Structure–function analysis of PorXFj, the PorX homolog from Flavobacterium johnsioniae, suggests a role of the CheY-like domain in type IX secretion motor activity

The type IX secretion system (T9SS) is a large multi-protein transenvelope complex distributed into the Bacteroidetes phylum and responsible for the secretion of proteins involved in pathogenesis, carbohydrate utilization or gliding motility. In Porphyromonas gingivalis, the two-component system PorY sensor and response regulator PorX participate to T9SS gene regulation. Here, we present the crystal structure of PorXFj, the Flavobacterium johnsoniae PorX homolog. As for PorX, the PorXFj structure is comprised of a CheY-like N-terminal domain and an alkaline phosphatase-like C-terminal domain separated by a three-helix bundle central domain. While not activated and monomeric in solution, PorXFj crystallized as a dimer identical to active PorX. The CheY-like domain of PorXFj is in an active-like conformation, and PorXFj possesses phosphodiesterase activity, in agreement with the observation that the active site of its phosphatase-like domain is highly conserved with PorX.


Overall structure of PorX Fj
In order to solve the structure of PorX Fj , the native protein as well as its Selenomethionine-labeled (SeMet) derivative were produced and purified by immobilized ion metal affinity and size-exclusion chromatographies.Diffracting crystals of both proteins grew in the P2 1 2 1 2 1 space group; the SeMet PorX Fj structure was solved using a SAD data set and then used as a model to solve a higher resolution structure of the native PorX Fj by molecular replacement.

The CheY-like domain
The CheY-like RD adopts the classical (α/β) 5 doubly-wound fold consisting of a central 5-stranded β-sheet, surrounded by five α-helices.The conserved active site is an acidic pocket formed by three acidic residues (D11, E12 and D54), T82, and K104.PorX Fj was not activated by phosphorylation before crystallization, and indeed no phosphoryl group attached to the conserved D54 residue is present in the electron density map.The putative binding site of the metal supposed to stabilize the phosphoryl group is clearly occupied by an ion that is octahedrally coordinated by the carboxylate oxygens of D11 and D54, the main chain-carbonyl oxygen of N56 and three water molecules (Fig. 3).The nature of the ion cannot be attributed unambiguously from inspection of the electron density.By comparison with the PorX Pg structure, and accordingly to the coordination number and the bond lengths between the ion and its ligands, a Mg 2+ ion was modelled in the structure.As no magnesium was added during the purification and crystallization steps, we can therefore speculate that the ion present in the structure was acquired intracellularly.Despite the absence of BeF 3 that mimics the activating phosphorylation of the RD, the side chains of the two PorX Fj highly conserved 'switch' residues T82 and Y101 are oriented towards the active site, in a conformation corresponding to the active state of RDs (Fig. 3).This confirms that binding of the Mg 2+ ion is likely sufficient to induce the conformational changes associated to RDs activation, while phosphorylation stabilizes this Mg 2+ -bound conformation, as previously proposed 36 .www.nature.com/scientificreports/

The HBD domain
The PorX Fj HBD folds as a three-helix bundle, and presents the same features as in the P. gingivalis PorX Pg structure: a 80° bend of the polypeptide chain at its N terminus, and a kink at the end of the first helix.In PorX Pg , this domain was proposed to interact with DNA 26 .However, the first hits returned by a structural similarity search within the Protein Data Bank (PDB) using the DALI server 37 correspond to domains involved in protein-protein interaction: the fibronectin-binding protein RevA 38 , the plakin domain of plectin 39 , or the BAG domain 40 for instance.

