Báo cáo khoa học: Structure of Streptococcus agalactiae serine⁄threonine phosphatase The subdomain conformation is coupled to the binding of a third metal ion pptx

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Structure of Streptococcus agalactiae serine⁄threoninephosphataseThe subdomain conformation is coupled to the binding of a thirdmetal ionMika K. Rantanen1, Lari Lehtio¨1, Lakshmi Rajagopal2, Craig E. Rubens2and Adrian Goldman11 Institute of Biotechnology, University of Helsinki, Finland2 Division of Infectious Disease, Children’s Hospital and Regional Medical Center, Seattle, WA, USAProtein phosphatases are primarily classified on thebasis of the type of the amino acid they dephosphory-late, serine ⁄ threonine phosphatases (STPs) act specific-ally on phosphoserine and phosphothreonine residues.Evolution has developed two main families of metallo-enzymes for this purpose, phosphoprotein phospha-tase P (PPP) and phosphoprotein phosphatase M(PPM) [1]. Based on sequence similarity, Streptococcusagalactiae STP (SaSTP) studied here belongs to aPPM subfamily called PP2C, because members of thissubfamily resemble human phosphoprotein phospha-tase 2C [1].Serine ⁄ threonine phosphorylation ⁄ dephosphoryla-tion is intimately linked with signaling events insidethe cell. Many STPs expand the scope of signaling byrecruiting additional domains into their structures.This is most common in PPP family enzymes, whereboth regulatory and targeting domains occur; forexample, in phosphoprotein phosphatase 5, the STPdomain is fused to four tetracotripeptide repeat pro-tein–protein interaction modules [2]. PPM ⁄ PP2Cenzymes may also have additional domains, for exam-ple, Arabidopsis thaliana ABI1 in which the catalyticdomain is fused to an EF-hand motif, and human STP(HsSTP), which has an additional 8 kDa a-helicaldomain at the C-terminus [3,4].The sizes of the catalytic domains of both PPP andPPM families are well conserved and structural studieshave revealed significant similarities between them [4–9]. Both utilize two b sheets to help the enzymes orientKeywordsdephosphorylation; serine ⁄ threoninephosphatase; signaling; Streptococcusagalactiae; structureCorrespondenceA. Goldman, Institute of Biotechnology,University of Helsinki, PO Box 65,00014, Helsinki, FinlandFax: +358 9191 59940Tel: +358 9191 58923E-mail: adrian.goldman@helsinki.fi(Received 31 January 2007, revised 20 April2007, accepted 25 April 2007)doi:10.1111/j.1742-4658.2007.05845.xWe solved the crystal structure of Streptococcus agalactiae serine ⁄ threoninephosphatase (SaSTP) using a combination of single-wavelength anomalousdispersion phasing and molecular replacement. The overall structure resem-bles that of previously characterized members of the PPM ⁄ PP2C STP fam-ily. The asymmetric unit contains four monomers and we observed twonovel conformations for the flap domain among them. In one of these con-formations, the enzyme binds three metal ions, whereas in the other itbinds only two. The three-metal ion structure also has the active site argin-ine in a novel conformation. The switch between the two- and three-metalion structures appears to be binding of another monomer to the active siteof STP, which promotes binding of the third metal ion. This interactionmay mimic the binding of a product complex, especially since the motifbinding to the active site contains a serine residue aligning remarkably wellwith the phosphate found in the human STP structure.AbbreviationsMtSTP, Mycobacterium tuberculosis serine ⁄ threonine phosphatase; PPM, phosphoprotein phosphatase M; PPP, phosphoproteinphosphatase P; SAD, single-wavelength anomalous dispersion; SaSTP, Streptococcus agalactiae serine ⁄ threonine phosphatase; STK,serine ⁄ threonine kinase; STP, serine ⁄ threonine phosphatase; TxSTP, Toxoplasma gondii STP.3128 FEBS Journal 274 (2007) 3128–3137 ª 2007 The Authors Journal compilation ª 2007 FEBStheir active site residues in a conformation where theybind active-site metal ions. The active-site ligands are,however, different; in PPP enzymes, histidine, aspar-tate and asparagine side chains bind the metal ions,whereas in PPM enzymes, aspartates and a glycinebackbone carbonyl coordinate the metal ions [4]. Theidentity of metal ions within the groups varies andstudies have sometimes shown slightly controversialresults [1]. The PPM ⁄ PP2C studied to date have beenshown to contain either Mg2+or Mn2+[1,4]. In addi-tion, in the crystal structure of Toxoplasma gondii STP(TxSTP; PDB code 2I44), the metals are modeled asCa2+ions (unpublished).Detailed biochemical analysis has revealed differ-ences between these enzymes. Only PPPs are inhibitedby the classical STP inhibitor okadaic acid [1].Although similar, the mechanisms of these enzymesare not identical, because PPM and PPP class enzymesbind their substrates differently. In the PPP family, thesubstrate phosphoryl group is bound directly to thetwo metal ions via its oxygen residues, whereasPPM ⁄ PP2C family enzymes bind the substrate indi-rectly, via hydrogen-bonding interactions between thephosphoryl group and water molecules liganded to themetal ions [4,10,11].The biochemistry of PPM ⁄ PP2C has been studiedextensively using the human enzyme as a model[4,10,11]. It relies on two divalent metal ions and anactivated bridging water molecule with a pKaof 7.5[11] to achieve catalysis, a common feature in hydro-lytic metalloenzymes [12]. One residue that appears totake part in catalysis in HsSTP is His62, which mayact as a general acid and protonate the phosphate as itleaves [11], but this residue is missing from the pro-karyotic Mycobacterium tuberculosis STP (MtSTP) [9];and from SaSTP. This implies that, in these enzymes,some other residue or a water