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Crystal structures of HpSoj–DNA complexes and the nucleoid-adaptor complex formation in chromosome segregation

Abstract ParABS, an important DNA partitioning process in chromosome segregation, includes ParA (an ATPase), ParB (a parS binding protein) and parS (a centromere-like DNA). The homologous proteins of ParA and ParB in Helicobacter pylori are HpSoj and HpSpo0J, respectively. We analyzed the ATPase activity of HpSoj and found that it is enhanced by both DNA and HpSpo0J. Crystal structures of HpSoj and its DNA complexes revealed a typical ATPase fold and that it is dimeric. DNA binding by HpSoj is promoted by ATP. The HpSoj–ATP–DNA complex non-specifically binds DNA through a continuous basic binding patch formed by lysine residues, with a single DNA-binding site. This complex exhibits a DNA-binding adept state with an active ATP-bound conformation, whereas the HpSoj–ADP–DNA complex may represent a transient DNA-bound state. Based on structural comparisons, HpSoj exhibits a similar DNA binding surface to the bacterial ParA superfamily, but the archaeal ParA superfamily exhibits distinct non-specific DNA-binding via two DNA-binding sites. We detected the HpSpo0J–HpSoj–DNA complex by electron microscopy and show that this nucleoid-adaptor complex (NAC) is formed through HpSoj and HpSpo0J interaction and parS DNA binding. NAC formation is promoted by HpSoj participation and specific parS DNA facilitation.


INTRODUCTION
Precisely replicated DNA segregation is necessary for accurate inheritance of genetic material (1). In bacteria, the partitioning (par) system (ParABS) is a highly-conserved process for the delivery and localization of chromosomes or low-copy-number plasmids in cells (2). The ParABS system comprises three elements: the NTPase motor protein (ParA: partitioning protein A); the centromere-binding protein (ParB: partitioning protein B); and a centromere-like DNA site (parS) (3,4). The general partitioning process involves specific binding of a parS site by ParB and assembly into a higher-order partitioning complex (2,4,5). The ParA dimer uses NTP-binding to trigger NTP hydrolysis, which drives segregation of newly-replicated ParB-parScontaining plasmids (6)(7)(8).
Plasmid DNA stability is maintained by the ParABS system (15). In bacterial chromosome-encoded ParABS, Soj (sporulation protein J) and Spo0J (stage 0 sporulation protein J) are the homologs of the ParA and ParB proteins, respectively. In Thermus thermophiles and Bacillus subtilis, the ATPase activity of Soj is elevated by Spo0J upon addition of DNA (7,13,16). Soj exists as a monomer in the absence of ATP, but is dimeric when ATP is present. ATP-bound Soj dimer exhibits nsDNA-binding ability (7,16). Several studies have shown that DNA binding by ParA is essential for DNA segregation (17,18).
In the ParMRC segregation system of R1 plasmid, it was found that parC manages plasmid molecule pairing, which requires binding of ParR to parC, and the ParR-parC pair complex is enhanced by the ParM-ATP complex (19). The DNA partitioning system of pB171 plasmid involves ParA and ParB with two parC sites. It has been suggested that ParB mediates the pairing complex and the higher-order complex comprising several DNA molecules, and the Nterminus of ParB was observed to be essential for ParBmediated pair formation (20). In the E. coli P1 ParABS system, ParB binds to parS DNA to form the pairing complex (the ParB-parS complex) before the nucleoprotein ParA-ParB-DNA complex is assembled (15,(21)(22)(23). ATP regulates these interactions and the parS DNA stabilizes the nucleoprotein complex (22). The dynamic interaction between plasmid-bound ParB and nucleoid-bound ParA of the ParA-ParB-DNA complex is defined as the nucleoidadaptor complex (NAC), which mediates cooperation between the plasmid and the nucleoid (22).
Two distinct models have been proposed for the ParABS system: (i) a filament model whereby the ParB partitioning complex (i.e. cargo, the ParB-parS DNA complex) is translocated by ParA, which is polymerized along the nucleoid to form a nucleoprotein filament (24,25); and (ii) a diffusion-ratchet model whereby the ParB-DNA cargo is associated with ParA via a concentration gradient of ParA-ATP in the nucleoid surface (26)(27)(28)(29). Two diffusion-ratchet mechanisms have been proposed: (a) a DNA-relay mechanism involving elastic dynamics of the chromosome involved in the translocation of the ParB partitioning cargo (30,31); and (b) a hitch-hiking mechanism whereby ParA assembles in high-density DNA regions (HDRs) to guide ParB partitioning cargo delivery (32). The diffusion-ratchet model suggests that ParA-ATP needs to undergo a conformational change into a DNA-binding competent state, ParA* 2 -ATP 2 , for binding to the nucleoid (26), resulting in ParA* 2 -ATP 2 attachment to the nucleoid (or localization at HDRs) to form a nucleoid matrix in which the association between plasmid-bound ParB and nucleoid-bound ParA is facilitated (26,27,29,32).
Two Walker-box DNA segregation systems were identified recently in archaeal plasmids and chromosomes, i.e., SegAB and AspA-ParBA machineries (33-36). The SegAB system was characterized in the thermophilic crenarchaeon S. solfataricus and consists of SegA and SegB proteins and a centromere-like region (34). SegA is an ortholog of the bacterial Walker-type ATPase ParA. Similar to the polymerization behavior of bacterial ParA, SegA assembles into polymers in the ATP-bound state. Archaea SegB is a sitespecific DNA-binding protein that interacts with SegA and affects SegA polymerization (34). The AspA-ParBA machinery is encoded by the plasmid pNOB8 hosted by Sulfolobus NOB8H2 strain (35). It comprises AspA (archaeal segregation protein A, a centromere-binding component), ParA (NTPase), and ParB (an atypical ParB) (35). Archaea AspA is a site-specific DNA-binding protein with no sequence homology to any characterized segregation protein (35). The pNOB8 ParA possesses nsDNA-binding ability (37), and a multifaceted DNA-binding mode has been identified from the structure of pNOB8 ParA-AMPPNP-DNA (37). The pNOB8 ParB exhibits nsDNA-binding activity and functions as an adaptor protein to interact with AspA, ParA and nsDNA (35).
The crystal structure of the HpSpo0J-parS DNA complex of the ParABS system of H. pylori was previously determined (2), revealing that HpSpo0J molecular spreading and DNA bridging is mediated by the HpSpo0J N-terminus. Here, we report the crystal structures of HpSoj and its DNA complexes. HpSoj non-specifically binds to DNA through a basic binding patch comprising Lys199, Lys227, Lys230 and Lys247, and directly binds within the DNA minor groove at Asn196, Lys199 and Val229. Furthermore, we used electron microscopy (EM) to monitor nucleoprotein formation among HpSpo0J, HpSoj and DNA, allowing us to propose a potential NAC complex for the partitioning system in H. pylori.