The PglZ domain
The PorX Fj C-terminal PglZ domain adopts the classical α-β-α fold of the alkaline phosphatase superfamily, which consists of a catalytic domain with a central β-sheet of six β-strands surrounded by six and four α-helices on each side, and a β-rich cap subdomain at the entrance of the active site (Fig. 4A).The active site coordinates two Zn 2+ ions: Zn1 is coordinated by the catalytic T271, D237, D414 and H415 residues while Zn2 is coordinated by residues H364, H499, D360 and a water molecule (Fig. 4A).Of note, the distance between the two ions is 5.4 Å, a distance much greater than the distance reported for any other alkaline phosphatase superfamily members 41 .
In the PorX Pg dimer, each monomer presents a distinct conformation of residues 359-367: in one monomer this region folds as an α-helix (conformation HH), while in the other it folds as an extended loop (conformation HL) 33 .As the HL conformation is not compatible with Zn2 binding, the monomer with this conformation is unlikely to be active.In contrast, the two monomers of the PorX Fj dimer adopt the HH conformation with two coordinated Zn 2+ ions and are therefore likely to be active.Comparison with the structure of an inactive PorX Pg mutant (T272A) crystallized in complex with pGpG reveals that residues interacting with the ligand are strictly conserved in PorX Fj , except Y332 that is replaced by T331 (Fig. 4B).However, the electron density around Y332 and its contacting pGpG purine group is poorly defined 33 , suggesting that this interaction is labile and could be not critical for the catalyzed reaction.

Purified PorX FJ has phosphodiesterase activity
PorX Pg was previously shown to have phosphodiesterase activity in vitro 33 .Enzymatic assays using the purified PorX Fj protein showed that it also hydrolyzes bis-p-nitrophenyl-phosphate (Fig. 5).Similarly to PorX Pg , this activity requires the presence of a divalent cation, but unlike PorX Pg , PorX Fj is active not only in presence of Zn 2+ , but also in presence of Cu 2+ and Mn 2+ (Fig. 5).The presence of the divalent cation is necessary but also sufficient for PorX Fj activity as a comparable activity was measured when the protein was phosphorylated or not prior to the reaction (Fig. 5).Such a broad metal specifity was also observed for the Sinorhizobium meliloti PhnA alkaline phosphatase 41 .However, comparison of the PorX Pg and PorX Fj structures highlights no significant structural difference in the ions pocket that could explain the different specificity of the two proteins.www.nature.com/scientificreports/SEC-MALS analysis showed that PorX Fj incubation with Zn 2+ induced dimerization, similarly to PorX Pg 33 (Fig. 2).Interestingly, dimers induced by phosphorylation (AcP + Mg 2+ ) and Zn 2+ eluted at different volumes, suggesting that they adopt different conformations, which confirms SAXS analysis of PorX Pg 33 .Moreover, a dimer/monomer equilibrium was observed when PorX Fj was incubated with Zn 2+ , which could be explained by a dilution effect as the SEC-MALS analysis was carried out with the column equilibrated in PBS only.Therefore, SEC analysis of PorX Fj dimerization induced by the different ions necessary for activity (Zn 2+ , Cu 2+ , and Mn 2+ ) was carried out with the column equilibrated with PBS supplemented with the corresponding metal solutions.As expected, full dimerization was observed in the presence of Zn 2+ and Cu 2+ (Fig. 6).A dimer/monomer equilibrium was still observed in the presence of Mn 2+ that could reflect a lower affinity of PorX Fj for this ion.The PorX Pg protein was shown to interact with PorL 24 , a component of the PorLM rotor 5,6,21,42 , a situation resembling the interaction of CheY with the FliN subunit of the flagellar C-ring.Bacterial two-hybrid (BACTH) assays showed that this is also the case of PorX Fj , which interacts with the cytoplasmic domain of the PorL homologue, GldL (GldL C , Fig. 7A).Interestingly, reminiscent of the CheY/FliN interaction, our analyses defined that the PorX Fj /GldL C interaction is mediated by the PorX Fj CheY-like RD (Fig. 7A).We further tested whether activation of the CheY-like domain of PorX Fj is required for GldL interaction.Figure 7B shows that while a phosphomimetic substitution of the phosphorylated D54 residue (D54E) maintains the interaction with GldL, a phosphoablative substitution (D54A) prevents PorX Fj -GldL interaction.

