 A).Moreover, the addition of parS notably increased CTP turnover to 10.2-folds as 41.6 ± 1.0 μM CTP / μM Hp ParB •hour −1 (Figure 2 A).Upon addition of nsDNA, the CTP hydrolysis of Hp ParB was slightly increased to 2-folds as 7.9 ± 0.1 μM CTP / μM Hp ParB •hour −1 .When adding in both the Hp ParA with parS or nsDNA, the CTPase activity of Hp ParB is not affected by Hp ParA significantly, no matter in the presence of parS or nsDNA.The CTPase activity of Hp ParB might be stimulated by parS specifically.
To investigate whether CTP is involved in the regulation of DNA binding by Hp ParB, we performed EMSA (Figure 2 B) and fluorescence polarization (FP) (Figure 2 C-F) using two 24-bp DNAs, parS -containing DNA ( parS ) and nsDNA.We observed two DNA binding modes in Hp ParB: specific and non-specific DNA binding.In the absence of CTP, Hp ParB binds parS , resulting in a band shift and the formation of a specific binding complex through specific DNA binding mode.However, there was only minimal binding observed with ns-DNA (Figure 2 B, lanes 2 and 4).The K d for the specific DNA binding mode with parS in the absence of CTP was calculated as 63.4 ± 5.6 nM by FP (Figure 2 C), while the binding affinity with nsDNA cannot be determined (Figure 2 E).In the presence of CTP, Hp ParB binds with both parS and nsDNA, resulting in a band shift with slowly migrating mobility species, nonspecific DNA complexes, which formation might be through the non-specific DNA binding mode of Hp ParB (Figure 2 B, lane 3 and 5).The K d values for the non-specific DNA binding mode in the presence of CTP were calculated as 231.7 ± 42.0 nM ( parS ) and 144.6 ± 28.8 nM (nsDNA) using FP (Figure 2 D and F), respectively.Based on these findings, we suggest that Hp ParB exhibits two DNA binding modes: specific binding in the absence of CTP and non-specific binding in the presence of CTP.These results demonstrate that CTP may regulate the DNA binding mode of Hp ParB, when CTP-unbound Hp ParB showing a preference for specific DNA binding, the CTP-bound Hp ParB favors non-specific DNA binding.Furthermore, in the absence of CTP, the Ct-Hp ParB binds to parS forming a specific binding complex (Figure 2 G, lane 7-10); however, in the presence of CTP, this complex was only observed with an excess of protein (Figure 2 G, lanes 3-6).In contrast, no non-specific DNA binding complex was observed either in the presence or absence of CTP.When binding to nsDNA, neither specific nor non-specific DNA binding complexes were observed, regardless of the existence of CTP (Figure 2 H, lanes 1-4 and lanes 7-10).The results demonstrated that the DBD of Hp ParB is responsible for specific DNAbinding; while the CTD of Hp ParB is involved in non-specific DNA binding.

Interactions between Hp ParA, Hp ParB, and DNA leading to Nucleoid-adaptor complex formation
We investigated the nucleoid-adaptor complex (NAC) formation by Hp ParA or Hp ParAD41A, Hp ParB and DNA using EMSA ( Supplementary Figure S3 ).The shifted bands with different mobility, NAC1 and NAC2, were observed and the amount elevated when the molar ratio of [ Hp ParA-nsDNA]:[ Hp ParB-parS ] was increasing ( Supplementary Figure S3 A, lanes 3-5).At the same time, the amount of [ Hp ParB-parS ] complex vanished ( Supplementary Figure S3 A, lane 5).The proteins compositions of the NAC1 and NAC2 contained both Hp ParA and Hp ParB proteins that are confirmed by the peptide mass fingerprinting (PMF) ( Supplementary Figure S3 B).The results indicate that the [ Hp ParB-parS ] complex tends to interact with [ Hp ParA-nsDNA] complex and to form NAC. The ATP hydrolysis deficient mutant, Hp ParAD41A, was anticipated to form a stable ATP-bound dimer than the Hp ParA to interact with Hp ParB; however, there was observed one shifted band as NAC3    Given the potential regulation of DNA binding mode by CTP on Hp ParB, we conducted further investigations into the role of CTP in NAC formation using EMSA with Cy3-labeled parS and Cy5-labeled nsDNA (Figure 3 C).We observed distinct DNA binding modes of Hp ParB on parS DNA in the absence and presence of CTP, as indicated by the presence of two shifted bands: a specific DNA complex and a