Protein expression and purification
The PCR product of the Hp1139 (HpSoj) gene (795 bp) from H. pylori 26695 genomic DNA was ligated into the BamHI and SalI restriction sites of expression vector pQE30 (Qiagen) that possesses a hexa-histidine tag at the N-terminus (13). The plasmid was then transformed into the expression host, E. coli strain SG13009. Recombinant HpSoj was grown in Luria-Bertani medium and induced overnight at 20 • C by adding 1 mM isopropyl-␤-D-1-thiogalactopyranoside (IPTG). The Ni-NTA system (GE Healthcare) was used to purify HpSoj with elution buffer (20 mM Tris pH 8.0, 500 mM NaCl, 110 mM imidazole, 5 mM MgCl 2 and 10% glycerol). The eluted proteins were applied to a Superose™ 12 gel filtration column (GE Healthcare) 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. HpSoj mutations (D41A, K199E, K230E, K199/230E and K199/227/230/247E) were generated by site-directed mutagenesis method and verified by DNA sequencing. Protein purification of these HpSoj mutants were similar to that of wild-type HpSoj.
PCR products of HpSpo0J and HpSpo0JN (residues 1-50) from H. pylori 26695 genomic DNA were ligated into the NcoI and XhoI restriction sites of the pET-28a(+) expression vector (Novagene) that contains a C-terminal hexa-histidine tag. The resulting two plasmids, pET-28a(+)-HpSpo0J and pET-28a(+)-HpSpo0JN, were transformed into E. coli strain BL21(DE3). The expression systems were incubated overnight at 37 • C in Luria-Bertani medium. Cultures were induced by adding 1 mM IPTG and incubating at 37 • C for three hours. Ni-NTA columns (GE Healthcare) were used to purify the two proteins. The eluted HpSpo0J and HpSpo0JN proteins were further purified by a Super-ose™ 12 gel filtration column (GE Healthcare).

DNA preparation
Double-stranded 24 and 55 bp parS DNA fragments were prepared using an equal molar ratio of two complementary oligonucleotides (the 16

ATPase activity assay
The ATPase activity of HpSoj and HpSojD41A was determined using the malachite green method (38 . The color was allowed to stabilize for 30 min before absorbances of the antimony-phosphomolybdate complex were detected at 850 nm. Absorbance at 850 nm resulted from non-enzymatic hydrolysis of ATP was subtracted from that of each reaction mixture. We created a standard curve using a serial dilution of potassium phosphate (0-400 M). A 850 values were converted into moles Pi produced per mole of protein using phosphate absorbance as a standard. The control experiments in the presence of DNA, HpSpo0J and HpSpo0JN alone and in combination but without HpSoj were performed. The ATPase activities of these control experiments were near zero or undetectable.

Microscale thermophoresis
HpSpo0JN proteins were labeled with the fluorescent amine-reactive red dye NT-647-NHS (NanoTemper Technologies) and then diluted to optimal fluorescence intensity (around 10,000 counts). Unlabeled HpSoj (214 M) was serially diluted 1:1 in protein buffer (20 mM MOPS pH 7.0, 5 mM MgCl 2 , 110 mM imidazole, 250 mM NaCl, 50 mM arginine, 50 mM glutamic acid, 1 mM ATP) with 0.05% Tween20 over 16 tubes, each containing 10 l protein. Aliquots were mixed with 10 l of labeled HpSpo0JN (33 nM) before being transferred into hydrophilic glass capillaries. Measurements were performed at 25 • C using 20% LED power and 60% MST power. In the negative control experiment, unlabeled lysozyme (250 M) was also serially diluted and mixed with labeled HpSpo0JN. The instrument used was a NanoTemper monolith NT.115Pico and the data were analyzed from triplicate experiments.

