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Genes to Cells (2005) 10, 1-11. doi:10.1111/j.1365-2443.2005.00817.x
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Crystal structure of the protein histidine phosphatase SixA in the multistep His-Asp phosphorelay

Keisuke Hamada1, Masato Kato1, Toshiyuki Shimizu1, Kentaro Ihara1, Takeshi Mizuno3 and Toshio Hakoshima1,2,*

1 Structural Biology Laboratory, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan
2 CREST, Japan Science and Technology Agency, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan
3 Laboratory of Molecular Microbiology, School of Agricultural Science, Nagoya University, Chikusa-ku, Nagoya 464-8061, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
The multiple histidine-aspartate phosphorelay system plays a crucial role in cellular adaptation to environments in microorganisms and plants. Like kinase-phosphatase systems in higher eukaryotes, the multiple steps provide additional regulatory checkpoints with phosphatases. The Escherichia coli phosphatase SixA exhibits protein phosphatase activity against the histidine-containing phosphotransfer (HPt) domain located in the C-terminus of the histidine kinase ArcB engaged in anaerobic responses. We have determined the crystal structures of the free and tungstate-bound forms of SixA at 2.06 Å and 1.90 Å resolution, respectively. The results provide the first three-dimensional view of a bacterial protein histidine phosphatase, revealing a compact {alpha}/ß architecture related to a family of phosphatases containing the arginine-histidine-glycine (RHG) motif at their active sites. Compared with these RHG phosphatases, SixA lacks an extra {alpha}-helical subdomain as a lid over the active site, thereby forming a relatively shallow groove important for the accommodation of the HPt domain of ArcB. The tungstate ion, which mimics the substrate phosphate group, is located at the centre of the active site where the active residue, His8, points to the tungsten atom in the mode of in-line nucleophilic attack.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Signalling pathways mediated by histidine kinases play a crucial role in cellular adaptation to environments in plants and microorganisms such as bacteria, yeasts, and fungi. These pathways are made up of at least two protein components having histidine or aspartate as their active residue, and were referred to as the two-component system. Recent biochemical work, however, has shown that some of these pathways contain more than two components that relay the phosphoryl group from the histidine to the aspartate and then from the aspartate to the next histidine (Burbulys et al. 1991; Wurgler-Murphy & Saito 1997; Mizuno 1998) (Fig. 1). These signalling pathways are commonly referred to now as the multistep histidine-aspartate (His-Asp) phosphorelay. Findings of new protein phosphatases (Hess et al. 1988; Ohlsen et al. 1994; Perego et al. 1994; Duncan et al. 1995; Missiakas & Raina 1997; Ogino et al. 1998) and a protein kinase inhibitor (Wang et al. 1997) suggest that one of the most important advantages of multistepping is its potential for providing additional regulatory checkpoints. Like serine/threonine and tyrosine kinase-phosphatase systems in eukaryotes, regulation of the phosphorelay is now known to occur at the level of the phosphotransfer by regulated dephosphorylation as well as at the level of phosphate input by control of histidine kinase activities. To date, five protein phosphatases have been characterized in the His-Asp phosphorelay. These phosphatases modulate the signal transduction pathways controlling sporulation, chemotaxis, and anaerobic response. Among them, SixA (Ogino et al. 1998) acts as the protein histidine phosphatase that dephosphorylates the phosphor-histidine in the sensor kinase ArcB (Fig. 1A), while RapA, RapB (Perego et al. 1994), Spo0E (Ohlsen et al. 1994) (Fig. 1B), and CheZ (Hess et al. 1988) (Fig. 1C) act as protein aspartate phosphatases that dephosphorylate the phosphate group bound to each aspartate of the response regulators. RapA and RapB are highly homologous (50% identity), but no significant sequence homology has been detected in any other pairs. A recent study by sequence analysis and molecular modelling of phosphoglycerate mutase (PGMase) superfamily containing a variety of phosphatases have shown that SixA and its homologues have a distant evolutionary relationship with the PGMase family and may act as phosphoprotein phosphatase (Rigden 2003).