molecule would act asthe general acid. HsSTP Arg33, conserved amongSTPs, has been proposed to take part in binding thephosphorylated protein substrate. The function ofother conserved residues near the active site remainsunclear [11]. Interestingly, MtSTP has been shown tobind a third metal ion near the active site [9]. A serineresidue that takes part in binding the third metal ion isnot conserved and the function of the third metal ionin MtSTP is unknown.Recently, serine ⁄threonine phosphorylation ⁄ dephospho-rylation has been shown to occur in many prokaryotes,where it modulates cellular activities analogously toevents found in eukaryotes. In Bacillus subtilis aPPM ⁄ PP2C STP activates sporulation transcriptionfactor [13,14]. M. tuberculosis contains many serine ⁄threonine kinases ( STKs), including serine⁄threonine pro-tein kinase G, which mediates survival of the bacteria[15]. Yersinia pseudotuberculosis and Yersinia enterocol-itica YpkA STKs induce the secretion of many Yopvirulence effector proteins [16,17], the Streptococcuspneumoniae stkP–strain has reduced infectivity in mice[18], and the Pseudomonas aeruginosa ppkA STK is nee-ded for virulence in mice [19]. S. agalactiae has an act-ive STP ⁄ STK system, which affects both the virulenceand morphology of the bacteria [20–22].The above-mentioned studies have thus shown thatserine ⁄ threonine signaling cascades are linked to thevirulence of organisms, leading to interesting possibilit-ies for rational drug design against these pathogens.Drugs targeting S. agalactiae signaling enzymes maycure many severe diseases, such as sepsis and menin-gitis, which threaten the lives of newborn babies andimmunocompromised adults. To support drug design,we need detailed structural information about the sign-aling proteins (STKs and STPs), and their complexeswith their downstream targets, which in S. agalactiaeinclude the response regulator CovR, adenylosuccinatesynthase and a family II inorganic pyrophosphatase(SaPPase) [20–22]. We have previously crystallized oneof the substrate molecules (SaPPase) [23]. Here wereport the crystal structure of SaSTP. The structurerevealed a third metal ion, as in MtSTP. However,unlike MtSTP, its presence correlates with binding ofanother STP monomer over the active site. This inter-action may resemble the dephosphorylated productcomplex.ResultsOverall structureThe structure of SaSTP was solved at 2.65 A˚resolu-tion. The model consists of residues (1–242) in all fourmonomers in the asymmetric unit. Additional residuesat the N-terminus (residues )4 to 0), introduced duringthe cloning step [23], were partly visible and the lastresidues of the polypeptide (residues 243–245) were notvisible in electron-density maps. SaSTP has an abbasandwich structure, consisting of two antiparallelb sheets packed against each other. The b sheets (b1and b2) are surrounded by a helices (Fig. 1A,B). Sim-ilar to other PPM ⁄ PP2C STPs, both b sheets consist ofantiparallel strands: these are formed by residues 1–8,120–125, 128–135, 186–190 and 233–240 in b1, and18–24, 30–38, 98–107, 110–116 and 175–180 in b2. b2is flanked by two long antiparallel a helices (43–61 and67–91). b1 is flanked by an a-helical region comprisedof three separate helices (192–195, 200–207and 213–226). The otherwise compact structure isM. K. Rantanen et al. Structure of S. agalactiae STPFEBS Journal 274 (2007) 3128–3137 ª 2007 The Authors Journal compilation ª 2007 FEBS 3129interrupted by a flap subdomain (132–174) (Fig. 1B).This flap has two short ahelices (138–144 and150–154). The four monomers found in the asymmetricunit are similar but not identical to each other, andthe largest deviations between them occur in this area(see below). Two conformations of the flap occur inthe crystal; monomers A and B share a similar confor-mation, as do monomers C and D. Notably, these twogroups also differ in metal content (see below).The rmsd ⁄ Ca values between different SaSTP mono-mers and known structures (PDB code 2I44) [4,9] showthat the SaSTP is a member of the PPM ⁄ PP2C STPfamily (Table 1). The core structure is well preservedin these enzymes. The rmsd values range between 1.16and 1.49 A˚for the core b sheets. For the whole struc-ture, the rmsd values are slightly higher, ranging from1.78 to 2.31 A˚. The evolutionarily less related TxSTPand HsSTP have remarkably different and larger flapdomains than SaSTP and MtSTP, which is why thenumber of aligned residues is lower (Fig. 1C). In addi-tion, both HsSTP and TxSTP have an extra strand inthe b sheets, so that the arrangement is 5 plus 6 [4](PDB code 2I44), and HsSTP also has a 75-residueC-terminal domain. Despite the low sequenceidentity (33%) between SaSTP and MtSTP, they areABCFig. 1. (A) Structure of the SaSTP (mono-mer C). The protein was drawn using a sec-ondary structure representation in which theprotein is colored from the N-terminus tothe C-terminus using a spectrum of colorsfrom blue to red. The three active site metalions (M1, M2 and M3) are shown as grayspheres. The N- and C-termini, and the twob sheets are labeled. (B) The structure hasbeen rotated through 90 ° around the y-axis.The flap subdomain is at right angles to thecore structure. (C) Comparison of SaSTP(magenta), TxSTP (cyan) and HsSTP (yellow).Metal ions are shown as spheres and coloredaccording to the corresponding protein. InSaSTP, the three metals are magnesium, thetwo metals in HsSTP are manganese and inTxSTP the two metals are calcium ions. Thisand the other figures were generated usingPYMOL [39] and GIMP (http://www.gimp.org).Table 1. rmsd values between different STP structures. Mono-mers A and D of SaSTP as well as the core b sheet of monomer Aare compared with A monomers of HsSTP (PDB code 