Fluorescence polarization binding isotherms
The equilibrium DNA binding assays of HpSoj was done by fluorescence polarization binding isotherms. parS24 DNA substrates were fluorescently labeled on the 5 end of DNA, which are enabled to measure the increase in fluorescence polarization of the HpSoj:DNA complex relative to the unbound DNA due to HpSoj binding. Serial dilutions of HpSoj proteins were made in 20 mM Tris-HCl pH 8.0, 175 mM NaCl, 5 mM MgCl 2 , 10% glycerol and 1 mM ATP before being incubated with 5 nM Cyanine-3 (Cy3)-labeled DNA at room temperature. DNA binding by HpSoj was determined by measuring the changes in fluorescence polarization using a Paradigm plate reader (Molecular Devices).  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.
The structural phases of HpSoj-ATP were determined by molecular replacement (MR) with Phaser (40), using TtSoj (PDB ID: 1WCV) (7) as a search model. Structural phases of HpSoj-ATP-DNA and HpSoj-ADP-DNA were solved by MR using HpSoj as the search model. Structural refinements were performed in Phenix (41), and structural model adjustment was carried out in COOT (42). X-ray diffraction data and structural refinements are summarized in Table 1.

Electron microscopy
For electron microscopy, parS550 (550 bp DNA with one parS site in middle), 180DNA (180 bp DNA) and 600DNA (600 bp DNA) were used. The parS550, 180DNA, and 600DNA fragments were amplified by PCR. The DNAonly control (Supplementary Figure S5) was examined by a Cytochrome C spreading technique (43). For the HpSpo0J:parS550 complex, we used 20 nM HpSpo0J and 2 nM parS550 in binding buffer (20 mM Tris pH 8.0, 150 mM NaCl, 5 mM MgCl 2 , 33 mM imidazole, 10% glyc-erol). For the HpSoj:180DNA complex, we used 80 nM HpSoj and 4 nM 180DNA in the presence of ATP (1 mM). HpSpo0J and parS550 DNA (molar ratio 10:1), HpSpo0J and 600DNA (molar ratio 10:1), or HpSoj and 180DNA (molar ratio 20:1) were incubated for 10 min at 37 • C. To induce NAC formation, the HpSpo0J-parS550 complex and the HpSoj-180DNA complex were mixed together and incubated for 6 h at 37 • C. The resulting protein-DNA mixture was placed on a clean Parafilm surface and was picked up on a carbon-coated grid before being negatively stained with 1% uranyl acetate. After the grid had been air-dried for one day, rotary shadowing was performed with the platinumpalladium source at an angle of 10 degree and a sample-tosource distance of 8 cm. The grid was coated with carbon and then visualized by electron microscopy. Images were captured by a Tecnai G2 Spirit TWIN (FEI Company) electron microscope at a magnification of ×15 000 at 80 kV. EM image quantifications were conducted using ImageJ. Image frames were randomly selected from different grids. The parS550, 180DNA, and 600DNA fragments were dis- tinguishable by length. Numbers of various complexes in images are listed in Table 2 and Supplementary Table S3.

Characterization of HpSoj
HpSoj belongs to the ParA superfamily. We present a multiple sequence alignment in Supplementary Figure S1 of the ParA superfamily from the bacterial par system (7,9,11,16,25) and of archaeal Sulfolobus NOB8H2 pNOB8 ParA (35). HpSoj shares a sequence identity of 22-48% with the ParA superfamily. In addition, proteins of the 'deviant' Walker A motif family with different biological functions (44)(45)(46)(47) are also shown in Supplementary Figure S1. HpSoj shares a sequence identity of 18-28% with the 'deviant' Walker A motif superfamily. We assayed the ATPase activity of HpSoj by the malachite green method (37) ( Figure 1A). HpSoj achieved ATP hydrolysis of 1.0±0.3 moles Pi released/mole HpSoj·hour −1 . The activities show slightly increase of 1.4and 1.3-fold with specific DNA (parS55) and non-specific DNA (55DNA), respectively. However, the ATPase activity of HpSoj with HpSpo0J reveals an increase of 3.8-fold using parS55 but only 1.2-fold with 55DNA. The ATPase activity of HpSoj was stimulated when HpSpo0J binds to parS55 and the HpSpo0J-parS complex is formed. In addition, the ATPase activities of HpSoj were detected by HpSpo0JN (the N-terminal domain only of HpSpo0J) with 3.7-and 3.1-fold stimulation in parS55 and 55DNA, respectively ( Figure 1A). Although HpSpo0JN does not contain the DNA binding domain it still can stimulate the ATPase activity of HpSoj. It suggested that HpSpo0JN enhances ATP hydrolysis of HpSoj. In addition, we conducted the MST experiment to detect the potential interaction between HpSoj and HpSpo0JN and a weak interaction was observed ( Figure 1B).
We assessed the DNA-binding ability of HpSoj by EMSA using parS24 and parS55 fragments and with or without nucleotide ADP or ATP ( Figure 1C and Supplementary Figure S2). We found that the DNA-binding ability of HpSoj is dependent on ATP. The DNA-binding were observed under ATP and ADP, at the concentrations of HpSoj of 0.4 and 1.5 mg/ml, respectively ( Figure 1C and Supplementary Figure S2). Meanwhile, HpSoj completely bound DNA at a molar ratio of 40:1 in the presence of ATP ( Figure 1C).

