Article No. jmbi.1999.3261 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 294, 9±15

COMMUNICATION

Phosphorylated Aspartate in the Structure of aResponse Regulator Protein

Richard J. Lewis1, James A. Brannigan1, KatarõÂna Muchova 2

Imrich BaraÂk2 and Anthony J. Wilkinson1*

1Structural Biology LaboratoryDepartment of ChemistryUniversity of York, YorkYO10 5DD, UK2Institute of Molecular BiologySlovak Academy of SciencesDuÂbravska cesta 2184251, Bratislava, SlovakRepublic

E-mail address of the correspondajw@yorvic.york.ac.uk

0022-2836/99/460009±7 $30.00/0

Phosphorylation of aspartic acid residues is the hallmark of two-component signal transduction systems that orchestrate the adaptiveresponses of micro-organisms to changes in their surroundings. Two-component systems consist of a sensor kinase that interprets environmen-tal signals and a response regulator that activates the appropriate physio-logical response. Although structures of response regulators are known,little is understood about their activated phosphorylated forms, due tothe intrinsic instability of the acid phosphate linkage. Here, we report thephosphorylated structure of the receiver/phosphoacceptor domain ofSpo0A, the master regulator of sporulation, from Bacillus stearothermophi-lus. The phosphoryl group is covalently bonded to the invariant aspartate55, and co-ordinated to a nearby divalent metal cation, with both speciesful®lling their electrostatic potential through interactions with solventwater molecules, the protein main chain, and with side-chains of aminoacid residues strongly conserved across the response regulator family.This is the ®rst direct visualisation of a phosphoryl group covalentlylinked to an aspartic acid residue in any protein, with implications forsignalling within the response regulator family.

# 1999 Academic Press

Keywords: response regulator; signal transduction; sporulation;phosphoaspartate; structure

*Corresponding author

Protein phosphorylation is used by all livingcells as a mechanism of signal transduction andmetabolic regulation. A variety of amino acid resi-dues serve as phosphate acceptors, and the struc-tural basis of phosphotyrosine and phosphoserine/threonine signalling is emerging from the determi-nation of phosphoprotein structures (Johnson &O'Reilly, 1996). Histidine and aspartate phos-phorylation are at the heart of signalling in the ubi-quitous ``two-component'' systems of bacteria thatorchestrate a range of adaptive responses, includ-ing chemotaxis, virulence, antibiotic resistance,metabolite utilisation and development (Hoch &Silhavy, 1995). Two-component systems are beingdiscovered in yeast, fungi and plants (Chang &Stewart, 1998). In response to changes in the extra-cellular environment, the ®rst component, a sensorkinase, uses ATP to phosphorylate one of its histi-dine residues. This phosphoryl group is sub-

ing author:

sequently transferred to an aspartic acid residue onthe second component, the response regulator.This event serves as a signal to an effector domainthat elicits a physiological response. The sharedcharacteristic of response regulator proteins issequence similarity spanning some 120 residues intheir receiver domains, implying a common struc-ture (Stock et al., 1989). The N-terminal receiverdomain of Spo0A is connected by a linker peptideto a C-terminal DNA-binding/transcriptional acti-vator (effector) domain (Grimsley et al., 1994). Asthe effector domain is almost fully active uponremoval of the receiver domain, either by proteo-lysis or mutation, the receiver domain is thoughtto inhibit the effector domain in the intact protein.Phosphorylation of aspartate 55 overcomes thisautoinhibition (Ireton et al., 1993). If a thresholdlevel of Spo0A-phosphate accumulates in the cell,transcription of genes required for spore formationis activated and sporulation proceeds (Spiegelmanet al., 1995; Burbulys et al., 1991; Stragier & Losick,1996).

# 1999 Academic Press

10 Phosphorylated Response Regulator Structure

The three-dimensional structure of the receiverdomain of Spo0A, residues 2-130, (N-Spo0A) fromthe moderate thermophile Bacillus stearothermophi-lus was solved from crystals grown in the presenceof calcium (Muchova et al., 1999). The overall foldof N-Spo0A matches those of the receiver domainsof other response regulators whose structures havebeen determined (Volkman et al., 1995; Baikalovet al., 1996; Sola et al., 1999) (Figure 1; Table 1)including the chemotaxis factors CheY (Stock et al.,1989) and CheB (Djordjevic et al., 1998), and thesporulation factor Spo0F (Madhusudan et al., 1996).Each has a doubly wound a/b structure with acentral ®ve-stranded parallel b-pleated sheet sur-rounded by ®ve a-helices (Figure 1). The site ofphosphorylation, Asp55 in Spo0A, is located at theC terminus of strand-b3 in a pocket containing acluster of residues strongly conserved across theresponse regulator family.