1A6Q),MtSTP (PDB code 1TXO), and TxSTP (PDB code 2I44). The rmsdcalculations were carried out usingSSM alignment program [38]. Val-ues in parentheses show the number of aligned residues.MtSTP (A˚) HsSTP (A˚) TxSTP (A˚)SaSTP (A) 1.82 (223) 2.31 (212) 2.05 (208)SaSTP (D) 1.78 (223) 2.24 (209) 2.19 (207)SaSTP (A) b sheet only 1.21 (70) 1.49 (70) 1.16 (62)Structure of S. agalactiae STP M. K. Rantanen et al.3130 FEBS Journal 274 (2007) 3128–3137 ª 2007 The Authors Journal compilation ª 2007 FEBSstructurally closely related, both having a core of twofive-stranded sheets put together in an identical manner.Active siteOverall, the active-site organization of SaSTP is verysimilar to that of the previously characterizedPPM ⁄ PP2Cs. This was expected because the residuesforming the active site are very well conserved and canbe easily identified by sequence alignment. There arefour monomers in the asymmetric unit in the SaSTPstructure, and we found two catalytic metal ions (M1and M2) in all the active sites (Fig. 2A). Based on thecoordination and lack of an anomalous signal, wemodeled the metal ions as Mg2+ions (see Experimen-tal procedures). Despite modest resolution of the struc-ture, we observed all the coordinating water moleculesof the M1 and M2 metal ions, including the watermolecule bridging them. This water molecule ⁄ hydrox-ide ion is proposed to perform nucleophilic attack onthe phosphorus atom of the substrate [4,9]. Similarlyto MtSTP [9], there is no likely candidate residue for ageneral acid in the active site. This suggests that awater molecule may fill this role. Arg13, which hasbeen proposed to participate in binding of the sub-strate, is in a similar position to the equivalent residuein HsSTP (Arg33), where it binds Pifound in the act-ive site [4]. There are, however, important differencesin the SaSTP monomers in this area (see below).In addition to the M1 and M2 metal ions, weobserved an additional metal ion (M3) in two mono-mers of the asymmetric unit (monomers C and D;Fig. 2A). M3 was also modeled as a Mg2+ion,based on the same principles as for M1 and M2 (seeABFig. 2. (A) Stereoimage of the active site ofSaSTP monomer C. Residues are shown ascombination of cartoon and stick models.Mg2+ions are shown as gray spheres andwater molecules as blue spheres. The den-sity around the metal binding site is shownin blue mesh. The (Fo–Fc) omit map, con-toured at 3r was calculated after removingall the solvent atoms from the model.Before map calculation, the stripped modelwas refined for 20 cycles withREFMAC5.Coordinations for the metal ions and forAsn160 are indicated by dashed lines. (B)Superimposition of monomers A and C ofSaSTP, and MtSTP monomer A [9]. Mono-mer C of SaSTP is magenta as in Fig. 2A,monomer A is blue and MtSTP is gray.Metal ions and nucleophilic water moleculesare shown as spheres and colored accordingto the protein to which they are bound. The‘additional’ metal, M3, is present in SaSTPmonomer C and in MtSTP. In SaSTP all themetals are Mg2+, whereas in MtSTP theyare Mn2+ions. Arg13 and Asn160 (Ser inMtSTP) are shown as sticks.M. K. Rantanen et al. Structure of S. agalactiae STPFEBS Journal 274 (2007) 3128–3137 ª 2007 The Authors Journal compilation ª 2007 FEBS 3131Experimental procedures). M3 is coordinated byAsp118, Asp192 and four water molecules (Fig. 2A).Asp192 thus bridges metals M1 and M3. The positionof M3 between the flap subdomain and the active siteis analogous to the third metal, Mn2+, found inMtSTP [9]. In order to bind the metal ion, the flapdomain moves away from the core and the main chainconformation, especially that of the first helix, changes(Fig. 2B). In MtSTP, in addition to Asp118 andAsp191 (192 in SaSTP), M3 is coordinated by both thehydroxyl and the carbonyl groups of Ser160. Theequivalent residue in SaSTP is Asn160, and it does notparticipate directly in metal coordination (Fig. 2A).Because most of the M3 ligands in SaSTP are watermolecules, M3 in SaSTP may be less tightly boundthan the M3 in MtSTP.Crystal contacts at the active siteThe differential occurrence of the metal ions correlateswith the conformation of the flap subdomain (132–174)and the ‘binding’ of adjacent STP monomers over theactive sites of monomers C and D, but not of A and B(Fig. 3A). For monomer C, which contains three metalions, the monomer ‘binding’ over the active site ismonomer A. Similarly, in monomer D the ‘binding’molecule is monomer B. This crystal packing induceschanges in the flap and near Arg13 (Fig. 3B), and corre-lates with binding of M3. Residues 147–157 of the flap,including a second short helix (150–156) in monomer A,bind to the active site of monomer C. The interactionsin this contact include two salt bridges, Arg12(C)–Glu151(A) and His41(C)–Glu152(A), and three directhydrogen bonds Ser14(C)–Glu152(A), Arg13(C)–Ser155(A), and Ile162(C)–Pro157(A) (Fig. 3B). Theseinteractions may give clues as to how SaSTP interactswith its substrate (see below). Another crystal contact isformed by the flap subdomain of monomer C, but thiscontact region only contains a single hydrogen bond:Gln147(C) hydrogen bonds to the carbonyl group ofHis226(A). In monomers A and B, where His41 doesnot participate in a salt bridge, the area around His41 ispoorly defined by the electron density.The Arg13 side chain shows two different conforma-tions in the active site. In the monomer A and B struc-tures, its conformation is similar to HsSTP, where itbinds phosphate (Fig. 2B) [4]. In monomers C and D,Arg13 adopts a new conformation; the side chain isrotated so that the guanidine group points away fromthe active site. Interestingly, Arg13 now binds a serineresidue (Ser155) in the monomer bound to the activesite (Fig. 3B). This feature might be related to