Overall structure of HpSoj
HpSoj exhibits Rossmann folding, which is a typical of nucleotide-binding protein (Figure 2A), and forms a dimer ( Figure 2B). HpSoj comprises a central seven-stranded ␤sheet (␤1-␤7) framed by ␣1-␣2 and ␣9-␣11 on one side and ␣3-␣8 on the other side ( Figure 2A). The ATP molecule in complex with the hexa-coordinated magnesium ion was wedged into a surface-exposed cavity formed by helices ␣1 and ␣9 and loops ␤1␣1 and ␣8␣9 (Figure 2A). Each HpSoj monomer binds to one ATP that is sandwiched between the interfaces of the dimer ( Figure 2B). The ATP is bound to loop ␤1␣1 (residues 11-19) of the conserved Walker A motif, and is shielded from the other monomer by loop ␤6␣7 (residues 155-158) ( Figure 2B). However, the highly conserved Lys12 residue of the neighboring monomer can interact with the ATP by extended hydrogen bonding (Supplementary Table S1). In addition, Gly13 and Gly14 from the neighboring monomer make van der Waal contacts with the ATP molecule (Supplementary Table S1). HpSoj forms a tight dimer with a buried surface area of 1440Å 2 , as calculated by PISA (48) ( Figure 2B). Dimerization of HpSoj involves seven hydrogen bonds and numerous hydrophobic interactions (Supplementary Table S2). Based on the crystal structures of ParA superfamily, there are also several hydrogen bonds and hydrophobic interactions exited in TtSoj, SpParA and TP228 ParA dimer interface. However, the conservation among these dimerization residues is low.
According to previous reports on Soj (7,10), a key functional aspartic acid residue (Asp41 in HpSoj) cocoordinates the water nucleophile (W Nu ) and initializes ATP hydrolysis, playing an important role in ATPase activity. Asp41 of HpSoj binds the W Nu and interacts with the ␥phosphate of ATP to initiate ATP catalysis ( Figure 2C), as found for Walker-type ATPases. The ATP-binding residues are all conserved and critical in the ParA superfamily (7,16), and corresponding residues in HpSoj are 14 GVGKTT 19 (the 'deviant' Walker A motif), Asp41, and Asp130 (Figure 2C). The ATP hydrolysis of HpSojD41A was reduced to 0.4 ± 0.2 mol Pi released/mole HpSojD41A·hour −1 . The P1 ParAD152N and TtSojD44A (corresponding to HpSojD41A) also have been reported and TtSojD44A mu-  tant was suggested as an ATP-hydrolysis deficient mutant (7,49).

HpSoj-DNA complexes
The overall structure of the HpSoj-ATP-DNA complex is shown in Figure 2D, and the ATP-binding site is detailed in Figure 2E. Both monomers of the HpSoj dimer equally contribute to DNA-binding, reinforcing the essential function of Soj dimerization. Each monomer binds DNA via the ␣8, ␣9 and ␣10 helices, with the DNA located in a furrowshaped basic patch formed by four key lysine residues: Lys199 (in ␣8), Lys227 (in ␣9), Lys230 (in ␣9) and Lys247 (in ␣10) ( Figure 3A). Among these DNA-binding residues, Lys227, Lys230 and Lys247 are conserved as either Lys/Arg in the bacterial ParA superfamily, and only Lys247 is threonine in CcParA (Supplementary Figure S1). Lys199 reveals sequence diversity as K/I/Q/E in the ParA superfamily (Supplementary Figure S1).
Residues Asn196, Lys199, and Val229 directly interact via hydrogen bonding with phosphate groups of the DNA backbone in the minor groove ( Figure 3A). Surprisingly, these DNA-binding residues are not conserved among the ParA superfamily (Supplementary Figure S1). Residues Asn196 and Val229 interact with DNA through the amine nitrogen of the main chain, whereas Lys199 interacts with it via the amine group of the side chain. These interactions between Soj protein and nsDNA in the minor groove are a common feature shared with other nsDNA-binding proteins (50,51). Furthermore, the continuous basic patch created by Lys199, Lys227, Lys230 and Lys247 in the HpSoj dimer is required for nsDNA-binding. Our sequence alignment (Supplementary Figure S1) reveals that these basic residues are partially conserved as either Lys/Arg in the bacterial ParA superfamily, so a continuous basic patch might be necessary for nsDNA binding.
The crystal structure of the HpSoj-ADP-DNA complex was determined at a resolution of 2.5Å. The ADP was located in the nucleotide binding site according to the F o -F c omit map at 3.0 sigma ( Figure 2F). Although ATP had been added in the crystallization setup, the hydrolyzed ADP was observed in the crystal ( Figure 2F). Two HpSoj-ADP dimers were bound to one DNA molecule, similar to the structure of the HpSoj-ATP-DNA complex (Supplementary Figure S3A). The overall structures and ATPbinding sites of the HpSoj-ATP-DNA and HpSoj-ADP-DNA complexes are similar to those of HpSoj ( Figure 2C, E, F and Supplementary Figure S3). Based on structural comparison, the root mean square deviation (r.m.s.d.) between the HpSoj-ATP and HpSoj-ATP-DNA complexes is 0.48Å (in C␣), between HpSoj-ATP and HpSoj-ADP-DNA it is 0.51Å (in C␣), and between HpSoj-ATP-DNA and HpSoj-ADP-DNA it is 0.35Å (in C␣). Previous study has shown that the average r.m.s.d. for proteins recognizing nsDNA is <1.0Å between unbound-and bound-DNA conformations (52). The overall B-factors of HpSoj-ADP-DNA and HpSoj-ATP-DNA are 59 and 85Å 2 , respectively ( Table 1). The observation that both HpSoj-ADP-DNA and HpSoj-ATP-DNA share similar structures may be due to ATP hydrolysis during crystallization, which could pre-vent any subsequent conformational change that might occur in the ADP-bound form.