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  		Figure 1  Protein phosphatases in the His-Asp phosphorelay. (A) The anaerobic response pathway of E. coli. (B) The sporulation control pathway of B. subtilis. KipI is a protein histidine kinase inhibitor for the sensor kinase KinA. (C) The chemotaxis pathway of E. coli.

 
ArcB is the sensor kinase for the anaerobic respiratory control (Arc) response. The cytoplasmic part of ArcB contains the histidine protein kinase (HPK), receiver and histidine-containing phosphotransfer (HPt) domains (Fig. 1A). The response regulator for ArcB is the transcription factor ArcA, which has a receiver domain, and in the phosphorylated form, functions as a repressor for the sdhCDAB operon. The SixA phosphatase activity toward ArcB has been shown to be specific to the histidine (His717) in the C-terminal HPt domain (Matsubara & Mizuno 2000). In the absence of oxygen, E. coli cells can utilize the TCA cycle and the electron transport system for ATP synthesis in the presence of an electron acceptor such as nitrate. Under such an anaerobic condition, SixA dephosphorylates ArcB, so that the phosphoryl transfer from ArcB to ArcA is interrupted. This event results in derepression of the sdhCDAB operon encoding the succinate dehydrogenase complex involved in the TCA cycle.

Here we report the first crystal structures of the protein histidine phosphatase SixA and the complex with a tungstate ion as the phosphate analogue. These structures allow us to identify the active site of SixA and show that SixA has structural features related with particular metabolic phosphatases. The molecular shape suggests how SixA interacts with the substrate HPt domain of ArcB.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Structure determination and overall structure

The crystal structure of the free form of SixA was determined by the multiple isomorphous replacement (MIR) method and refined to 2.06 Å resolution. The tungstate-bound form was solved by molecular replacement using the free form structure and refined to 1.90 Å resolution. The present model consists of 156 residues out of 161 residues. Five C-terminal residues could not be modelled because of the poor electron density. A summary of the structural statistics is given in Table 1.


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  		Table 1  Crystallographic analysis
 
SixA has an {alpha}/ß architecture that comprises a six-stranded ß-sheet (ß1-ß6), five {alpha}-helices (H2-H3 and H5-H7), and two short 310-helices (H1 and H4) (Fig. 2A). The ß-sheet is formed by the four-stranded (ß1-ß4-ß2-ß3) parallel and two-stranded (ß5, ß6) anti-parallel associations and is sandwiched between a pair of long {alpha}-helices (H2, H3) on one side and another pair of long {alpha}-helices (H5, H6) on the other side, forming extensive hydrophobic cores. The active residue His8, which was identified by mutation study (Ogino et al. 1998), is located at the carboxyl end of strand ß1.



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  		Figure 2  Structure of the protein histidine phosphatase SixA. (A) Stereo view of SixA by ribbon drawing. The side chain of the active residue His8 is shown with a stick model, and the {alpha}-helices (green), 310-helices (blue) and ß-strands (red) are labelled. (B) Sequence and secondary structure elements of SixA and the amino acid sequence alignment with its homologues; E. coli, Haemophilus influenzae, Synechococcus PCC7002 (Swiss-Prot number Y400-SYNP2), Aquifex aeolicus (Protein Information Resource number G70400 [GenBank] ), Synechocystis PCC6803 (Swiss-Prot number Y400-SYNY3), Anabaena PCC7120 (EMBL number AJ012408 [GenBank] ) and Mycobacterium tuberculosis (Swiss-Prot number YC76-MYCTU). Conserved residues are highlighted in yellow (hydrophobic residues), blue (basic residues), pink (acidic residues), and light blue (polar residues). Four basic residues necessary for the phosphatase activity are indicated by asterisks at the top. (C) Conserved residues shown in (B) are highlighted on the molecular surface of SixA. The viewpoint of the molecule and the colouring schemes are the same as in (A) and (B), respectively.