Discussion
In P. gingivalis, the T9SS activity was shown to be regulated in part by the TCS composed of the histidine sensor PorY and the response regulator PorX Pg .The mode of action of PorX Pg remains elusive, notably on how its phosphodiesterase activity and its phosphorylation status influence T9SS activity.In addition, its potential interaction with DNA is controversial.It was recently shown that PorX Pg activation is induced by phosphorylation of its RD, resulting in dimerization of the protein, and the structure of active, dimeric PorX Pg was solved 33 .
In this study, we solved the crystal structure of PorX Fj , the F. johnsoniae PorX homolog.Comparison with the PorX Pg structure strongly suggests that the two proteins share similar functions.Indeed, PorX Fj possesses phosphodiesterase activity, it crystallized as a dimer identical to the active PorX Pg dimer, the CheY-like RD adopts  www.nature.com/scientificreports/an active-like conformation, and the active site of the PglZ phosphatase-like domain is highly conserved with PorX Pg .Interestingly, crystallization is sufficient to promote dimerization of PorX Fj without prior activation by phosphorylation, as previously observed 44 .We propose that PorX Fj dimerization relies on the binding of the Mg 2+ ion to the PorX Fj RD, inducing its active-like conformation, in conjunction with high concentration conditions occurring during the crystallization process that promote molecular contacts.PorX Pg dimerization was not observed in the absence of activation, probably due to a less stable dimer than PorX Fj .Unfortunately, there is no monomeric PorX structure that could provide clues to the dimerization process, particularly if conformational changes are involved.
Similarly to PorX Pg , PorX Fj lacks any canonical DNA-binding motif, which further questions about the ability of PorX, or PorX Fj , to directly interact with DNA.Rather, PorX Pg interacts with the extracytoplasmic function (ECF) sigma factor SigP, that itself interacts with DNA 25 .Only one homolog of SigP is present in F. johnsoniae (GenBank: PZQ89414.1).Further investigation would be necessary to analyze the putative interaction of PorX Fj with the SigP homolog, and more generally to assess its involvement in T9SS gene regulation.In addition to its interaction with SigP, PorX Pg was shown to interact with the cytoplasmic domain of PorL, a component of the PorLM motor that uses the proton-motive force (PMF) to energize effector secretion through the T9SS 5,6,24,45 .Our results demonstrated that this interaction is conserved in F. johnsioniae, suggesting that PorX Fj may regulate T9SS activity.In addition, we showed here that the PorX Fj CheY-like domain is sufficient to mediate interaction with the PorL homolog, GldL.The interaction of a CheY-like protein with a multiprotein complex is well documented in the case of the regulation of the flagellum rotation, in which the chemotaxis CheY protein interacts with FliM and FliN, two components of the C-ring 32,46 .Binding of phosphorylated CheY to FliM/N induces a tilting movement of the C-ring that repercutates onto FliG to reverse the direction of motor rotation 32,[47][48][49][50][51] .Interestingly, mutagenesis of the conserved phosphorylable aspartate residue D54 further showed that a substitution preventing D54 phosphorylation prevents PorX Fj binding to GldL.Taken together, we propose that the PorX Fj phosphorylation status controls association to and dissociation from the GldLM complex.We further speculate that this interaction regulates the activity of the motor as a response of an environmental cues or of PglZ domain activity.In the case of the flagellum, CheY association to the C-ring controls the reversible switch between clockwise and counterclockwise rotation, hence enabling cells to swim towards favorable chemical habitats 50,51 .In F. johnsioniae, the PMF-dependent activity of the T9SS GldLM rotor powers effector transport through the outer membrane but also the movement of the SprB adhesin at the cell surface, hence supporting gliding 5,6,45,52 .One may hypothesize that PorX Fj may have a function comparable to CheY by switching the T9SS motor to different conformations allowing to control effector secretion or adhesin displacement.PorX Fj may thus control the speed of gliding or the gliding direction.Further studies should be performed to better understand the contribution of PorX Fj , and of its CheY, HBD and PglZ domain in gliding motility, T9SS gene regulation and SigP interaction.

Bacterial strains, media, chemicals and growth conditions
Escherichia coli K-12 strain DH5a was used for all cloning procedures, T7 strain for protein production, and BTH101 for bacterial two-hybrid assays, E. coli cells were grown in Lysogeny Broth (LB) or Turbo broth supplemented with antibiotics when necessary (kanamycin 50 µg mL −1 , ampicillin 100 µg mL −1 ).Expression from pLIC03 and BACTH vectors was induced with 1 mM and 0.5 mM of isopropyl β-d-1-thiogalactopyranoside (IPTG), respectively.