Structural differences among HpSoj and its DNA complexes
Structural comparison of HpSoj-ATP and its DNA complexes ( Figure 3D) revealed that they share a similar overall structure, with r.m.s.d. values less than 1Å. PyMOL (53) calculations revealed that helix ␣7 rotates 5.1 • and participates in dimer interactions via residues Glu161/Leu165 (Supplementary Table S2), and that loop ␤6␣7 is involved in ATP binding through residues Glu156/Phe157/Phe158 ( Figure 2C and E). Also, we found that helix ␣9 rotates 4.6 • and is involved in DNA binding ( Figure 3B), and that loop ␣8␣9 is involved in ATP binding ( Figure 3B). The dimer interfaces of the HpSoj-ATP, HpSoj-ATP-DNA and HpSoj-ADP-DNA complexes are 1440, 1349, and 1313 A 2 , respectively, based on PISA calculation (48). Given the disparity in interface area between DNA-unbound andbound HpSoj dimer, the monomer-monomer relationship may have to be adjusted in order to interact properly with DNA. Our PyMOL calculations (53) revealed a difference of 3.6 • in the angle between ␣4 of the dimer for HpSoj-ATP and HpSoj-ATP-DNA. Several hydrogen bonds present in the HpSoj-ATP dimer interaction were absent in the HpSoj-ATP-DNA complex, all of which were located in ␣2 and ␣7 including the hydrogen bonds of Gln43-Gln11 and Ser48-Glu161 (Supplementary Table S2).

Non-specific DNA binding
Protein can non-specifically bind with DNA using positively-charged and surface-exposed residues to interact with the negatively-charged phosphate of the DNA backbone (17). HpSoj dimer binds DNA through a continuous basic binding patch formed by Lys199, Lys227, Lys230 and Lys247. Based on the sequence alignment and the structural comparisons, the four corresponding basic residues in TtSoj are Arg182/Arg194/Arg208/Arg211, in BsSoj they are Arg189/Lys201/Arg215/Arg218, and in SpParA they are Lys230/Arg253/Lys256/Lys273 (Supplementary Figure S1). Mutagenesis of BsSoj revealed that Arg189 and Arg218 are involved in DNA-binding and mediate the interaction between BsSoj and the nucleoid (17). In SpParA, Lys256A and Lys273/274A mutation abrogated DNA-binding ability (10,54). Thus, the continuous basic patch may be important for DNA-binding among the bacterial ParA superfamily.
Based on our HpSoj-ATP-DNA complex structure, HpSoj dimer binds nsDNA through a continuous basic binding patch formed by residues Lys199, Lys227, Lys230, Lys247, as well as Asn196, and Val229 ( Figure 3A). Lys199/Asn196 and Lys230/Val229 interact with DNA by clamping the minor groove. Asn196 and Lys199 from ␣8 exhibit low sequence homology among bacterial ParA (Supplementary Figure S1). Asn196 interacts with DNA through the amine nitrogen of the main chain, whereas Lys199 interacts with DNA via the amine group of the side chain. Val229 and Lys230 of ␣9 are conserved among bacterial ParA ( 229 V-K/R-L/V-A/S-E 233 ) ( Supplementary Figure S1). Val229 interacts with DNA through the amine ni- trogen of the main chain, but Lys230 does not directly interact with DNA. Lys199 and Lys230 promote DNA binding by inducing their neighboring residues, Asn196 and Val229 respectively, to form hydrogen bonds via their main chains with DNA in the center of the DNA minor groove ( Figure  3A). Loop ␣8␣9 exhibits sequence consensus among bacterial ParA ( 224 M/V-I-P-K/R-S/N 228 ) ( Supplementary Figure S1). Pro226 and Ser228 directly interact with the adenosine of ATP through their main chains ( Figure 2C and Supplementary Table S1). These loop ␣8␣9 interactions with ATP have also been observed in TtSoj (corresponding to residues Pro207/Arg208/Asn209) (7) and SpParA (corresponding to residues Lys252/Arg253/Ser254) (10).
Lys227 is highly conserved in the ParA superfamily (Supplementary Figure S1). Interestingly, Lys227 is located at the core of the DNA binding surface, but its side chain is oriented away from the DNA and it does not directly interact with DNA ( Figure 3A). Moreover, this residue is embedded inside the dimer interface and interacts with the ribose of ATP through a water molecule ( Figure 2C and Supplementary Table S1). Thus, significantly, Lys227 might play an important role in linking ATP-and DNA-binding in the HpSoj dimer ( Figure 3B). The orientation of the Lys227 side chain is important for adjusting loop ␣8␣9 to interact with ATP. In the presence of DNA, Lys227 is located at the core of the HpSoj DNA-binding surface, at a distance of 4.3-7.2Å from the DNA molecule. In addition, Lys247 exhibits sequence homology among the bacterial ParA superfamily (except for TtSoj: Thr228) (Supplementary Figure  S1), and is located 4.9-8.0Å from DNA, so it might play an interactive role in the DNA binding of bacterial ParA. Thus, based on our analyses of these residues, ␣8, loop ␣8␣9 and ␣9 are important functional regions for ATP-and DNAbinding and they link these two functions in the ParA superfamily.
Based on the crystal structure of the HpSoj-ATP-DNA complex ( Figure 3A), four key lysine residues (Lys199, Lys227, Lys230 and Lys247) are involved in the nonspecific DNA binding. The HpSoj mutants, K199E, K230E, K199/230E and K199/227/230/247E, have been isolated and purified. Meanwhile, their non-specific DNA binding were measured by the fluorescence polarization (FP) and shown in Figure 3C. The K d of non-specific DNA binding of wildtype HpSoj is calculated to be 122 nM. However, the non-specific DNA binding of the mutants are decreased and the K d for K199E, K230E and K199/230E are 187, 215, 308 nM, respectively. Nevertheless, the K d of mutant K199/230/230/247E is undetectable. Indeed, it suggested that Lys199, Lys227, Lys230 and Lys247 play an important role in the non-specific DNA binding of HpSoj.