In analysing the electron density maps, we werepuzzled by the presence and persistence of a well-de®ned tetrahedral feature, close to Asp55. Thiscould not be accounted for by protein atoms orany conventional arrangement of solvent mol-ecules, but it is consistent with a phosphorylgroup, covalently attached to the carboxylategroup of Asp55 and situated alongside a calciumion (Figure 2(a) and (b)). The latter occupies a simi-lar position to the Mg2� and Ca2� in the crystalstructures of CheY (Stock et al., 1993) and Spo0F(Madhusudan et al., 1996), respectively.

The structure reported here is the ®rst of a phos-phoryl group covalently attached to an asparticacid residue in a protein. In searching the Cam-bridge Structure Database, we found only onecrystal structure of a compound containing a car-boxyl group linked to a monoesteri®ed phosphate,that of phenylcarbamoyl phosphate (Weichsel &Lis, 1992). This is surprising, in view of the wide-spread use of acid phosphates as model systemsfor probing nucleophilic displacement reactions in

Table 1. Summary of data collection and re®nementstatistics

Space group P212121

Unit cell dimensions (AÊ ) a � 69.3, b � 73.0, c � 114.5Resolution range (AÊ ) 30-2.0Number of reflections

(total/unique) 173,534/39,774Rsymm (overall/last shell

2.03 to 2.00 AÊ ) 0.085/0.293Completeness (%) 99.9I/s(I) 17.5 (5.2)Rcryst (%) 20.0Rfree (%) 25.3rms deviation of bonds (AÊ ) 0.011rms deviation of angles (deg.) 1.8rms deviation of dihedrals (AÊ ) 0.032Average B-factor for main-

chain/side-chain atoms (AÊ 2) 20.2/26.0Average B-factor for solvent

molecules/Ca2� (AÊ 2) 33.4/15.7

enzymes (Koshland, 1952; DiSabato & Jencks, 1961;Herschlag & Jencks, 1990). The carboxyl phosphategeometry is the same in both the small moleculeand protein structures. The phosphoryl moiety andthe calcium ion in N-Spo0A make an extended setof complementary interactions, with each other,with the surrounding protein and with solventmolecules, so ful®lling their hydrogen bondingand electrostatic potential (Figure 2(b) and (c)). Thenet result is that the cation and phosphoryl moi-eties draw together the side-chains of the ®ve mosthighly conserved residues within the response reg-ulator family, through direct interactions in thecase of Asp10, Asp55, Thr84 and Lys106, andthrough a water-bridged interaction in the case ofAsp9. Since the remaining ligands are provided bywater molecules and protein backbone groups,divalent cation and phosphate co-ordination arelikely to be similar in all response regulators.

The presence of phosphorylated aspartate inheterologously expressed N-Spo0A protein wassurprising, and presumably the result of either pro-miscuous sensor kinase activity (Stock et al., 1995)or the action of small molecule phosphodonors(McCleary & Stock, 1994). Response regulatorsdephosphorylate spontaneously with half-timesthat vary from seconds for CheY-phosphate tohours for Spo0F-phosphate (Zapf et al., 1998). Sincepuri®cation and crystallisation of N-Spo0A tookplace over several days, the evident stability of theacid phosphate may be due to (i) the fact thatN-Spo0A is from the moderate thermophileB. stearothermophilus whose phosphorylated formmay be more stable at room temperature(Muchova et al., 1999); for example, the phosphory-lated form of the response regulator DrrA from thehyperthermophilic Thermotoga maritima has a half-life of three minutes at 80 �C and 24 hours at 25 �C(Goudreau et al., 1998), (ii) the replacement of thepresumed intracellular cofactor Mg2�, by Ca2�,which will alter the metal co-ordination numberfrom six to seven, lengthen the metal-ligand bonddistances and almost certainly perturb reactivity(Lukat et al., 1990) and (iii) the presence of the apo-lar side-chains of Ile57, Ala85 and Phe86, whichpartially close off the active site and restrict theaccess of water for in-line attack at the phosphorusatom. Substitutions of residues topologically equiv-alent to Ile57 in CheB and Spo0F have profoundeffects on the dephosphorylation kinetics (Zapfet al., 1998; Stewart, 1993).