Plasmid construction
The sequence encoding full length PorX Fj (Fjoh_2906) was amplified from F. johnsoniae genomic DNA (ATCC17061/DSMZ2064) using the following primers: 5ʹ-CCT GTA CTT CCA ATC AAT GGA TAA GAT AAG AAT ACT TTG GGT CG and 5ʹ-CCG TAT CCA CCT TTA CTT TAT TAT TTA GGG TTA AAT ACC AAA AACGG.The sequence was cloned into pLIC03 (kindly provided by the BioXtal company, Marseille) using the In-Fusion technology (Takara), following the manufacturer protocol.The pLIC03 expression vector is a pET-28a + derivative (Novagen) carrying a cassette coding for a His 6 tag and a Tobacco Etch Virus (TEV) protease-cleavage.
BACTH vectors producing TssF, TssG and GldL C fused to the Bordetella adenylate cyclase T18 or T25 domains have been previously published 6,43 .BACTH plasmid producing PorX Fj and PorX Fj domains fused to the T18 and T25 domains were constructed by restriction free (RF) cloning 53 using oligonucleotides CGC CAC TGC AGG GAT TAT AAA GAT GAC GAT GAC AAG GAT AAG ATA AGA ATA CTT TGG GTC GAT GAT GAG and CGA GGT CGA CGG TAT CGA TAA GCT TGA TAT CGA ATT CTAG TTA TTT AGG GTT AAA TAC CAA AAA CGG AAT AAT CAT TTC (T18-PorX Fj ), CGC CAC TGC AGG GAT TAT AAA GAT GAC GAT GAC AAG GAT AAG ATA AGA ATA CTT TGG GTC GAT GAT GAG and CGA GGT CGA CGG TAT CGA TAA GCT TGA TAT CGA ATT CTAG TTA TTT TTG GTA ATC TAA TGT TGT TTT TTC TGT AAT CAGTC (T18-CheY), CGC CAC TGC AGG GAT TAT AAA GAT GAC GAT GAC AAG GAA TTC CGC AAA ATC TCG ATG GAA TTAGC and CGA GGT CGA CGG TAT CGA TAA GCT TGA TAT CGA ATT CTAG TTA TTT TGG AGC AAA CCA GTC TTC GTA ATT TC (T18-HBD), and CGC CAC TGC AGG GAT TAT AAA GAT GAC GAT GAC AAG GCA GAT AAA CCA ATT CAA TCT CAT AAT TTA TTT AAA GAA TTA GTTG and CGA GGT CGA CGG TAT CGA TAA GCT TGA TAT CGA ATT CTAG TTA TTT AGG GTT AAA TAC CAA AAA CGG AAT AAT CAT TTC (T18-PglZ) (sequences annealing on the target plasmids in italics).Briefly, the DNA fragment was amplified using primers that introduced extensions annealing to the target vector.The double-stranded product of the first PCR has then been used as primer for a second PCR using the target vector as template.PCR products were then treated with DpnI to eliminate template plasmids and transformed into DH5a-competent cells.Substitutions were introduced by site-directed mutagenesis using complementary oligonucleotides bearing the desired mutation (CTT TGA CAT TGT TTT TCT TGCCGAA AAT ATG CCG GGA ATG and CAT TCC CGG CAT ATT TTC GGCAAG AAA AAC AAT GTC AAA G for D54A, CTT TGA CAT TGT TTT TCT TGAGGAA AAT ATG Vol:.( 1234567890

Protein production, purification and analysis
PorX Fj was produced in E. coli T7 cells cultured in Turbo Broth medium at 37 °C.At OD 600nm of 0.6-0.8,porX Fj expression was induced by adding 1 mM IPTG and the bacterial growth was pursued for 18 h at 17 °C.Cells were harvested by centrifugation at 4000×g for 10 min, resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 250 µg mL −1 lysozyme, 1 mM PMSF) and frozen overnight at − 20 °C.After thawing, 20 µg mL −1 of DNase and 1 mM of MgSO 4 were added, and cells were lysed by sonication.The pellet and soluble fractions were separated by centrifugation at 16,000×g for 30 min, and the His 6 -tagged protein was purified from the soluble fraction by immobilized metal ion affinity chromatography using a 5 mL HisTrap crude (GE Healthcare) Ni 2+ -chelating column equilibrated in buffer A (50 mM Tris-HCl p H8.0, 300 mM NaCl, 10 mM imidazole).The protein was eluted with buffer A supplemented with 250 mM imidazole and further purified by size exclusion chromatography (HiLoad 16/60 Superdex 200 prep grade, GE Healthcare) equilibrated in 10 mM HEPES pH 7.5, 500 mM NaCl.Selenomethionine-labeled (SeMet) PorX Fj was produced and purified with the same protocol as the native PorX Fj , except that the cells were grown in SeMet minimal medium 54 .