Protein-protein interactions of HpSoj-DNA complexes
We observed HpSoj dimer-dimer interactions at several locations, but the DNA-binding region is preserved in the DNA-unbound state ( Figure 3E). Dimer-dimer interactions were observed in three regions of HpSoj-DNA complexes (I, II and III in Figure 3E). The buried surface areas of these regions were calculated for HpSoj-ATP-DNA and HpSoj-ADP-DNA as 295 and 319Å 2 (region I), 392 and 558Å 2 (region II), and 317 and 305Å 2 (region III), re-spectively. Among the three interfaces, region II contributes most to protein-protein interactions. Region II is mainly formed by helix ␣4 that displays diverse sequence and length among the ParA superfamily (Supplementary Figure S1). The calculated buried surface area of region II is 488Å 2 for HpSoj-ATP alone. After DNA binding, HpSoj-ATP-DNA adopts an active state with fewer protein-protein interactions. HpSoj-ADP-DNA may adopt a relaxed state and return to the DNA-unbound state, so it has a similar protein-protein interaction area as that of HpSoj-ATP.
Superimposition of HpSoj-ATP and the HpSoj-ATP-DNA complexes relative to the AB dimer (Supplementary Figure S3B) revealed an ∼9-10Å deviation between the CD dimers (C␣ of Thr98 in ␣4, and C␣ of Lys207 in ␣8). After DNA binding, dimer-dimer interactions affect the relationship of the AB and CD dimers to induce a molecular shift in the CD dimer (Supplementary Figure S3B). Although the HpSoj dimer only has one DNA-binding site, it possesses numerous dimer-dimer interaction regions to recruit HpSoj molecules ( Figure 3E and Supplementary Figure S3B).
The protein-protein interactions of HpSoj dimer might play an essential role in facilitating HpSoj localization to high-density DNA regions (HDRs, Supplementary Figure  S4A) in the H. pylori ParABS system. It has previously been reported that the DNA-binding activity of ParA is essential for localization of all components of the partitioning machinery to HDRs and for a stable HDR-bound ParA state (32). Accordingly, the HpSoj-ATP-DNA structure might mimic the stable and active HDR-bound state (Supplementary Figure S4A). In the multifaceted DNA-binding site of pNOB8 ParA-DNA, each ParA dimer is surrounded by a dense DNA substrate (Supplementary Figure S4B) (37). The sole DNA-binding site in our HpSoj-DNA complex structure ( Figure 3E), HpSoj dimers can be recruited by protein-protein interactions to engage several DNA substrates (Supplementary Figure S4A).
In the HpSoj-DNA complex, the continuous basic patch of HpSoj is exposed for DNA binding (Figure 4D). The pNOB8 ParA and its DNA complex reveal a different dimer conformation (35,37). The corresponding DNA-binding residues of HpSoj (Asn196, Lys199, Lys227, Val229, Lys230, Lys247) in pNOB8 are Arg210/Ala214/Arg257/Lys258/Glu259/Val288, but Arg257/Lys258/Glu259/Val288 are missing from the crystal structure of the ParA-AMPPNP-DNA complex (PDB ID: 5U1J). In the pNOB8 ParA-AMPPNP-DNA complex, the two monomers of the ParA dimer need to be rotated to expose basic residues (Arg52, Lys58, Lys85, Lys218, Lys221 and Lys 270) for optimal DNA binding. HpSoj region III (responsible for dimer-dimer interactions; Figures  3E and 4C) corresponds to the DNA-binding residues of pNOB8 ParA. Notably, the electrostatic surface distribution of the DNA-binding region of HpSoj is predominantly basic, whereas it is evenly distributed among basic/acidic in the pNOB8 ParA-AMPPNP-DNA complex (PDB ID: 5K5Z) ( Figure 4D and E).