 
Putative SixA homologues found in the amino acid sequence databases of SWISSPROT and PIR exhibit similar sizes but relatively low identity. The most homologous example is one from Haemophilus influenzae, which displays a 35% identity (Fig. 2B). The conserved residues are spread throughout the entirety of each protein molecule, but most of these charged residues mapped on the three-dimensional structure are localized at the region around His8 (Fig. 2C), while most of the hydrophobic residues are at core of the fold. In contrast, the residues in helix H5 and strand ß6 are poorly conserved. These secondary elements are not close to the active site where His8 is located.

Unlike most eukaryotic protein phosphatases (Barford et al. 1994; Goldberg et al. 1995; Griffith et al. 1995; Hof et al. 1998), SixA exhibits a compact {alpha}/ß structure as described above. We found that low-molecular-weight protein tyrosine phosphatase (LMW PTPase) (Su et al. 1994) consists of a similar number of residues forming an {alpha}/ß core in which the arrangement of some helices and strands corresponds to that of SixA. However, there is no similarity in topology or sequence. Small {alpha}/ß folds are also found in Cdc25 (Fauman et al. 1998) and VHR (Yuvaniyama et al. 1996) but these are more distant from that of SixA than the LMW PTPase structure.

Structural comparison of SixA with the RHG phosphatases

SixA has the arginine-histidine-glycine (RHG) motif at its active site. This RHG signature is found in phosphatases possessing a common catalytic mechanism involving a covalent phosphohistidine intermediate. SixA shares limited homology with the RHG phosphatase domains found in ubiquitous PGMase, eukaryotic fructose-2,6-bisphosphatase (F26BPase), prostatic acid phosphatase (PAPase), E. coli periplasmic phosphatase, and glucose 1-phosphate phosphatase. The crystal structures of yeast PGMase (Campbell et al. 1974) rat F26BPase (Hasemann et al. 1996), and rat PAPase (Schneider et al. 1993) have been determined. Despite a relatively poor homology (~14%) between SixA and these RHG phosphatases, these enzymes share a similar fold of the phosphatase core domains (Fig. 3A). This similarity is consistent with the results by homology modelling (Rigden 2003). Compared with SixA, each of these RHG phosphatases has an extra {alpha}-helical subdomain that covers the active site. The extra {alpha}-helical subdomain of yeast PGMase is primarily formed by a long inserted segment between helices H4 and H5 and two inserted segments at loops ß1-H2 and H6-ß5. In both rat F26BPase and rat PAPase, the extended C-terminal tail also contributes to formation of the subdomain (Fig. 3B). Excluding the insertion and deletion sites, the superposition of SixA on the phosphatase core domains of yeast PGMase, rat F26BPase, and rat PAPase results in the best fit with root mean square (rms) deviations ranging from 2.7 Å to 3.5 Å for corresponding C{alpha}-carbon atoms.



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  		Figure 3  The related RHG phosphatases. (A) Comparison of the RHG phosphatase folds found in SixA and the related enzymes. Ribbon representation of the enzymes with the corresponding RHG phosphatase folds in light green. Each active histidine residue is shown as a ball-and-stick model. The extra helical domains forming the active sites of the RHG phosphatase domains are coloured in brown (yeast PGMase), blue (rat F26BPase domain of the bifunctional enzyme), and red (rat PAPase). (B) Comparison of the linear arrangement of structural elements and insertion. Helices and ß-strands are represented with arrows and rectangles, respectively. Inserted segments forming the helical domain are coloured as in A. (C) Left, a general shape match between the convexity of the ArcB HPt domain (with ribbon representation) and the grooved molecular surface of SixA (with surface representation). The active histidine residue (His717) of the HPt domain is shown as a ball-and-stick model. Right, the {alpha}-helical subdomain of yeast PGMase forms a deep pocket for binding to its small substrate, 3-phosphoglycerate. The extra helical domain is coloured in yellow. The active histidine residues of SixA and PGMase are coloured in blue-green.