nonspecific DNA complex, with different mobility on the PAGE (Figure 3 C Furthermore, we explored the NAC formation under the influence of CTP.In the absence of CTP, we observed that the [ Hp ParB-parS ] complex interact with the [ Hp ParA-nsDNA] complex, resulting in the formation of a shifted band (NAC1) (Figure 3 C, lane 6).However, in the presence of CTP, instead of NAC1, we observed a shifted band with similar mobility to that of the non-specific DNA complex of Hp ParB (Figure 3 C, lane 5).Additionally, the amount of free DNA was greater than that of the reaction condition in the absence of CTP (Figure 3 C, lane 6).For further investigations, we have performed the EMSA using the ATP-hydrolysis deficient mutant ( Hp ParAD41A) (Figure 3  ).This result from Hp ParAD41A (Figure 3 D) is different from that of Hp ParA (Figure 3 C).For Hp ParA, in the presence of CTP, NAC1 was not observed but the [ Hp ParB-parS ] complex and free DNA were found.In the absence of CTP, we observed NAC1 formation, and the [ Hp ParB-parS] complex was observed but no free DNA was left.Since the ATP hydrolysis activity of Hp ParAD41A is half that of Hp ParA, NAC3 can be observed in the presence of CTP.Because the Hp ParA dimer is required for DNA binding, Hp ParA dissociated from the nsDNA after hydrolyzing ATP; therefore, more free DNA was observed (Figure 3 C, lane 5).Meanwhile, the interaction of Hp ParA and Hp ParB was abolished, and the NAC1 was not observed (Figure 3 C, lane 5).We suggest that the Hp ParB might interact with Hp ParA more efficiently in the presence of CTP, promoting the ATP hydrolysis activity of Hp ParA.Consequently, the ATP-hydrolyzed Hp ParA dimer might dissociate into monomers and be released from the nsDNA, resulting in an increased amount of free DNA.
Overall structures of Hp ParAD41A and the Hp ParAD41A-DNA complex The water nucleophile (W Nu ) binds to Asp41 of Hp ParA and interacts with the γ-phosphate of ATP to initiate ATP catalysis.To investigate the relationship between W Nu and ATP hydrolysis, we solved the crystal structure of Hp ParAD41A in complex with A TP, Hp ParAD41A-A TP (Figure 4 A).The overall structure of the Hp ParAD41A is similar to that of the Hp ParA ( 19 ), with a root mean square deviation (r.m.s.d.) of 0.4 Å (in C α).In the ATP binding pocket, the electron density map of the γ-phosphate of ATP clearly revealed a complete and unhydrolyzed ATP molecule (Figure 4 B).We can clearly observe the W Nu located and coordinated between the γ-phosphate and Asp41 in the wild type ( 19 ), while the W Nu cannot be observed in the Hp ParAD41A-ATP.This suggests that Asp41 likely captures the essential W Nu and plays a crucial role in initiating ATP hydrolysis.As a result, Hp ParAD41A exhibits reduced ATP hydrolysis activity and functions as a deficient mutant in ATP hydrolysis.The detailed interactions of Hp ParAD41A and ATP are listed in Table 2 .
We also determined the overall structure of the Hp ParAD41A in complex with ATP and DNA, named Hp ParAD41A-DNA, that is shown in Figure 4 C.In Hp ParA-DNA structures, four lysine residues, Lys199, Lys227, Lys230 and Lys247, have been reported to be involved in non-specific DNA binding ( 19 ), with Lys199 and Lys230 directly binding to DNA.In the Hp ParAD41A-DNA complex, an additional interaction with DNA involving Lys227 was observed (Figure 4 D).This conserved Lys227 residue is positioned at the core of the DNA binding surface, between the DNA backbone (4.0 Å) and ATP (3.6 Å).It suggests its importance in connecting the two major functions of Hp ParA.Since Hp ParAD41A is deficient in ATP hydrolysis ability, Lys227 may interact with the DNA backbone, resulting in the loss of the connection between ATP and DNA binding.
The Hp ParAD41A-DNA complex displays a higher resolution of 2.6 Å compared to that of the Hp ParA-ATP-DNA complex structure (3.4 Å).ATP hydrolysis promotes the dissociation of the dimer into monomers.The ATP-hydrolysis deficient mutant Hp ParAD41A exhibits low ATP hydrolysis activity and remains in a dimeric state while preserving DNA binding.Therefore, the Hp ParAD41A-DNA complex may adopt a stable conformation with bound DNA than that of Hp ParA.