themechanism of the enzyme (see Discussion).DiscussionOverall structureThe structure of SaSTP is very similar to that ofMtSTP, with an rmsd ⁄ Ca of 1.8 A˚overall (Table 1).All the secondary structural elements are conservedbetween these enzymes, but there is variance, partic-ularly in the conformation of the flap subdomain (seebelow). Interestingly, other PP2C-family STPs solvedto date show variable numbers of strands in their corestructure. HsSTP contains an additional b strand atthe N-terminus extending the b1 sheet by one strand(Fig. 1). TxSTP also contains a similar, albeit muchshorter, extra strand. With respect to SaSTP andMtSTP, there are other clear differences in the TxSTPstructure at the first two N-terminal helices, of whichthere are three in TxSTP. The helices are also longer.The major difference within the PPM ⁄ PP2C STP struc-tural family resides in the flap subdomain (Fig. 1C).The flap in TxSTP contains the antiparallel strands inaddition to the two helices found in SaSTP andMtSTP. Furthermore, the flap is in a totally differentconformation. In HsSTP, the flap consists of practi-cally a single helix, because the second helical elementinvolves only couple of residues.The MtSTP crystal structure revealed for the firsttime a third metal ion in the active site of aPPM ⁄ PP2C STP and a flap domain conformationvery different to that in HsSTP [9]. This raises thequestion of whether the difference in conformation ofthe flap domain is related to binding of the thirdmetal ion or the substrate. The ligands for Mn3 inMtSTP were Asp118, Asp191 and Ser160, the lastcoming from the flap domain [9]. There are homolog-ous residues to Asp118 and Asp191 in HsSTP(Asp146 and Asp239) and, of course, in SaSTP(Asp118 and Asp192). Our SaSTP crystal structureprovides clear evidence that the flap subdomain is amobile element. Furthermore, its conformation islinked with the binding of the third metal ion sug-gesting, like Pullen et al. [9], that rearrangement ofthe flap domain in HsSTP might lead to binding of athird metal ion. The implications of the M3 bindingand flap subdomain conformations to the catalyticmechanism are discussed below.The role of the third metal in catalysisIn MtSTP [9], Ser160 takes part in binding M3 metal byforming two of the coordinating interactions. In SaSTP,this residue has been replaced by Asn160 (Fig. 2B). Thisresidue is in two different conformations, depending onStructure of S. agalactiae STP M. K. Rantanen et al.3132 FEBS Journal 274 (2007) 3128–3137 ª 2007 The Authors Journal compilation ª 2007 FEBSthe overall conformation of the flap subdomain. Theconformations are correlated with the presence orabsence of M3, but Asn160 does not bind directly toM3. Asn160 is, however, the closest flap domain resi-due to the M3 binding site. Because the M3 bindingsite is linked to the conformation of the flap, M3might also exist in eukaryotic enzymes such as HsSTP.However, it was not observed in that structure [4], andan even larger movement of the flap domain would berequired than the one observed in SaSTP. It wouldalso require that the Asp146–Lys165 ion pair locatedin a position similar to the M3 site in SaSTP would bebroken.However, the most important reason to doubt thepresence of an M3 site in eukaryotic STPs concernsthe presence of a general acid. In HsSTP, His62 hasbeen shown to be the general acid protonating theleaving group [11], but this residue is missing fromprokaryotic STPs. However, our structure and are-evaluation of the data presented by Pullen et al. [9]Fig. 3. (A) Stereoimage showing the packinginteractions between selected monomers(A and C) that mimics substrate binding.Monomer C (magenta) binds a peptide fromthe loop of monomer A (blue) to the activesite. Metal ions are shown as gray spheres.Notably, monomer A binds two metals,whereas monomer C binds three. (B) Close-up view of crystal contact indicating thepotential position of phosphate based onHsSTP. Coloring is as in (A) except that thenucleophilic (Wn) and putative general acid(WH) water molecules are shown as bluespheres, and residues participating in theinteractions are shown as sticks. The directinteractions across the interface are indica-ted by dashed lines. The phosphate found inHsSTP was modeled into SaSTP by aligningthe HsSTP and SaSTP monomers activesites. In the alignment metal ions M1 andM2, Wn, and carboxylate groups of Asp36,Asp192 and Asp231 were used. Alignmentof 12 atoms resulted in an rmsd value of0.3 A˚. As shown, the modeled position ofthe phosphate from HsSTP is close toSer155(A) and Arg13(C). Also, the generalacid in HsSTP, His62, is shown as stickswith gray carbon atoms.M. K. Rantanen et al. Structure of S. agalactiae STPFEBS Journal 274 (2007) 3128–3137 ª 2007 The Authors Journal compilation ª 2007 FEBS 3133suggest that a water molecule may play this role inprokaryotic STPs. First, WH(Fig. 2A) is $4A˚awayfrom the phosphate modeled by transferring thecoordinates from the HsSTP structure. Because WHiscoordinated to the M3 metal, its pKavalue should besignificantly less than 14, and so should be capable ofprotonating the phosphate leaving group. This is alsoconsistent with the binding of M3 in SaSTP when aputative substrate-mimic loop binds into the active site(see below). Second, when the residues binding themetal ion in MtSTP are mutated, the Kmvalue formetal binding was little changed. These mutationsincluded the S160A mutation, making the MtSTPbinding site similar to SaSTP. As our structure shows,this sequence does indeed bind metal ion at M3 – andthe largest change they report in all their mutants atthis site is less than a factor of four greater than wild-type (3.7–14.6 mm) [9]. Consequently, their measure-ments of MtSTP activity