 
SixA has a relatively open active site located alongside a prominent shallow groove. SixA dephosphorylates a phosphate group bound to the active histidine residue of the HPt domain of ArcB (Ogino et al. 1998). The crystal structure of the ArcB HPt domain has revealed that the HPt domain has a somewhat kidney-shaped structure formed by a four-helix bundle core fold, with the active histidine residue located at the middle of the helix D (Kato et al. 1997). There is a general shape match between the grooved molecular surface of SixA and the convexity of the ArcB HPt domain (Fig. 3C). In contrast, each extra {alpha}-helical subdomain of the RHG phosphatases forms a deep pocket at the active site to trap their small substrate.

The active site

The active residue His8 is surrounded by three loops, the long loop between strand ß1 and helix H2 (loop ß1–H2), which contains the short helix H1, and two short loops, loops ß2–H3 and ß4–H6. The {alpha}/ß arrangement directs all the N termini of four {alpha}-helices toward the carboxyl end of the parallel strands. In particular, helices H3 and H6 point their N termini to His8. The dipole moments aligned along these {alpha}-helices likely contribute to attracting the negatively charged phosphate group of the substrate protein. In addition to the dipole moments, the highly positively charged active site is due to localized basic residues at the site. Interestingly, the overall molecular surface is mostly negatively charged, indicating that the electric field around the SixA pushes the negatively charged phosphohistidine toward the active site (Fig. 4A). These basic residues likely play some role in recruiting and binding the substrate phosphate group as well as in stabilizing the transition state. The RHG (residues 7–9) motif located at the carboxyl end of strand ß1 forms the active site together with conserved Asp18, Arg21, Ser51, Arg55, and His108 (Figs 2B and 4B). In the free form of SixA, four water molecules linked by hydrogen bonds form a tetrahedral mimic of a phosphate group. In the tungstate-bound form, these water molecules are replaced with the tungstate ion, which is a good mimic of the substrate phosphate (Fig. 4C) (Heo et al. 2002). No significant structural change of the active site was observed between the free and tungstate-bound forms.




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  		Figure 4  The active site of SixA. (A) Electrostatic molecular surfaces of SixA. The regions of the surface that have positive electrostatic potentials are in blue, and those having negative potentials are in red (blue = 10kBT, red =–10kBT, where kB is Boltzmann's constant and T is the absolute temperature). The tungstate ion is shown with a stick model. Acidic and basic residues are labelled. (B) Stereo view of the active site of the free form of SixA. The side chains are shown as stick models with large labels, using colour codes; carbon in brown, oxygen in red, and nitrogen in blue. Main-chain tracings are shown with tubes in light green. Hydrogen bonds are indicated by dotted lines. Water molecules are shown with small labels (Wats). Four water molecules (Wat1-Wat4), which are replaced by the tungstate ion in the tungstate-bound form, are linked by yellow dotted lines. Residues whose main-chains participate in hydrogen bonding interactions are indicated with small labels at the main-chain tracings. (C) Stereo view of the active site of the tungstate-bound form of SixA. (D) A close-up stereo view of the residues interacting with the tungstate ion at the active site. Hydrogen-bonding and ion-pairing interactions are indicated by dotted lines and broken lines, respectively. The bonding distances are also shown. The contact between the tungsten atom and the N{varepsilon} nitrogen atom of His8 is indicated by an arrow. (E) Superposition of the active site residues between SixA (carbon in brown and nitrogen in blue) and each RHG phosphatase domain of yeast PGMase (dark yellow), rat F26BPase (blue), and rat PAPase (red).