The Hp P arAD41A-DNA-Hp P arBN10 complex, a potential nucleoid-adaptor complex The interaction between ParB to ParA has been mapped to the extreme N -terminus of ParB (45)(46)(47), as shown in Supplementary Figure S1 B, which presents the N -terminus sequence alignment of the ParB superfamily.To investigate the molecular mechanism of NAC formation, we determined the crystal structure of the Hp ParAD41A-DNA in complex with residues 1-10 ( 1 MAKNKVLGRG 10 ) of the Hp ParB N -terminus ( Hp ParBN10), referred to as the Hp ParAD41A-DNA-Hp ParBN10 complex (Figure 5 A).The overall structure revealed that one Hp ParAD41A dimers binds to one 24-bps DNA, exhibiting the same architecture as the Hp ParAD41A-DNA complex.Additionally, each Hp ParAD41A binds to one Hp ParBN10 peptide (Figure 5 A), as confirmed by the electron density map ( Supplementary Figure S4 ).The Hp ParBN10 is located near the dimer interface but is inclined towards two loops, loop The N -terminal tail of the ParB family, which contains lysine / arginine residues, is essential for stimulating the AT-Pase activity of ParA ( 15 , 18 , 48 ).In Tt Spo0J, the Arg10 mutant fails to stimulate the ATP hydrolysis of Tt Soj ( 18 ).In Bs Spo0J, L ys3 and L ys7 play an important role in regulating the ATPase activity of Bs Soj ( 17 ).The corresponding residues in Hp ParB are Lys5 and Arg9 ( Supplementary Figure S1 B).Arg9 of Hp ParB interacts with the residues Lue50, Gly51 and Phe52 in the L23 region of Hp ParAD41A.Moreover, Arg9 is oriented towards the phenyl group of Hp ParAD41A Phe52, forming a specific cationπ interaction between the amino group and the phenyl group (Figure 5 B).This interaction represents the strongest among noncovalent interactions involving a positively charged cation and negatively charged electron cloud of π systems ( 49 ).To investigate the role of the Hp ParB Arg9 in the interaction between Hp ParA and Hp ParB we measured the binding ability between the mutant peptide, Hp ParBN10R9A, and Hp ParAD41A using MST.The result showed that the Hp ParBN10R9A peptide is unable to interact with Hp ParAD41A as the Hp ParBN10 peptide (Figure 3 B).The cationπ interactions between Arg9 and Phe52 may play a crucial role in the interaction between Hp ParB and Hp ParA.
Furthermore, the main chain of Hp ParBN10 Arg9 interacts with Hp ParAD41A Gln79 at L34 (Table 3 ) , providing an additional interaction that stabilizes the Hp ParBN10.When superimposing the four protomers of the      6 C).Both complexes exhibit an ATP-sandwich dimer conformation and possess a similar continuous basic patch responsible for DNA binding.In Hp ParA, this patch is formed by L ys199, L ys227, L ys230, L ys247, while in TP228 ParA it is formed by Asn148, Arg169, Lys174 and Lys191 ( 19 ).This suggests that Hp ParA and TP228 ParA likely share a similar DNA binding site (Figure 6 C).The Hp ParBN10 binding site (named as H site) is clamped by two loops of Hp ParAD41A, L23 and L34 (U-shape region) (Figure 6 A).In contrast, the TP228 ParB fragment binding site (named as T site) is situated near the groove of the TP228 ParA dimer interface, close to α7 and α8 (Figure 6 C).Both ParB binding sites are located in the vicinity of the dimer interface but exhibit a slight displacement with a distance of 5.3 Å (Figure 6 C).