Dimerization analysis
For the Size-Exclusion Chromatography Multi-Angle Light Scattering (SEC-MALS) analysis, the purified PorX Fj (1.6 mg mL −1 ) was incubated for 1 h at RT with 20 mM acetyl phosphate (AcP) + 10 mM MgCl 2 , or 20 mM AcP, or 10 mM MgCl 2 , or 100 µM ZnCl 2 .The samples were loaded on a Superdex 200 Increase 10/300 GL column (GE Healthcare) equilibrated in PBS at a flow rate of 0.6 mL min −1 , using an Ultimate 3000 HPLC system (Fischer Scientific).Detection was performed using an eight-angle light-scattering detector (DAWN8, Wyatt Technology) and a differential refractometer (Optilab, Wyatt Technology).

Crystallization, data collection and processing
The purified PorX Fj and SeMet PorX Fj were concentrated to 10 and 12 mg mL −1 , respectively.Crystallization trials were performed using the sitting-drop vapor-diffusion method at 293 K in 96-well Swissci-3 plates, with Stura Footprint (Molecular Dimensions), Wizard I and II (Rigaku), Structure I and II (Molecular Dimensions) and JCSG + (Qiagen) screens, and using Tecan and Mosquito (TTP Labtech) robots to fill in the plates and dispense the drops, respectively.PorX Fj crystals appeared in condition No. 20 from Structure I screen (0.2 M calcium acetate, 0.1 M sodium cacodylate pH 6.5, 18% PEG 8000).SeMet PorX Fj crystals appeared in several condition, and after optimization 55 , the final crystallization conditions were 0.2 M ammonium sulfate, 0.05 M sodium acetate, 0.05 M sodium citrate pH 5.0-6.0,10-30% PEG 2000.Crystals were mounted in cryo-loops (Hampton CrystalCap Magnetic) and were briefly soaked in crystallization solution supplemented with 20% (v/v) polyethylene glycol.The crystals were flash-cooled in a nitrogen-gas stream at 100 K using a home cryocooling device (Oxford Cryosystems).
Native diffraction data of PorX Fj and single wavelength anomalous dispersion (SAD) data of SeMet PorX Fj were collected to 2.0 Å and 2.3 Å resolution, respectively, on beamline Proxima-1 at the Soleil synchrotron (Paris, France).The data sets were integrated with XDS and scaled with SCALA 56 from the CCP4 suite 57 .Heavy atom substructure determination of SeMet PorX Fj , phase calculations and density modification were performed using HySS 58 , Phaser 59 and Parrot 60 , respectively, as implemented in the Phaser SAD pipeline from the CCP4 suite.A partial SeMet PorX Fj model was built automatically in Buccaneer 61 , completed manually in COOT 62 , and was subsequently used as model for molecular replacement with MOLREP 63 to solve the structure of native PorX Fj .Refinement, correction, and validation of the structure were performed with autoBUSTER 64 , COOT, and Molprobity 65 , respectively.Data collection and refinement statistics are reported in Table 1.

Bacterial two-hybrid assays.
Bacterial two-hybrid assays were conducted as previously described 43 .The proteins or domains to be tested were fused to the isolated T18 and T25 catalytic domains of the Bordetella adenylate cyclase.After introduction of the two plasmids producing the fusion proteins into the BTH101 reporter strain, plates were incubated at 28 °C for 24 h.Three independent colonies for each transformation were inoculated into 600 μL of LB medium supplemented with ampicillin, kanamycin, and IPTG (0.5 mM).After overnight growth at 28 °C, 15 μL of each culture were spotted onto LB plates supplemented with ampicillin, kanamycin, IPTG, and X-Gal and incubated at 28 °C.Controls include interaction assays with TssF and TssG, two T6SS protein partners from enteroaggregative E. coli 43 unrelated to the T9SS.The experiments were done at least in triplicate and a representative result is shown.