 
The tungstate ion interacts with four conserved basic residues, two histidines (His8 and His108) and two arginines (Arg7 and Arg55). These residues participate in hydrogen bond networks involving the side chains and main chains of other residues and bridging water molecules, implying that the geometry of the active site may be rigid. SixA traps the tungstate ion at the centre of the active site where the active residue His8 points to the tungsten atom with the ring N{varepsilon} nitrogen atom (2.5 Å) by anchoring the anionic oxygen atoms of tungstate to Arg7 and Arg55 through ion pairs and/or hydrogen bonds (Fig. 4D). This geometry implies an in-line nucleophilic attack by His8, with the two arginines possibly delocalizing the charges of the transition state expected during the phosphoryl transfer to His8. His108, which is in contact with one of the tungstate oxygen atoms through its ring N{delta} nitrogen atom, is a candidate for the general acid. Superposition of the active site residues between SixA and each RHG catalytic domain reveals that the relative positions and side-chain conformations of two histidines (His8 and His108) and two arginines (Arg7 and Arg55) are well conserved, suggesting an identical catalytic mechanism for these enzymes (Fig. 4E). The essential roles of these conserved residues in the catalytic actions are documented by mutation studies of these RHG phosphatases (Tauler et al. 1990; Lin et al. 1992).


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Under certain anaerobic respiratory growth conditions, SixA plays a crucial role in modulating the ArcB-ArcA signalling for inducing the sdh operon expression in E. coli cells. The SixA activity toward ArcB is specific to His717 in the HPt domain. The present structure provides the first three-dimensional details of the bacterial protein histidine phosphatase. SixA has the active site pocket located alongside a relatively shallow groove, which may be important for the accommodation of the substrate ArcB HPt domain folded into a four-helix bundle. Moreover, the active site pocket would fit to the phosphate group bound to the protruded active histidine His717 of the HPt domain.

We originally built the docking model of the binary complex between SixA and the HPt domain of ArcB to investigate their binding specificity (Fig. 5). The HPt domain is docked to SixA so as to make an in-line attack of His8 of SixA to the phosphate group that is linked to His717 of the HPt domain and overlaid on to the tungstate ion. In this model, the helix D of the HPt domain is accommodated by the grooved molecular surface of SixA as described above (Fig. 3C), enabling insertion of the active residue His717 of helix D into the active site of SixA. The active histidine residue of the HPK domain is located at a four-helix bundle formed by a parallel packing arrangement with two anti-parallel helices contributed from each monomer (Tomomori et al. 1999). The helix bundle of the HPK domain can fit to the groove of SixA, while the HPK helices have no apparent convexity that matches with the groove of SixA. As described below, this docking model between HPK domain and SixA produces unfavourable contacts or looses favourable contacts found in the HPt-SixA model.



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  		Figure 5  A structural model of the binary complex between SixA and the HPt domain of ArcB. (Left) Overall structure of the complex is shown with ribbon model and the side chains of contact residues are shown with stick model. The active residue of SixA, His8, binds to His717 of the HPt domain through the mediation of PO4. Each of two binding sites (Site 1 or Site 2) is enclosed by a circle. (Right panels) Close-up views of the contact sites (Site 1 and Site 2) are shown. Hydrogen-bonding and ion-pairing interactions between two proteins are shown as dotted lines.

 
We found two close contact sites in our HPt-SixA docked model. In the contact site, Site 1 in Fig. 5, Lys718 and Tyr696 from the HPt domain of ArcB stick their side chains toward SixA. These residues may make polar interactions such as hydrogen bonding and salt bridging with SixA residues including an acidic residue Asp14 as shown in Fig. 5. Interestingly, Lys718 and Tyr696 are not conserved in other HPt domains and the HPK domain of ArcB. Moreover, Lys718 and Tyr696 are replaced by acidic glutamic acids in the HPK domain of ArcB and in the HPt (P1 domain) of CheA, respectively. These acidic residues are unfavourable for binding to SixA because of repulsion between negative charges. Thus, the contact site including Lys718 and Tyr696 in the HPt domain presumably contributes to the specific binding between SixA and the HPt domain of ArcB.

In the other contact site, Site 2 in Fig. 5, Gln672 and Tyr673 in the helix B of the ArcB HPt domain exist in the vicinity of Asn26, Glu30 and Lys156 of SixA enough for making interaction with each other. These residues are replaced with the non-polar residues valine and proline in the HPK domain. Therefore, it is conceivable that the helix B of the ArcB HPt domain may be an important architecture for specific interactions between SixA and the ArcB HPt domain. The structural inspection provides a clue to identify the specificity-determinant residues of SixA in the further experimental investigation.