In the Hp ParAD41A-DNA-Hp ParBN10 complex, Arg9 of Hp ParBN10 plays a direct role in the interaction between Hp ParA and Hp ParB (Figure 5 A and B).The corresponding residue of Arg9 in TP228ParB is Arg19 ( Supplementary Figure S1 B).Arg19 has been shown to stimulate the ATPase activity of TP228 ParA and is suggested to function as an arginine finger ( 15 ).In the TP228 ParA-AMPPMP-ParB complex structure, Arg19 is positioned close to the γ-phosphate of AMPPNP, with a distance of approximately 10 Å and likely stabilizes the transition state of TP228 ParA during ATP hydrolysis ( 50 ).In the Hp ParAD41A-DNA-Hp ParBN10 complex, the distance between Arg9 of Hp ParBN10 and ATP is relatively far, approximately 20 Å.However, it cannot be ruled out that Arg9 serves as the arginine finger to interact with L23 (residues 50-56), which connects to the α2 (residues 45-49) (Figure 5 ( 19 ).We observed that Hp ParAD41A has a tendency to prolong the ATP-bound state when in dimer form.Additionally, we utilized fluorescence polarization to determine the DNA binding affinities of Hp ParA and Hp ParAD41A (Figure 1 ).The Hp ParAD41A mutant exhibits inferior ATP hydrolysis and DNA binding capabilities.The Hp ParA-DNA and Hp ParAD41A-DNA complexes share similar overall structures, featuring an ATP binding pocket and a readily exposed continuous basic DNA binding patch (Figure 4 ).Therefore, these complexes exist in a DNA-binding competent state (ParA* 2 -ATP 2 ) and undergo parallel DNA binding processes.In the Tt Soj and the δ 2 superfamilies, the mutants Tt SojD44A and the δ 2 D60A display decreased ATP hydrolysis in ATPase activity assay ( 8 ) but increased DNA binding in EMSA ( 18 ,51 ) compared to their wild-type counterparts.We propose that the conversion efficiency of ParA* 2 -ATP 2 of Hp ParAD41A may be slower than that of Hp ParA, indicating that the activation energy barrier of Hp ParAD41A is higher than that of Hp ParA.Asp41 may be critical for the precise formation of the ParA* 2 -ATP 2 conformation, necessary to overcome the energy barrier.Once the Hp ParAD41A-DNA complex is formed, it may maintain a more stable conformation than Hp ParA and undergo a steady interaction with the N -terminus of Hp ParB.
ParB can interact with both specific and non-specific DNA and both DNA binding modes are essential for chromosome partition ( 13 , 25 , 52 , 53 ).Our EMSA results (Figure 2 B, G and  H) reveal that Hp ParB exhibits specific DNA binding in the absence of CTP, while favoring non-specific DNA binding in the presence of CTP.The specific DNA-binding occurs via the DBD domain; whereas the non-specific DNA-binding involves in the CTD domain.In Ct-Hp ParB-parS complex, the Hp ParB binds specifically to DNA through residues Arg159, Asn164, Lys190, Arg215 and Glu218 located in the DBD domain ( 11 ).In the CTD domain of Bs ParB, a lysine-rich surface composed of K252, K255, K256 and K259 has been probed as the nonspecific DNA binding surface ( 54 ).The four corresponding basic residues are K263, K267, K270 and R274 in Hp ParB.
The ParB superfamily undergoes conformational changes depending on the binding of CTP and parS , exhibiting open and closed conformations ( 33 ,36 ).In the Ct-Hp ParB-parS complex without CTP, α3 in the NTD and α4 in the DBD fold as a hairpin and the NTDs of ParB dimer do not occur domain-swapping, resulting in an open conformation.Conversely, in the Bs ParB-CDP complex, α3 swings out by 103.1º ( Supplementary Figure S5 B) and the NTDs of ParB dimer undergo domain-swapping, resulting in a closed conformation.Previous reports have indicated that in the native state of ParB, the predominant orientation of the NTD and DBD is tethered together.Additionally, we observed that the addition of parS enhances the CTPase activity of Hp ParB (Figure 2 A), similar to that is observed in Bs ParB.We propose that parS binding of ParB serves two functional roles in the ParAB S system: (i) loosening the NTD to facilitate domain-swapping and CTP binding, thus promoting the formation of the closed conformation and (ii) assisting in exposing the extreme N -terminus of ParB, allowing it to interact with the ParA-DNA complex.