The outstanding question concerning responseregulators is the nature of the conformationalchange upon phosphorylation that underlies theswitching mechanism. To address this question, wehave compared the structures of N-Spo0A � pho-sphate and the unphosphorylated forms of CheY(Stock et al., 1993) and Spo0F (Madhusudan et al.,1996) by least-squares superposition of theirb-sheet atoms. The average pair-wise rms� � 0.8 AÊ

based on 30 Ca atoms. These comparisons revealthat the loops around the active site connecting theb-strands to the a-helices take up a variety of

Figure 1. The overall fold of N-Spo0A � phosphate.The chain is colour ramped from the N (blue) to the Cterminus (red) with Ca2� in grey. The amino acidsequences of N-Spo0A from B. stearothermophilus andB. subtilis are closely similar, with 98 identities (includ-ing all the residues of the active centre) and 24 conser-vative substitutions (Muchova et al., 1999). RecombinantN-Spo0A was puri®ed from E. coli cells overexpressinga truncated spo0A gene as described (Muchova et al.,1999). Following disruption of the cells by sonication,the protein was puri®ed in steps of precipitation inammonium sulphate, phenyl-Sepharose chromatographyand MonoQ ion-exchange chromatography in Tris-buf-fered solutions at pH 7.5. Protein puri®ed in this wayproduced a mixed population of monomers and dimers,as judged by gel-®ltration chromatography. Orthorhom-bic crystals of N-Spo0A were grown overnight in hang-ing drops by vapour diffusion from Mops buffer(pH 6.5), 225 mM CaCl2. These crystals are in theorthorhombic space group P212121. No attempt wasmade to phosphorylate or dephosphorylate the proteinprior to crystallisation. It was only after the crystalstructure had been solved that we performed MALDI-TOF mass spectrophotometric analyses of the materialused for crystallisation, revealing the presence of twopeaks in the spectrum separated by �80 mass units,consistent with a covalently attached phosphoryl group.Current experiments are aimed at reconciling theunphosphorylated monomer and phosphorylated dimerexpected from studies in solution with the phosphory-lated monomer in the orthorhombic crystal form. Thestructure of N-Spo0A was initially solved in a hexagonalcrystal form by MAD phasing of selenomethionylderivative data extending to 2.5 AÊ resolution, whichallowed a model comprising residues 2-106 to be built(unpublished results). The orthorhombic structuredescribed here was solved by molecular replacementusing this partial co-ordinate set as a search model. Thisstructure was re®ned preferentially because higher-resol-ution data, extending to 2.0 AÊ spacing, were available(Muchova et al., 1999). The four independent moleculesof the asymmetric unit were easily positioned in MOL-REP (Vagin & Teplyakov, 1997) and the electron densitymaps were improved by 4-fold non-crystallographicsymmetry (NCS) averaging and solvent ¯attening usingthe CCP4 program DM (CCP4, 1994). At this point,the electron density corresponding to the phosphorylgroup and the calcium was clear. The model was builtand re®ned by maximum likelihood proceduresimplemented in REFMAC (CCP4, 1994), strict NCS

Phosphorylated Response Regulator Structure 11

conformations, thus making it hard to pin-pointstructural changes arising directly fromphosphorylation. One signi®cant feature of thesecomparisons, however, is the dramatic reorienta-tion of the Thr84 side-chain, whose hydroxylgroup has to move by �4.5 AÊ in order to contactthe phosphoryl group (Figure 3(a)). This move-ment is inevitably accompanied by alterations ofthe conformation of Phe103, which packs onto theactive site-distal face of Thr84 in N-Spo0A � phos-phate (Figure 3(b)). In CheY (Figure 3(b)) and otherresponse regulators, this side-chain is simplyoriented away from the core of the molecule (Stocket al., 1989, 1993; Volkman et al., 1995; Baikalovet al., 1996; Sola et al., 1999; Djordjevic et al., 1998;Madhusudan et al., 1996).