in the presence of 100 mmMn2+actually reflect enzyme activity with three metalions present, and so the fact that mutations at the M3site do not change kcatis unsurprising. There is prob-ably no change in active site contents.The WHin prokaryotic enzymes is located on theopposite side of the substrate phosphate in comparisonwith His62 in HsSTP (Fig. 3B), but at similar distancesfrom the phosphate found in HsSTP structure. Thenucleophilic water bridging metals M1 and M2 are ata reasonable distance (3.8 A˚) from the phosphorousatom when compared with the HsSTP structure. Thephosphate is bound indirectly to the metal ions viawater molecules and the Wn–P–O angle (146°) is rea-sonably close to optimal when the roughness of theanalysis is taken into account.SaSTP has been shown to dephosphorylate three dif-ferent substrates. These are a family II inorganic pyro-phosphatase, a response regulator CovR, and a purinebiosynthesis protein PurA [20–22]. Although the exactsite of phosphorylation in these proteins is not known,it has been shown that SaPPase is phosphorylated at aserine residue [20]. Interestingly, we observed a crystalcontact in SaSTP structure which involves the flap sub-domain in the adjacent monomer. This contact corre-lates with binding of the M3 and, intriguingly, places aserine residue (Ser155) from a neighboring monomerclose to the active site (Fig. 3B). An approximate loca-tion for the Pican be obtained by superimposing theHsSTP structure [4], which contains a phosphate ionin the active site on SaSTP. When we did so, we foundthat Ser155 is located rather close to the phosphatewith an Oc-P distance of only 3.8 A˚.At the crystal contact of SaSTP, Ser155 also formsa hydrogen bond to the very same residue (Arg13),which is responsible for binding the phosphoserineresidue in vivo [11]. This interaction suggests that thecrystal structure might mimic a product complex.Although it is possible that the serendipitous interac-tion is only an artifact, it is apparently strong enoughto appear in the crystal and adjust the enzyme in aninduced-fit-like manner: the conformation of the flapdomain changes, M3 metal is introduced and Arg13turns so that it is binding the serine residue in the ‘sub-strate’. To our knowledge, this is the first time that thebinding of a protein component at the active site hasbeen described for a STP. Given that the interactionwould resemble that of the actual complex, we wereable to identify a sequence motif of the substrate mole-cule among the actual substrates of SaSTP. The motif,[ED]-hydrophil-X(1,2)-[ST]-X-P, allows similar inter-actions to those described here and is present in theS. agalactiae PPase, kinase and adenylosuccinate syn-thase, but not in S. agalactiae CovR ⁄ CsrR. The motifis located at the surface of the SaPPase (Rantanen,unpublished), and superposition of the prolines allowssuperposition of the serine Ocs – but not the rest ofthe putative motif. We are currently attempting to con-firm the site of phosphorylation using biochemicalmethods.Experimental proceduresData collection and processingCloning, expression, purification, crystallization and dataprocessing of the native protein have been described else-where [20,23]. The reported 2.65 A˚native data set wasprocessed using the program xds [24] and the crystal wasassigned to space group P21212 with four monomers perasymmetric unit (Table 1). We also produced selenomethio-nine-labeled protein, because molecular replacement withthe best available molecular replacement probe (MtSTP [9]with 33% sequence identity) was not successful. The sele-nomethionine-labeled protein was produced using the sameconstruct and purification protocol as for wild-type.Expression was, however, performed in M9 minimal med-ium (6.0 g Na2HPO4, 3.0 g KH2PO4, 1.0 g NH4Cl, 0.5 gNaCl per L), supplemented with 2 mm MgSO4, 0.2%glucose, 0.5 · 10)3% thiamine, 0.1 mm CaCl2, and100 lgÆmL)1ampicillin. Just before induction, we addedtwo mixtures of amino acids to shut down the biosyntheticpathways leading to methionine: lysine, threonine and phe-nylalanine at a concentration of 100 lgÆL)1and leucine,isoleucine and valine at a concentration of 50 lgÆL)1. Sim-ultaneously, we added selenomethionine at a concentrationof 60 lgÆL)1, and after 15 min, we started induction byadding 1 mm isopropyl thio-b-d-galactoside. Cells weretransferred to 25 °C and harvested after 16 h. PurificationStructure of S. agalactiae STP M. K. Rantanen et al.3134 FEBS Journal 274 (2007) 3128–3137 ª 2007 The Authors Journal compilation ª 2007 FEBSand crystallization were performed as reported previously[20,23]. The protein yield was, however, considerably lowerthan for wild-type SaSTP (40 mgÆL)1) [23]; we obtainedonly 5 mg of the protein per L of culture. MS analysis anda fluorescence scan showed that labeling the protein hadsucceeded (data not shown). Suppliers for reagents were asfollows: ampicillin, thiamine and glucose, Sigma-Aldrich(St Louis, MO ⁄ YA-Kemia OY, Helsinki, Finland);Na2HPO4,KH2PO4and CaCl2, JT Baker (Phillipsburg,NJ); NH4Cl, NaCl and MgSO4, Sigma-Aldrich; and aminoacids, Merck ⁄ Calbiochem (Darmstad, Germany).The labeled SaSTP crystal diffracted to 2.50 A˚resolu-tion. We collected single-wavelength anomalous dispersion(SAD) data at the selenium peak wavelength (0.97935 A˚)and analyzed it using xds [24] (Table 2). Data were collec-ted at 100 K using radiation from the European Syncho-tron Radiation Facility tunable-wavelength beam lineID23-1. A total of 225° data were collected with 0.5° oscil-lation per frame. Data had an overall Rmerged(F) of 6.9% inspace group P212121(Table 1) [25]. Crystallization condi-tions were identical between native and selenomethionine-labeled crystals, but although the