The ParB superfamily comprises three domains: NTD, DBD, and CTD domains, each with unique functions, including ParA interaction / CTP-binding, parS binding, and dimerization / non-specific DNA binding, respectively.The Based on these results, we propose a molecular mechanism model of ParA, ParB and parS in the ParAB S system during chromosome partition (Figure 7 ).In the CTP-unbound state (Figure 7 A), ParB adopts an open conformation and specifically binds with parS .One of the two conserved CTPbinding motifs, GxxRxxA, plays an important role in molecular interactions, enabling ParB to spread, bridge, and condense DNA after binding to parS .In the CTP-bound state (Figure 7 B), ParB assumes a closed conformation and binds non-specifically to DNA and allows ParB to entrap and slide along the distal region from the parS site on the chromosomal DNA.ParB binds to parS not only facilitates CTP binding and promotes the formation of the closed conformation but also assists in exposing the extreme N -terminus of ParB, allowing it to interact with the ParA-ATP-DNA complex.Upon exposure of the N -terminus, both the parS -bound (Figure 7 A) and nsDNA-bound (Figure 7 B) of ParBs can interact with ParA through the novel cationπ interaction between a con- • C and grew to a dimensions of 0.05 × 0.02 × 0.01 mm.Hp ParAD41A-DNA and Hp ParAD41A-parS -Hp ParBN10 crystals were grown using Hp ParA (4 mg / ml) with 10 mM ATP as an additive.The Hp ParAD41A in 20 mM Tris-HCl (pH 8.0), 200 mM NaCl, 44 mM Imidazole, 5 mM MgCl 2 , and 10% Glycerol were mixed with parS at a molar ratio of 5:1 and for the Hp ParAD41A-DNA-Hp ParBN10 crystals, Hp ParBN10 were added in at a molar ratio of 1:8.The mixture was incubated at 25 • C for 10 min.The reservoir solution of Hp ParAD41-DNA and Hp ParAD41A-DNA-Hp ParBN10 contained 25% ethylene glycol and 0.1 M MES (pH 5.6), 25% ethylene glycol, respectively.The crystals were obtained after 1-2 days incubated at 25 • C and grew to a dimensions of 0.3 × 0.2 × 0.2 mm.

a
Values in parentheses are for the highest-resolution shell.b R merge = |I −< I > | / I, where I is the observed intensity and < I > is the average intensity from multiple observations of symmetry-related reflections.c R = |F obs −F calc | / F obs , where F obs and F calc are the observed and calculated structure factor amplitudes, respectively.d R free was calculated with 5% of the total number of reflections randomly omitted from the refinement.e Protein data bank identifiers for co-ordinates.

Figure 1 .
Figure 1.DNA Binding of Hp ParA and Hp ParAD41A.DNA binding by Hp ParA ( A ) and Hp ParAD41A ( B ) to a 24-bp nsDNA was measured by FP binding isotherms and plotted against protein concentration (0-10 μM).All measurements were reported in triplicate and error bars represent the standard deviation of the mean; the solid lines represent fitting curves to the Michaelis-Menten equation.

Figure 2 .
Figure 2. CTP h y droly sis and DNA binding of Hp ParB.( A ) CTP h y droly sis of Hp ParB measured by colorimetric detection of inorganic phosphate using malachite green method.Error bars represent the standard error of the mean ( n = 3).( B ) EMSA for Hp ParB DNA binding.Specific and non-specific DNA binding of Hp ParB against parS and nsDNA w as analyz ed without or with CTP.T he binding affinity of Hp ParB to the parS in the absence ( C ) and presence ( D ) of CTP w as measured b y FP and plotted against protein concentration (0-10 μM).T he binding affinity of Hp ParB to the nsDNA in the absence ( E ) and presence ( F ) of CTP was measured by FP and plotted against protein concentration (0-10 μM).All measurements are reported in triplicate and error bars represent the standard deviation of the mean; the solid lines represent fitting curves to the Michaelis-Menten equation.The parS ( G ) and nsDNA ( H ) binding of Ct-Hp ParB in the absence and presence of CTP.The Ct-Hp ParB and DNA controls are shown in lane 1 and 2 in ( G ) and lanes 5 and 6 in ( H ), respectively.
, lanes 1 and 2).Specifically, Hp ParB exhibited specific DNA binding in the absence of CTP, while non-specific DNA binding occurred in CTP presence.In contrast, the DNA binding ability of Hp ParA remained unaffected by CTP (Figure 3 C, lanes 3 and 4), as evidenced by the presence of shifted bands ( Hp ParA-nsDNA) with similar mobility regardless of the existence of CTP.
D) instead of Hp ParA.For Hp ParAD41A, both with CTP and without CTP (Figure 3 D, lanes 5 and 6), we observed NAC3 (nucleoid-adaptor complex 3) formation, the [ Hp ParB-parS ] and [ Hp ParAD41A-nsDNA] complex.Furthermore, in the presence of CTP, the [ Hp ParB-parS ] complex and free DNA were not observed (Figure 3 D, lane 5); however, in the absence of CTP, the [ Hp ParB-parS ] complex was observed but not free DNA (Figure 3 D, lane 6

Figure 3 .