The bacterial and archaeal partitioning system
The partitioning system in most bacteria involves two proteins (ParA and ParB) and a cis-acting centromere-like parS DNA (55). Two partitioning systems have been described in archaea; the SegAB system and AspA-ParBA that comprise different key functional proteins (36). We superimposed the monomer structures of HpSoj and TtSoj ( Figure  4A) and found that they share a similar overall structure, with an r.m.s.d. of 0.8Å (C␣ atom). Structural differences between the HpSoj and TtSoj monomers mainly occur in ␣4 and loops ␣2␣3 and ␣8␣9 ( Figure 4A). Loop ␣2␣3 and ␣4 are involved in dimer-dimer interactions (Supplementary  Table S2), and loop ␣8␣9 participates in ATP-binding (Supplementary Table S1). HpSoj has longer ␣4 (residues 99-111) and loop ␣8␣9 (residues 211-224) regions than TtSoj (Supplementary Figure S1). However, the dimer relationships of both HpSoj and TtSoj are quite similar ( Figure  5A and B). Based on the crystal structure of the HpSoj-ATP-DNA complex, we postulate a possible DNA-binding patch in TtSoj created by residues Arg182, Arg208, Arg211 and Thr228 ( Figure 5B). Significantly, a basic patch can also be observed for SpParA (Lys230, Arg253, Lys256 and Lys273) ( Figure 5C) and for TP228 ParA (Asn148, Arg169, Lys174 and Lys191) ( Figure 5D), which also correspond to the DNA-binding basic patch of HpSoj. These findings suggest that bacterial ParA superfamily might have a similar nsDNA-binding mode as that HpSoj has.
In the archaeal SegAB system, the ATPase SegA is polymerized upon binding ATP, and the site-specific DNAbinding protein SegB promotes SegA polymerization (34). In the archaeal AspA-ParBA machinery, pNOB8 ParA-AMPPNP forms a polymer in the absence of DNA but not in the presence of DNA (37). HpSoj does not form a polymer, regardless of the presence of nucleotide or DNA. HpSoj and pNOB8 ParA reveals different monomer relationships of their dimers ( Figure 4B). Remarkably, there is a significant discrepancy in DNA binding between HpSoj and pNOB8 ParA ( Figure 4C); HpSoj dimer binds one DNA at the base of the molecule, whereas the pNOB8 ParA dimer binds two DNA molecules at two sides of the molecule ( Figure 4C). HpSoj and pNOB8 ParA also have different DNA-binding surfaces; 504Å 2 for the sole DNA-binding site of HpSoj and 238 and 194Å 2 for the two pNOB8 ParA DNA-binding sites ( Figure 4C), as calculated by PISA (48). The solvent content of the HpSoj-ATP-DNA and pNOB8 ParA-AMPPNP-DNA complexes are 58% and 45%, respectively. The pNOB8 ParA-AMPPNP-DNA complex contains multiple DNA-binding sites and the low solvent content induces tight molecular packing between pNOB8 ParA and the DNA (Supplementary Figure S4B). Thus, structural comparison of HpSoj-ATP-DNA and pNOB8 ParA-AMPPNP-DNA complexes demonstrates that the mode of nsDNA-binding might differ between bacteria and archaea. Despite this difference, they might share a similar DNA segregation mechanism to drive movement of the ParB-DNA cargo, which does not require ParA polymerization.

Nucleoid-adaptor complex (NAC) formation
To investigate ParABS nucleoid-adaptor complex (NAC) formation in H. pylori, we used EM to directly capture HpSpo0J and HpSoj in complex with DNA ( Figure 6). We used parS550 for specific DNA-binding by HpSpo0J and 180DNA as a substrate for HpSoj to probe the HpSpo0J-HpSoj-DNA complex based on the differing DNA lengths. We assessed [HpSpo0J:parS550] experiment, a total of 203 conformational complexes were counted, including 70.9% of the single HpSpo0J:parS550 complex ( Figure 6A), 12.8% of the HpSpo0J:parS550 paired complex ( Figure 6B), and 16.3% of a higher-order complex ( Figure 6C) ( Table 2). However we observed NAC complex formation when we co-incubated [HpSpo0J:parS550] (the partitioning complex, cargo) with [HpSoj-180DNA] (the nucleoid-bound complex) ( Figure 6F-H). We counted a total of 173 conformational complexes in this latter experiment (