crystals were still primit-ive orthorhombic, the unit cell parameters and space grouphad changed with respect to the native data (Table 2).We used the programs shelxc, shelxd and shelxe inthe hkl2map program suite to solve the structure of SaSTP[26,27]. We found 12 sites, which suggested two monomersin the asymmetric unit as expected from the Matthews coef-ficient (Vm¼ 2.4 A˚3ÆDa)1) [28]. Correlation coefficientsfrom shelxd were 50 and 28%, and the Patterson figure ofmerit was 19 (resolution cut-off 3.0 A˚). Subsequent phasingwith shelxe resulted in a correlation coefficient of 54%and allowed us to build a model of the protein into theelectron density map. The model was, however, ratherincomplete; 20% of the residues were either missing ormodeled in a wrong conformation (checked against thefinal coordinates) due to the poor quality of the maps, andrefinement with refmac5 [29] stalled at R-factors of 26 and30%. This may be due to real disorder in the crystal as themean B-factor from the refinement was rather high(78.5 A˚2) – as was the Wilson B-factor (77 A˚2). It shouldbe noted that the data did not show signs of twinning andother possible space groups were also tested for refinement.The structure that we obtained using SAD-phasing is repor-ted only to clarify the structure solution process – thenative data set was ultimately used to derive the structurethat we present here (see below).We used the best model we built to the experimental elec-tron density as a model in molecular replacement to the2.65 A˚native data (Table 2) [23]. Molecular replacementwas performed using the molrep [30] program of the ccp4package [30,31] using the ccp4i graphical interface [32]. TheMatthews coefficient [28] indicated four monomers in theasymmetric unit (Vm¼ 2.7 A˚3ÆDa)1) and molrep was ableto find all of them. The rotation searches initially hinted atthe possible presence of the protein in two different confor-mations: two rotations had Rf⁄ sigma values of 9.6, andthe following two had lower values (7.8 and 6.7). InitialR-factor after the translation searches was 49.4% with acorrelation coefficient of 0.394.Model building and refinementWe refined the monomers using refmac [29] to an R-factorof 19.7% (for 5% test reflections Rfree¼ 27.1%). Modelbuilding between refinement cycles was done with coot[33]. Because of the modest resolution and large asymmetricunit, we used strict NCS constraints to keep all four mono-mers similar at the beginning of the refinement. Duringrefinement, it became apparent that there were differencesbetween the molecules and finally NCS restraints with thedefault medium weight was applied between monomers Aand B as well as between C and D. The stereochemistry ofthe final model is good as indicated by the Ramachandranplot calculated with procheck [34]: 94.6% of the residuesare in the most favored region, the rest of the residues inother allowed regions (Table 3).PPM class STPs are metalloenzymes and we found 2–3metal ions bound near the active site in all monomers. Weassigned these ions as Mg2+for the following reasons. Thecrystallization solution contained 0.2 m magnesium acetate,making Mg2+the most likely candidate. The coordinationis octahedral, which is typical for Mg2+[35–37] and thecoordinating distances (1.83–2.47 A˚) are close to those seenfor Mg2+. We did not find any peaks in the anomalousmaps at the metal binding sites (calculated at wavelengthsof 0.979 and 0.933 A˚) excluding the other metal ion,Mn2+, seen in PPM STPs. Furthermore, the B-factors ofTable 2. Data collection statistics for native [23] and selenomethio-nine-labeled STP. Values in parentheses are for the highest resolu-tion shell.Native SelenomethionineSpace group P21212P212121Wavelength (A˚) 0.933 0.97935Unit-cellparameters (A˚)a ¼ 91.8,b ¼ 139.0,c ¼ 86.7a ¼ 49.1,b ¼ 74.9,c ¼ 137.3Resolution (A˚) 20–2.65 (2.7–2.65) 20–2.5 (2.60–2.50)Reflections measured 169847 (7368) 160419 (18194)Unique reflections 32767 (1731) 33057 (3688)Completeness (%) 99.5 (97.5) 97.8 (98.1)Redundancy 5.2 (4.3) 4.9 (4.9)I ⁄ r (I) 11.7 (3.7) 14.2 (3.2)s-norm ⁄ s-ano 1.02 (1.04) 1.26 (1.00)Rmerged-Fa(%) 11.5 (43.7) 6.9 (49.8)aRmergedÀF¼PAIðh;P ÞÀ AIðh;QÞ¼ =0:5PðAIðh;P ÞÀ AIðh;QÞÞ, whereAI¼ffiffiIpif I ‡ 0 AI¼ÀffiffiIpif I <0. P and Q are two subsets of data[25].M. K. Rantanen et al. Structure of S. agalactiae STPFEBS Journal 274 (2007) 3128–3137 ª 2007 The Authors Journal compilation ª 2007 FEBS 3135the metal ions are close to the B-factors of the coordinatingresidues (16–42 versus 27–40 A˚2).AcknowledgementsWe would like to thank Professor Arto Annila forfunding. We acknowledge the European SynchotronRadiation Facility for provision of synchrotron radi-ation and we would like to thank Didier Nurizzo forassistance in using beam line. This study was suppor-ted by grants from the Sigrid Juselius Foundation, andfrom the Academy of Finland (grant number 1105157)to AG, who is a member of the Biocentrum Helsinkiresearch organization. It was also supported by theNational Institutes of Health, Grant # RO1 AI056073to CER and CHRMC Basic Science Steering Commit-tee Award to LR.References1 Barford D, Das AK & Egloff M-P (1998) The structureand mechanism of protein phosphatases: insights intocatalysis and regulation. 