Figure 3.The interaction among Hp ParA, Hp ParB and DNA.Microscale thermophoresis binding measurements of Hp ParA and Hp ParAD41A with Hp ParB-N variants are shown in ( A ) and ( B ), respectively.( A ) The fluorescence-labeled Hp ParBN10 peptide was mixed with serially diluted Hp ParA.( B ) The fluorescence-labeled Hp ParBN10 ( •) and Hp ParBN10R9A ( ) were mixed with serially diluted Hp ParAD41A.( C ) EMSA for Hp ParB-parS complex and Hp ParA-nsDNA complex binding.The Hp ParB and Hp ParA were incubated with Cy3-labeled parS and Cy5-labeled nsDNA in the presence or absence of CTP, separately.The preincubated Hp ParB-parS complex and Hp ParA-nsDNA complex were further incubated together in the presence or absence of CTP and detected by EMSA.( D ) EMSA for Hp ParB-parS complex and Hp ParAD41A-nsDNA complex binding.The experiments were conducted as that in ( C ) with the replacement of Hp ParA with Hp ParAD41A.
α2 α3 (L23) and β3 β4 (L34).The binding of Hp ParBN10 may not affect the formation of the ATP-sandwich dimer or the Hp ParA-DNA complex.The Hp ParAD41A-DNA-Hp ParBN10 complex suggests that the DNA-bound Hp ParA dimer is capable of interacting with Hp ParB.The Hp ParBN10 is clamped by the two flexible loops, L23 and L34, of Hp ParAD41A (Figure 5 A).Although the two loops (L23 and L34) exhibit low sequence homology among ParA superfamily (residues 51-82, Supplementary Figure S1 A), L23 consists mostly of charged residues ( 51 GFRRDKIDYD 60 ).Meanwhile, the Hp ParBN10 contains three positively charged residues ( 1 MAKNKVLGRG 10 ), leading to electrostatic interactions involved in the Hp ParAD41A and Hp ParBN10 interactions.The Lys3, Val6, Leu7 and Arg9 of Hp ParBN10 are the primarily interaction residues (Figure 5 B), while the remaining parts are exposed to the solvent.Leu7 and Arg9 are conserved in bacterial ParB ( Supplementary Figure S1 B).

Figure 4 .
Figure 4. Crystal str uct ures of Hp ParAD41A and its DNA complex.( A ) Hp ParAD41A monomer.The str uct ure of Hp ParAD41A-ATP monomer comprises ele v en α-helices ( α1-α11) and se v en β-strands ( β1-β7).ATP is sho wn as a stick and the magnesium ion is sho wn as a y ello w sphere.( B ) T he ATP-binding site of the Hp ParAD41A-ATP complex.The F o -F c omit electron density maps of ATP are contoured at 3.0 σ and shown as a mesh.The ATP-interaction residues from two monomers of the dimer are shown as sticks and are colored green and pink, respectively.( C ) The Hp ParAD41A-DNA comple x.T he o v erall str uct ure of the Hp ParAD41A-DNA comple x is sho wn as a ribbon model.T he monomers of the dimer are colored green and pink, respectiv ely.T he DNA molecule bound to the tw o dimers is colored wheat.( D ) T he correlated positions of K227, ATP and DNA of the Hp ParA-DNA and Hp ParAD41A-DNA complex.The DNA binding residues K227, ATP and DNA are illustrated.Residues K227 and K227' from each monomer of the dimer are colored green and cyan for Hp ParA-DNA and Hp ParAD41A-DNA, respectively.

Figure 5 .
Figure 5.The str uct ure of the Hp ParAD41A-DNA-Hp ParBN10 complex.( A ) The overall str uct ure of the Hp P arAD41A-DNA-Hp P arBN10 complex.Each monomer of the Hp ParAD41A dimer is shown as ribbon and colored in pink and green, respectively.The corresponding Hp ParBN10 peptides of each Hp ParAD41A monomer are shown in sticks and colored in pink and green, respectively.The DNA molecule is depicted in wheat.( B ) The binding site of Hp ParBN10 in the Hp ParAD41A-DNA-Hp ParBN10 complex.The Hp ParBN10 is shown as sticks and colored in magenta.The Hp ParAD41A is shown as ribbon and colored in grey, and the residues involved in the interaction of Hp ParBN10 are shown as sticks, labeled and colored in green.The interactions between Hp ParAD41A and Hp ParBN10 are indicated as dashed lines.