Phosphodiesterase activity assay
PorX Fj phosphodiesterase activity was measured using bis-p-nitrophenyl phosphate (bis-pNPP, Sigma-Aldrich) as substrate, essentially as previously published 33  (Schmitz et al. 2022).Briefly, purified PorX Fj was phosphorylated by incubation for 1 h in the presence of 20 mM of acetyl-phosphate and 10 mM of MgCl 2 , before being desalted on a 7-kDa Zeba™ Spin desalting column (ThermoFischer Scientific).50 μL of 2 μM purified phosphorylated or non-phosphorylated PorX Fj in 50 mM Tris-HCl pH8, 150 mM NaCl were mixed with 50 μL of 50 mM Tris-HCl pH8.5, 150 mM NaCl, 10 mM bis-pNPP supplemented, or not, with 100 μM of MgCl 2 , 100 μM of CaCl 2 , 100 μM of CuCl 2 , 100 μM of MnCl 2 or 100 μM ZnCl 2 in a 96-well microplate (transparent, flat bottom, Nunc).The release of p-nitrophenol was monitored for 180 min at 37 °C by measuring the absorbance at 405 nm (A 405 ) using a TECAN microplate reader.A control experiment with bis-pNPP only was also performed to measure the spontaneous hydrolysis, and the values of bis-pNPP spontaneous hydrolysis were subtracted to the measures of PorX Fj activity.Bis-pNPP activity was calculated from the slope and reported as A 405 per minute.The experiments were done in triplicate.

Figure 1 .
Figure 1.Overall structure of PorX Fj .(A) Structure of the PorX Fj monomer ; the CheY-like N-terminal, the HBD and the PglZ C-terminal domains are shown in cyan, white and purple, respectively; the N-and C-termini are labelled.(B) Comparison of the PorX Fj (left) and PorX Pg (right) dimer structures; For each structure, molecules A and B are shown in light and dark grey, respectively.

Figure 3 .
Figure 3.The PorX Fj CheY-like N-terminal domain.Overlay of the PorX Fj (cyan), active CheY (orange; PDB: 1FQW) and inactive CheY (yellow; PDB: 2CHE) structures.For clarity purpose, only the side chains of the CheY 'switch' residues (corresponding to PorX Fj T82 and Y101) are displayed.The Mg 2+ ion and the water molecules are shown as black and red spheres, respectively, and the hydrogen bonds are indicated by dashed black lines.

Figure 4 .
Figure 4.The PorX Fj PglZ C-terminal domain.(A) The catalytic and cap subdomains are shown in magenta and pink, respectively.The two Zn 2+ ions (Zn1 and Zn2) are indicated as black spheres.Enclosed is a close-up view of the Zn 2+ ions coordination, with the water molecule and the hydrogen bonds displayed as a red sphere and dashed black lines, respectively.(B) Overlay of the ligand-binding region of PorX Fj (magenta) with PorX Pg (grey) in complex with pGpG (yellow).The Zn 2+ ions present in PorX Fj and PorX Pg are shown as black and grey spheres, respectively.The side chains of PorX Pg residues in contact with pGpG and of the corresponding PorX Fj residues are indicated.For clarity purpose, only PorX Fj residues are labelled, and the molecules are slightly rotated compared to panel (A).

Figure 7 .
Figure 7.The activated form of the PorX Fj CheY-like domain interacts with GldL.Bacterial two-hybrid assay.BTH101 reporter cells producing the indicated domains/proteins (A, same color code as in Fig. 1: CheY domain, cyan; HBD, grey; PglZ domain, magenta) and CheY D54 phosphomimetic (D54E) and phosphoablative (D54A) variants (B) fused to the T18 and T25 domain of the Bordetella adenylate cyclase were spotted on X-Gal-IPTG reporter LB agar plates.The blue color of the colony reports interaction between the two partners.Controls include T18 and T25 fusions to TssF and TssG, two T6SS proteins from enteroaggregative E. coli that interact but unrelated to the T9SS 43 .
CCG GGA ATG and CAT TCC CGG CAT ATT TTC CTCAAG AAA AAC AAT GTC AAA G for D54E; mutagenized codon in italics, mutagenized bases underlined).All plasmids have been verified by DNA sequencing (Eurofins).

Table 1 .
Data collection and refinement statistics of PorX Fj .*Values in parentheses are for the highestresolution shell.