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Nature 376, 745–753.6 Griffith JP, Kim JL, Kim EE, Sintchak MD, ThomsonJA, Fitzgibbon MJ, Fleming MA, Caron PR, Hsiao K& Navia MA (1995) X-Ray structure of calcineurininhibited by the immunophilin–immunosuppressantFKBP12–FK506 complex. Cell 82, 507–522.7 Egloff M-P, Cohen PTW, Reinemer P & Barford D(1995) Crystal structure of the catalytic subunit ofhuman protein phosphatase 1 and its complex withtungstate. J Mol Biol 254, 942–959.8 Egloff M-P, Johnson DF, Moorhead G, Cohen PTW,Cohen P & Barford D (1997) Structural basis for therecognition of regulatory subunits by the catalytic sub-unit of protein phosphatase 1. EMBO J 16, 1876–1887.9 Pullen KE, Ng HL, Sung PY, Good MC, Smith SM &Alber T (2004) An alternate conformation and a thirdmetal in PstP ⁄ Ppp, the M. tuberculosis PP2C-familySer ⁄ Thr protein phosphatase. Structure 12, 1947–1954.10 Jackson MD & Denu JM (2001) Molecular reactions ofprotein phosphatases – insights from structure andchemistry. Chem Rev 101, 2313–2340.11 Jackson MD, Fjeld CC & Denu JM (2003) Probing thefunction of conserved residues in the serine ⁄ threoninephosphatase PP2Calpha. Biochemistry 42, 8513–8521.12 Christianson DW & Cox JD (1999) Catalysis by metal-activated hydroxide in zinc and manganese metallo-enzymes. Annu Rev Biochem 68, 33–57.13 Yang X, Kang CM, Brody MS & Price CW (1996)Opposing pairs of serine protein kinases and phospha-tases transmit signals of environmental stress to activatea bacterial transcription factor. Genes Dev 10, 2265–2275.14 Adler E, Donella-Deana A, Arigoni F, Pinna LA &Stragler P (1997) Structural relationship between a bac-terial developmental protein and eukaryotic PP2C phos-phatases. Mol Microbiol 23, 57–62.15 Walburger A, Koul A, Ferrari G, Nguyen L, Prescian-otto-Baschong C, Huygen K, Klebl B, Thompson C,Bacher G & Pieters J (2004) Protein kinase G frompathogenic mycobacteria promotes survival withinmacrophages. Science 304, 1800–1804.16 Hakansson S, Galyov EE, Rosqvist R & Wolf-Watz H(1996) The Yersinia YpkA Ser ⁄ Thr kinase is translocatedTable 3. Structure refinement statistics for SaSTP against the nativeP21212 and SAD data. SAD data collected at 0.97935 A˚wavelength.Native SelenomethionineSpace group P21212P212121Resolution range (A˚) 19.68–2.65 10–2.50NCS restraints A to B & C to D noneNumber of reflectionsRwork⁄ Rfreea31351 ⁄ 1568 15989 ⁄ 799Atoms (total ⁄ water ⁄ metal) 7780 ⁄ 298 ⁄ 10 3508 ⁄ 16 ⁄ 7Rwork(%) ⁄ Rfree(%) 19.7 ⁄ 27.1 26.1 ⁄ 29.9rmsd bond length (A˚) 0.010 0.021rmsd bond angle (O) 1.3 2.78Mean B-value (A˚2) 42.1 78.5Mean B-value (A˚2), metalions29.8 95.0Mean B-value (A˚2), watermolecules28.2 66.5Ramachandran plotResidues in most favoredregion (%)94.6 71.9Residues in additionallyand generouslyallowed regions5.4 25.3aRwork¼ (P|Fobs) Fcalc|)/(P|Fobs|), where Fobsand Fcalcareobserved and calculated structure factor amplitudes, respectively.Rfreeis an R-factor for an unrefined subset of the data (5% of thedata).Structure of S. agalactiae STP M. K. Rantanen et al.3136 FEBS Journal 274 (2007) 3128–3137 ª 2007 The Authors Journal compilation ª 2007 FEBSand subsequently targeted to the inner surface of theHeLa cell plasma membrane. Mol Microbiol 20, 593–603.17 Galyov EE, Hakansson S, Forsberg A & Wolf-Watz H(1993) A secreted protein kinase of Yersinia pseudotuber-culosis is an indispensable virulence determinant. Nature361, 730–732.18 Echenique J, Kadioglu A, Romao S, Andrew PW &Trombe MC (2004) Protein serine ⁄ threonine kinaseStkP positively controls virulence and competence inStreptococcus pneumoniae. Infect Immun 72, 2434–2437.19 Wang J, Li C, Yang H, Mushegian A & Jin S (1998) Anovel serine ⁄ threonine protein kinase homologue ofPseudomonas aeruginosa is specifically inducible withinthe host infection site and is required for full virulencein neutropenic mice. J Bacteriol 180, 6764–6768.20 Rajagopal L, Clancy A & Rubens CE (2003) A eukar-yotic-type serine ⁄ threonine kinase and phosphatase inStreptococcus agalactiae reversibly phosphorylate aninorganic pyrophosphatase and affect growth, cell segre-gation and virulence. J Biol Chem 278, 14429–14441.21 Rajagopal L, Vo A, Silvestroni A & Rubens CE (2005)Regulation of purine biosynthesis by a eukaryotic-typekinase in Streptococcus agalactiae. Mol Microbiol 56,1329–1346.22 Rajagopal L, Vo A, Silvestroni A & Rubens CE (2006)Regulation of cytotoxin expression by converging eukar-yotic-type and two-component signalling mechanisms inStreptococcus agalactiae. Mol Microbiol 62, 941–957.23 Rantanen MK, Lehtio¨L, Rajagopal L, Rubens CE &Goldman A (2006) Crystallization and preliminary crys-tallographic analysis of two Streptococcus agalactiaeproteins: the family II inorganic pyrophosphatase andthe serine ⁄ threonine phosphatase. Acta Crystallogr F 62,891–894.24 Kabsch W (1993) Automatic processing of rotation dif-fraction data from crystals of initially unknown sym-metry and cell constants. J Appl Crystallogr 26, 795–800.25 Diederichs K & Karplus PA (1997) Improved R-factorsfor diffraction data analysis in macromolecular crystal-lography. Nat Struct Biol 4, 269–275.26 Sheldrick G & Schneider T (1997) SHELXL: high-reso-lution refinement. Methods Enzymol 277, 319–343.27 Pape T & Schneider TR (2004) HKL2MAP: a graphicaluser interface for macromolecular phasing withSHELX-programs. J Appl Crystallogr 37, 843–844.28 Matthews BW (1968) Solvent content of protein crys-tals. J Mol Biol 33 , 491–497.29 Murshudov GN, Vagin AA & Dodson EJ (1997) Refine-ment of macromolecular structures by the maximum-like-lihood method. Acta Crystallogr D 53, 240–255.30 Vagin AA & Teplyakov A (1997) MOLREP: an auto-mated program for molecular replacement.J Appl Crys-tallogr 30, 1022–1025.31 CCP4 (1994) The CCP4 suite: programs for proteincrystallography. Acta Crystallogr D 50, 760–763.32 Potterton E, Briggs P, Turkenburg M & Dodson EJ(2003) A graphical user interface to the CCP4 programsuite. Acta Crystallogr D 59, 1131–1137.33 Emsley P & Cowtan K (2004) Coot: model-buildingtools for molecular graphics. Acta Crystallogr D 60 ,2126–2132.34 Laskowski RA, MacArthur MW, Moss DS & ThorntonJM (1993) PROCHECK: a program to check the stereo-chemical quality of protein structures. J Appl Crystal-logr 26, 283.35 Harding MM (2000) The geometry of metal–ligandinteractions relevant to proteins. II. Angles at the metalatom, additional weak metal–donor interactions. ActaCrystallogr D 56, 857–867.36 Harding MM (1999) The geometry of metal–ligandinteractions relevant to proteins. Acta Crystallog D 55,1432–1443.37 Harding MM (2001) Geometry of metal–ligand inter-actions in proteins. Acta Crystallogr D 57, 401–411.38 Krissinel E & Henrick K (2004) Secondary-structurematching (SSM), a new tool for fast protein structurealignment in three dimensions. Acta Crystallogr D 60,2256–2268.39 Delano WL (2002) The PyMOL Molecular GraphicsSystem. Delano Scientific, Palo Alto, CA.M. K. Rantanen et al. Structure of S. agalactiae STPFEBS Journal 274 (2007) 3128–3137 ª 2007 The Authors Journal compilation ª 2007 FEBS 3137[...]