asymmetry unit in the Hp ParAD41A-DNA-Hp ParBN10 complex ( Supplementary Figure S5 A), only Hp ParBN10 Arg9 is located precisely in the same position, while the remaining part of Hp ParBN10 exhibits different conformations due to the peptide's flexibility.Hp ParBN10 Arg9 is firmly positioned and might play a crucial role in the interaction between Hp ParA and Hp ParB.Consequently, we propose that the key regions for the interaction between Hp ParA and Hp ParB are L23 and L34 of Hp ParA, and the highly conserved Arg9 of Hp ParB, respectively.Structural comparison of Hp ParAD41A, TP228 ParA and pNOB8 ParA complexes The Hp ParAD41A-DNA-Hp ParBN10 complex represents the first ParA, ParB and DNA ternary complex (NAC) within the ParAB S superfamily.In this study, we determined the binding sites for both Hp ParBN10 and DNA (Figure 5 A).Another complex, the Salmonella Newport TP228 ParA-AMPPMP-ParB complex (PDB: 5U1G, ( 50 )), involves ParA and ParB but lacks DNA.In the Hp ParAD41A and TP228 ParA complexes, the ParB fragments consists of 10 residues ( Hp ParBN10) and 19 residues (TP228 ParBN19) peptides, respectively.The structural superimposition of the monomers from the Hp ParAD41A and TP228 ParA complexes is presented in Figure 6 A, with an r.m.s.d. of 2.3 Å (119 of 170 C α atoms).The Hp ParA and TP228 ParA monomers contain of 264 and 211 amino acids, respectively .Notably , the TP228 ParA contains a deletion in a loop region (residues 49-55), which corresponds to the loop α2 α3 (L23), α3, loop α3 β3, β3 and loop β3 β4 (L34) (residues 51-82) in Hp ParAD41A, referred to as the U-shape region (Figure 6 A).This U-shape region, also observed in other bacterial ParA proteins like Tt Soj (residues 54-80) ( 18 ) and Sp ParA (residues 84-116) ( 8 ), is not involved in ATP hydrolysis or DNA binding.The structural superimposition of Hp ParAD41A-DNA-Hp ParBN10 and TP228 ParA-AMPPMP-ParB complexes shows an r.m.s.d. of 3.6 Å (281 of 367 C α atoms) (Figure

Figure 6 .
Figure 6.Str uct ural comparison of Hp P arAD41A-DNA-Hp P arBN10 complex with TP228 P arA-AMPPMP-P arB and pNOB8 P arA-DNA comple x es.Superimposition of the Hp ParAD41A monomer with TP228 ParA monomer ( A ) and pNOB8 ParA monomer ( B ), respectively.The str uct ural differences are labeled as L23, α3, β3 and L34 in Hp ParAD41A (U-shape region), which are colored in green, cyan, and purple-blue for Hp ParAD41A, the corresponding region of TP228 ParA and pNOB8 ParA, respectively.The ATP are labeled and shown as sticks.The Hp P arAD41A-DNA-Hp P arBN10 complex is str uct urally superimposed with the TP228 P arA-AMPPMP-P arB complex ( C ) and pNOB8 ParA-AMPPNP-DNA complex ( D ), respectively.( C ) The interaction region of Hp ParAD41A dimer and Hp ParBN10 is labeled as H site and colored green.The interaction region of TP228 ParA dimer and ParB fragment is labeled as T site and colored in cyan.The Hp ParBN10 and TP228 ParB fragments are shown as sticks and colored green and cyan, respectiv ely.T he Hp ParAD41A bound DNA is sho wn as a ribbon and colored in green.( D ) T he Hp P arAD41A-DNA-Hp P arBN10 and pNOB8 ParA-AMPPNP-DNA comple x es are colored in green and purple-blue, respectiv ely, as w ell as their bound DNAs are sho wn as ribbons and colored accordingly.The Hp ParBN10 are represented as sticks and colored green.