DISCUSSION
In this study, we determined the ATPase activity of HpSoj and found that it is promoted by HpSpo0J and DNAbinding. We solved the crystal structures of HpSoj and its DNA-bound complexes, HpSoj-ATP-DNA and HpSoj-ADP-DNA. From our EM data, we observed possible interactions among HpSoj, HpSpo0J, and DNA and propose potential NAC formation for the H. pylori ParABS system.
HpSoj behaves typically as other ParAs revealing a weak ATPase that is stimulated by DNA and HpSpo0J/ParB. The ATPase activity of HpSoj is stimulated by the HpSpo0J-parS complex. When HpSpo0J binds to parS55 and the HpSpo0J-parS complex is formed the N-terminal domain of HpSpo0J might be exposed based on the crystal structure of the HpSpo0J-parS complex (2). And then the N-terminal domain might interact with HpSoj to promote the ATPase activity. In addition, the ATPase activ-ity of HpSoj was stimulated by HpSpo0JN in the presence of DNA. Although HpSpo0JN does not contain the DNA binding domain it still can stimulate the ATPase activity. This phenomenon also has been reported that the SopB AT-Pase activation domain exists at its N-terminus, which does not have the DNA binding domain either (56). Several previous functional studies demonstrated that the N-terminal domain of ParB likely contributes to the ParA/ParB interaction and to promote ParA ATPase activity (7,16,57,58). A weak interaction between HpSoj and HpSpo0JN was detected by MST and it perhaps due to their dynamic and transient association. Single-molecule and 3D structured illumination microscopy studies (17,18,31,32) have indicated that the nsDNA-binding ability of ParA is critical in DNA segregation. The HpSoj dimer binds to one DNA molecule, whereas the archaeal Sulfolobus pNOB8 ParA dimer binds to two DNA molecules via multifaceted interactions (37) ( Figure 4C). The DNA-binding regions of HpSoj and pNOB8 ParA differ, involving different non-conserved residues, perhaps because of species variation or because the DNA-binding of ParA is non-specific. Regarding the electrostatic surface of the ParA dimer, a basic patch environment and a proper binding curvature are required for nsDNA binding.
Previous biochemical studies identified that ParA binds with ATP to form a dimer and then undergoes a slow conformational change into the 'ParA* 2 -ATP 2 ' state that is stable and, based on tryptophan fluorescence assays on P1 ParA, precise for DNA binding (26). A continuous and basic DNA-binding patch (comprising residues Lys199, Lys 227, Lys 230 and Lys247) is created when HpSoj is dimerized in the ParA* 2 -ATP 2 state. However, there is no significant conformational difference between HpSoj-ATP and the HpSoj-ATP-DNA complex, as is the case for other nsDNA-binding proteins (52). Since the DNAbinding patch is on the HpSoj dimer surface and readily exposed for DNA binding, a further conformational change is not necessary. In the absence of ATP, HpSoj was not stable and the crystallization was not succeeded. Since the threedimensional structure of apo-HpSoj (no nucleotide) is not available we cannot rule out the possibility of a different conformation among apo-HpSoj, HpSoj-ATP and HpSoj-ATP-DNA. In addition, multiple conformations of HpSoj-ATP might exist in solution as that reported in P1 ParA (26).
The pNOB8 ParA, which resembles bacterial ParA, possesses nsDNA-binding ability (37), and a multifaceted DNA-binding mode. The pNOB8 ParB contains an Nterminal domain (residues 1-320) that corresponds to bacterial ParB (182-336 residues), albeit with low sequence homology (35). The pNOB8 ParB binds DNA in a nonspecific manner; however, bacterial ParB specifically binds DNA at the parS site (35). These Walker-box partitioning systems are ubiquitous and exist in both bacteria (ParABS) and archaea (SegAB and AspA-ParBA), though each exhibits their own particular mechanisms. In contrast, the DNA-binding patch of pNOB8 ParA is buried inside the dimer so monomer rotation is necessary to expose its basic residues for optimal DNA binding (37). Residues Lys199, Lys 227, Lys 230 and Lys247 form the basic DNA-binding patch of HpSoj, with the corresponding residues in pNOB8 ParA being Ala214, Glu250, Ile253 and Lys270. The HpSoj basic residues form a DNA-binding patch that harbors one DNA molecule, whereas the dispersed pNOB8 ParA basic residues result in a multifaceted DNA-binding surface, together suggesting that the DNA-binding mechanism of bacterial ParA is significantly different from that of archaeal ParA.
The HpSoj-ADP-DNA complex presented a similar conformation to that of HpSoj-ATP-DNA. Thus, the HpSoj-ADP-DNA complex might represent a transitional state in which the ATP has been hydrolyzed to ADP but the DNA remains bound. Interaction of HpSoj with HpSpo0J stimulates ATPase activity ( Figure 1A) and may accelerate dimer dissociation and DNA release. In Salmonella newport TP228, ParB interacts with the dimer interface of ParA, which may induce ParA to dissociate from DNA, resulting in segregation along the nucleoid matrix (37).