Unlike the HPt domain, the active residue (aspartate) of the receiver domains is located at the bottom of the deep active site pocket and a phosphate group bound to the active aspartate is partially buried inside the pocket. Therefore, phospho-aspartate of receiver domains is structurally unable to reach the SixA active site. This would be a reason why SixA is inactive for phospho-aspartate in the receiver domain of ArcB.

SixA would have to compete with the receiver domain of ArcA, the cognate response regulator of ArcB, for the HPt domain of ArcB. How is this competition of phosphate flow regulated? Unfortunately, we do not have much data about this point. Probably, regulation at the transcription level of SixA may be critical for modifying the phosphate flow. Structurally, the active site of SixA seems to be more easily accessible to the phosphor-His residue of the ArcB HPt domain than the active site of the receiver domain that has the active aspartate residue inside the deep pocket. Therefore, SixA could easily interfere with the phosphate flow to ArcA.

The SixA {alpha}/ß architecture related to a family of the RHG phosphatases suggests an evolutional relationship between this enzyme and the RHG enzymes in metabolic pathways. The conserved active site constitutes an essential platform for the phosphoryl transfer reaction, where a catalytic action similar to the RHG phosphatases may take place. A structural relationship between a protein phosphatase and metabolic enzymes has been found in eukaryotic proteins. For example, the catalytic domain of cell cycle control phosphatase Cdc25A (Fauman et al. 1998) displays a small {alpha}/ß fold identical to a metabolic enzyme rhodanese and a unique eukaryotic arsenate resistance enzyme ACR2 with a common active site containing the Cys-(X)5-Arg motif. These unexpected similarities between protein phosphatases and metabolic enzymes may reflect the process by which enzymes in metabolic pathways evolved in order to improve the function of the signal transduction systems.

The HPt domains are found in several microbial histidine kinases. Moreover, the HPt domain exists as an isolated protein such as yeast Ypd1p and Arabidopsis AHP1–AHP3 (Suzuki et al. 1998). In these organisms, unidentified SixA homologues may exist. Recently, a protein (14 kDa) that exhibits a histidine phosphatase activity has been isolated from rabbit liver. This enzyme, which shows no apparent sequence similarity to SixA, is ubiquitously expressed in mammalian tissues and is found to be localized in neurones in C. elegans (Klumpp et al. 2002). It is likely that genomic projects would reveal several candidates for protein histidine phosphatases, which may not be a SixA homologue. Further structural studies of these candidates as well as those of SixA homologues are essential for full understanding of His-Asp signalling pathways.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Protein preparation, crystallization and data collection

Protein expression and purification were carried out as previously described (Hamada et al. 1999). In brief, SixA (1–161 residues) was overproduced in E. coli BL21 (DE3) containing expression vector that codes the gene of SixA (Ogino et al. 1998). The protein was purified by an ammonium sulphate fractionation and column chromatography using DEAE-Sepharose FF, Mono-Q and Sephacryl S-100 HR (Amersham Biosciences). SixA crystals were obtained by vapor-diffusion method using a reservoir containing 6% PEG6K, 40 mM CaCl2, 100 mM Mes-Na (pH 6.6) at 10 °C. Diffraction data for native and derivative crystals were collected at 288 K on a Rigaku imaging-plate area-detector (R-AXIS IIc) using Cu-K{alpha} radiation and processed by the accompanying software PROCESS. The crystals diffract to 2.06 Å and belong to space group P212121 with unit cell dimensions of a = 39.26 Å, b = 48.62 Å, c = 83.18 Å, having one molecule in the crystallographic asymmetric unit (Hamada et al. 1999). Crystals of the tungstate-bound SixA were obtained by soaking native crystals into a solution containing 2 mM WO4Na2, 14% PEG6K, 40 mM CaCl2, 100 mM Mes-Na (pH 6.6) for 1 day at 10 °C. For diffraction studies, the crystals were flash-frozen in an oil solution containing 1 : 1 molar ratio of Paraton-N and Paraffin Oil (Hampton Research). Intensity data for this complex crystal were collected at 100 K on the imaging-plate area-detector (R-AXIS IV) and processed with the programs DENZO and SCALEPACK (Otwinowski & Minor 1997). The crystal of the complex is almost isomorphous to the free form with unit cell dimensions of a = 39.13 Å, b = 47.74 Å, c = 81.96 Å, and diffracted to 1.90 Å resolution.