A).Alternatively, Arg9 of Hp ParBN10 may simply contribute to the binding affinity between Hp ParA and Hp ParB.The archaeal pNOB8 ParA-AMPPNP-DNA complex (PDB: 5U1J, ( 50 )) involves ParA and DNA but lacks ParB.This complex reveals a multifaceted DNA-binding site, and each ParA dimer is surrounded by a dense DNA substrate.The structural superimposition of the Hp ParAD41A and pNOB8 ParA monomer is presented in Figure 6 B, with an r.m.s.d. of 4.7 Å (154 of 208 C α atoms).Additionally, the superimposition of the Hp ParAD41A-DNA-Hp ParBN10 and pNOB8 ParA-DNA structure (Figure 6 D) demonstrates that the Hp ParA dimer binds one DNA molecule through a basic patch at the base of the dimer, while the pNOB8 ParA dimer binds two DNA molecules through a groove of basic patch at the two sides of dimer interface (Figure 6 D).Therefore, the ParB binding site of Hp ParAD41A coincides with the DNA binding site of pNOB8 ParA.This indicates that the ParB binding site of ParA might differ between bacterial and archaeal species.Bacterial Hp ParA and TP228 ParA exhibit the same DNA binding mode and possess a similar ParB interaction region near the dimer interface.However, the archaeal pNOB8 ParA adopts a different DNA binding mode, and the nature of ParB binding of archaea remains unknown, potentially differing from that in bacteria ParA.The bacterial and the archaeal ParA family likely have distinct ParB interaction regions.Base on the Hp ParAD41A-DNA-Hp ParBN10 struc-ture, we can conclude that Hp ParB interacts with DNAbound Hp ParA ([ParA-ATP]*).Hp ParB binding may stimulate the ATP hydrolysis of Hp ParA and trigger its dissociation from the nucleoid, thereby promoting the partitioning of the chromosome / plasmid.Discussion Hp ParAD41A is an ATP hydrolysis deficient mutant with lower ATP hydrolysis (0.4 ± 0.2 mol Pi released / mole Hp ParAD41A •hour −1 ) compared to Hp ParA wild type (1.0 ± 0.3 moles Pi released / mole Hp ParA •hour −1 ) NTD and DBD domains are connected by α3, whose orientation could be regulated by CTP binding, determining the open or closed conformation of ParB.A flexible linker (residues 233-245 in Hp ParB), with varying consensus among the ParB superfamily, aids in building a DNA-storing chamber for nonspecific DNA binding between the DBD and CTD domains.The CTP molecule acts as a molecular switch, controlling the DNA binding mode during chromosome partition.Furthermore, we determined the crystal structure of the Hp ParAD41A-DNA-Hp ParBN10 complex, which mimics the nucleoid-associated complex (NAC), and the Hp ParBN10 peptide is positioned near the dimer interface of Hp ParAD41A without interfering with its ATP and DNA binding site.However, the ATP-bound and DNA-bound ParA dimer is required for ParB interaction.Previously, in the TP228 ParA-AMPPNP-ParB complex (PDB: 5U1G) ( 50 ), ParA is in the AMPPNP-bound state without DNA, interacting with the ParB N -terminus fragment (residues 15-23) and it has suggested that Arg19 of TP228 ParB functions as an arginine finger, stimulating the ATPase activity of ParA ( 15 ).Both ParB binding sites are located near the dimer interface, however, Hp ParA at H site in the U-shape region and TP228 ParA at T site around α7-α8.In the Hp ParAD41A-DNA-Hp ParBN10 complex, the corresponding residue of Arg19 is conserved Arg9 in Hp ParBN10, which interacts with Hp ParAD41A Phe52 through a novel cation-π interaction which may be significant for Hp ParA and Hp ParB interaction.Bacterial Hp ParA and TP228 ParA exhibit the same DNA binding mode and possess a similar ParB interaction region near the dimer interface.However, the archaeal pNOB8 ParA adopts a different DNA binding mode, the ParB binding site of Hp ParAD41A coincides with the DNA binding site of pNOB8 ParA.This indicates that the ParB binding site of ParA might differ between bacterial and archaeal.

Figure 7 .
Figure 7. Molecular mechanism model of ParAB S system in chromosome partition.The ParA and ParB proteins in the ParAB S system exhibit distinct functional states and interactions.ParB is composed of three domains: the N -terminal domain (NTD), the DNA binding domain (DBD), and the C -terminal domain (CTD) and colored green.Specific DNA binding mode in open conformation and non-specific DNA binding mode in closed conformation of ParB regulated by CTP are shown in ( A ) and ( B ), respectively.The ParA-ATP-DNA complex is shown and colored purple-blue.Both the specific DNA complex ParB-parS ( A ) and non-specific DNA complex ParB-DNA ( B ) might be interacted with ParA-ATP-DNA to form the nucleoid-adaptor complex (NAC), NAC-s ( C ) and NAC-ns ( D ), respectively.

Table 2 .
The interaction residues of ATP and Mg ion in Hp ParAD41A str uct ure

Table 3 .
The interactions between Hp ParBN10 and Hp ParAD41A