... maps at the metal binding sites (calculated at wavelengths ˚ of 0.979 and 0.933 A) excluding the other metal ion, Mn2+, seen in PPM STPs Furthermore, the B-factors of FEBS Journal 274 (2007) 3128–3137 ª 2007 The Authors Journal compilation ª 2007 FEBS 3135 Structure of S agalactiae STP M K Rantanen et al Table 3 Structure refinement statistics for SaSTP against the native ˚ P21212 and SAD data SAD data... for an unrefined subset of the data (5% of the data) 8 a 9 the metal ions are close to the B-factors of the coordinating ˚ residues (16–42 versus 27–40 A2 ) 10 Acknowledgements We would like to thank Professor Arto Annila for funding We acknowledge the European Synchotron Radiation Facility for provision of synchrotron radiation and we would like to thank Didier Nurizzo for assistance in using beam line... Crystallization and preliminary crystallographic analysis of two Streptococcus agalactiae proteins: the family II inorganic pyrophosphatase and the serine ⁄ threonine phosphatase Acta Crystallogr F 62, 891–894 Kabsch W (1993) Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants J Appl Crystallogr 26, 795–800 Diederichs K & Karplus PA (1997)... 26, 283 35 Harding MM (2000) The geometry of metal ligand interactions relevant to proteins II Angles at the metal atom, additional weak metal donor interactions Acta Crystallogr D 56, 857–867 36 Harding MM (1999) The geometry of metal ligand interactions relevant to proteins Acta Crystallog D 55, 1432–1443 37 Harding MM (2001) Geometry of metal ligand interactions in proteins Acta Crystallogr D 57,... indicated four monomers in the ˚ asymmetric unit (Vm ¼ 2.7 A3 ÆDa)1) and molrep was able to find all of them The rotation searches initially hinted at the possible presence of the protein in two different conformations: two rotations had Rf ⁄ sigma values of 9.6, and the following two had lower values (7.8 and 6.7) Initial R-factor after the translation searches was 49.4% with a correlation coefficient of. .. 2–3 metal ions bound near the active site in all monomers We assigned these ions as Mg2+ for the following reasons The crystallization solution contained 0.2 m magnesium acetate, making Mg2+ the most likely candidate The coordination is octahedral, which is typical for Mg2+ [35–37] and the ˚ coordinating distances (1.83–2.47 A) are close to those seen for Mg2+ We did not find any peaks in the anomalous... metalactivated hydroxide in zinc and manganese metalloenzymes Annu Rev Biochem 68, 33–57 Yang X, Kang CM, Brody MS & Price CW (1996) Opposing pairs of serine protein kinases and phosphatases transmit signals of environmental stress to activate a bacterial transcription factor Genes Dev 10, 2265– 2275 Adler E, Donella-Deana A, Arigoni F, Pinna LA & Stragler P (1997) Structural relationship between a. .. Regulation of purine biosynthesis by a eukaryotic-type kinase in Streptococcus agalactiae Mol Microbiol 56, 1329–1346 Rajagopal L, Vo A, Silvestroni A & Rubens CE (2006) Regulation of cytotoxin expression by converging eukaryotic-type and two-component signalling mechanisms in Streptococcus agalactiae Mol Microbiol 62, 941–957 Rantanen MK, Lehtio L, Rajagopal L, Rubens CE & ¨ Goldman A (2006) Crystallization... between the molecules and finally NCS restraints with the default medium weight was applied between monomers A and B as well as between C and D The stereochemistry of the final model is good as indicated by the Ramachandran plot calculated with procheck [34]: 94.6% of the residues are in the most favored region, the rest of the residues in other allowed regions (Table 3) PPM class STPs are metalloenzymes and... R-factors of 26 and 30% This may be due to real disorder in the crystal as the mean B-factor from the refinement was rather high ˚ ˚ (78.5 A2 ) – as was the Wilson B-factor (77 A2 ) It should be noted that the data did not show signs of twinning and other possible space groups were also tested for refinement The structure that we obtained using SAD-phasing is reported only to clarify the structure solution process . Structure of Streptococcus agalactiae serine⁄threonine phosphatase The subdomain conformation is coupled to the binding of a third metal ion Mika K implications of the M3 binding and flap subdomain conformations to the catalyticmechanism are discussed below. The role of the third metal in catalysisIn MtSTP
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Xem thêm: Báo cáo khoa học: Structure of Streptococcus agalactiae serine⁄threonine phosphatase The subdomain conformation is coupled to the binding of a third metal ion pptx, Báo cáo khoa học: Structure of Streptococcus agalactiae serine⁄threonine phosphatase The subdomain conformation is coupled to the binding of a third metal ion pptx, Báo cáo khoa học: Structure of Streptococcus agalactiae serine⁄threonine phosphatase The subdomain conformation is coupled to the binding of a third metal ion pptx