Structure determination and refinement

Heavy atom derivatives were prepared by soaking crystals for 1 day in solutions of either 1 mM Na2IrCl6 or 1 mM UO2 (NO3)2, for 12 days in 0.5 mM NaAu(CN)2, and for 4 days in 0.2 mM Pb(CH3COO)2. Multiple isomorphous replacement phases to 2.5 Å were calculated with MLPHARE (CCP4, 1994) and were improved using DM (Cowtan & Main 1996). A model was built into the MIR electron density maps with the program O (Jones et al. 1991) and refined by simulated annealing with the program X-PLOR (Brünger 1992). Using the obtained structure of the free form, the structure of the tungstate-bound form was solved by the molecular replacement with the program AMoRe (Navaza 1994) and refined with the program CNS (Brünger et al. 1998). The present structure consists of 156 residues out of 161 residues. Five C-terminal residues could not be modelled because of poor density, indicating flexibility. As defined in PROCHECK (Laskowski et al. 1993), no residue was in the disallowed regions of the Ramachandran plots. In both crystal forms, a Ca2+ ion was found to bridge two symmetry-related SixA molecules. Four of the octahedral coordination ligands were water molecules and two were the main-chain carbonyl groups of Asp65 and Gly99, one of which was a symmetry-related molecule. Since both of the residues are located on the molecular surface away from the active site, we believe that the Ca2+ ion helps in the crystallization of the protein, but has no functional significance for the enzyme activity. The ribbon and surface representation of the proteins were drawn using the program MOLSCRIPT (Kraulis 1991) and GRASP (Nicholls et al. 1991), respectively.

Model building studies

The initial docking model between SixA and the HPt domain of ArcB was built using the program O in consideration of molecular complementarity and proper arrangement of two active histidine residues. The active site of SixA is located at the grooved molecular surface, which would match with the convexity of the kidney-like shape of the ArcB HPt domain (Kato et al. 1997) (Fig. 3C). The ArcB helix D was docked into the groove so as to fit the modelled phospho-His residue of ArcB to the active His residue of SixA. Then, the model was refined by the CNS program without x-ray terms for side-chain energy minimizations and removal of steric clashes along the interface. Compared with the docking model between the ArcB HPt domain and SixA model, the CheA HPt domain (Mourey et al. 2001) is also docked to SixA as similar manner.


   Acknowledgements
 
This work was supported by Grants in Aid for Scientific Research (KAKENHI) from the MESSC of Japan to TM (10179104) and TH (09308025, 10359003, 10179103–4). KH, MK and KI were supported by research fellowships for Young Scientists from the JSPS. The work was also supported by a Protein 3000 project on Signal Transduction from the MESSC of Japan.


   Footnotes
 
Accession Numbers: Atomic coordinates have been deposited with the Protein Data Bank under accession code 1UJB for the free form and 1UJC for the tungstate-bound form of SixA.

Communicated by: Hiroji Aiba

* Correspondence: E-mail: hakosima{at}bs.naist.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Campbell, J.W., Watson, H.C. & Hodgson, G.I. (1974) Structure of yeast phosphoglycerate mutase. Nature 250, 301–303.[CrossRef][Medline]

CCP4 (Collaborative Computational Project, 4) (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 760–763.[CrossRef][Medline]

Cowtan, K.D. & Main, P. (1996) Phase combination and cross validation in iterated density-modification calculations. Acta Crystallogr. Sect. D Biol. Crystallogr. 52, 43–48.[CrossRef][Medline]

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Received: 26 August 2004
Accepted: 26 October 2004




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