Concerted movements of residues correspondingto Thr84 and Phe103 have been implicated in sig-nalling by other studies (Usher et al., 1998;Djordjevic et al., 1998). In Spo0F, a key role forThr82 in controlling the conformational exchangeof His101 between buried and exposed positions issuggested by NMR data (Feher & Cavanagh,1999). In chemotaxis, it has been proposed that theconformation of Tyr106 governs the signalling sta-tus of CheY, because mutations of Tyr106 and/orThr87, leading to exclusively inward or exclusivelyoutward orientations of the aromatic side-chain atthis position, lead to hyperactive and inactive phe-notypes, respectively (Ganguli et al., 1995; Zhuet al., 1997). In the structure of CheB, the corre-sponding Phe104 is directed outwards with itsside-chain forming an integral component of ahydrophobic core formed by the packing of thereceiver domain onto the C-terminal effectordomain (Djordjevic et al., 1998). The outward toinward repositioning of Phe104 accompanyingphosphorylation could destabilise these interdo-main interactions and lead to release of the effectordomain and realisation of its methylesteraseactivity. The data presented here provide structuralevidence for a mechanism in which concerted

restraints were maintained for the main-chain atom pos-itions. Successive rounds of rebuilding and re®nementwere interspersed until re®nement converged. Thecalcium and phosphoryl species, built into the mapswhen the conventional crystallographic R-factor fellbelow 25 %, re®ned with full occupancies and with tem-perature factors similar to those of surrounding residuesin all four independent molecules. The calcium-liganddistances (2.4-2.7 AÊ ) fall within the range of valuesobserved in other calcium complexes. Water moleculeswere added in the programs ARPP and REFMAC, andmaintained unless they failed to satisfy standard criteria.The current model contains 3952 protein atoms includ-ing the aspartyl phosphate groups, 385 water moleculesand four calcium ions: 98.8 % of all residues fall in theallowed region of the Ramachandran plot.

Figure 2. The active site of N-Spo0A � phosphate. (a) Stereo view of the initial electron density map displayed onselected atoms from the ®nal re®ned model in the vicinity of the aspartyl phosphate (left). The map is calculated usingcoef®cients 2Fobs ÿ Fcalc using 4-fold non-crystallographic symmetry-averaged density modi®ed phases from the initialmolecular replacement solution. In these unbiased maps, electron density de®ning the phosphoryl group, the calciumion (green) and neighbouring water molecules is clear. (b) Stereo view of an Fobs ÿ Fcalc electron density omit map con-toured at 3.5s displayed around Asp55, the phosphoryl group and the calcium ion in the re®ned co-ordinate set. Thismap was calculated with the occupancies of the side-chain atoms of residue 55, the calcium ion and the nearby watermolecules set to zero. Atoms are coloured according to type; C (cyan), O (red), N (blue), P (yellow), Ca (grey) and water(green). (c) Stereo view orthogonal to (b). Hydrogen bonds are shown as broken lines, AP denotes the phosphorylatedAsp. The calcium ion is seven co-ordinate, its ligands being three water molecules, carboxylate oxygen atoms of Asp10and Asp55, a phosphoryl oxygen atom and the main-chain carbonyl oxygen atoms of Ile57. The phosphoryl oxygen

12 Phosphorylated Response Regulator Structure

Figure 3. Concerted movements of active site Thr andaromatic side-chains on phosphorylation. (a) Compari-son of the active sites of N-Spo0A � phosphate (cyanwith phosphorus in yellow), Spo0F (green) and CheY(pink). Oxygen atoms are coloured in red and thehydroxyl oxygen atoms of the threonine residues havebeen slightly enlarged. The opposing directions of thethreonine side-chain in the presence and absence of thephosphoryl group are apparent. (b) A comparison ofAsp55, Thr84 and Phe103 of Ca2�-bound N-Spo0A� phosphate (cyan) with the corresponding Asp57,Thr87 and Tyr106 of Mg2�-coordinated CheY (pink),emphasising the concerted movements uponphosphorylation. The structures were superimposed onthe main-chain atoms of 112 residues with an rms� of1.5 AÊ .

Phosphorylated Response Regulator Structure 13

movements of conserved active-site residuesinitiates signal propagation through the protein fol-lowing aspartate phosphorylation.

There is further evidence for the important roleof Thr84 in signalling from the structure of asecond, hexagonal crystal form of N-Spo0A inwhich aspartate 55 is not phosphorylated (unpub-

atoms make interactions with the Ca2�, the side-chains of Thr8and Ala85, and a water molecule. Phosphorylation is believeattack of the carboxylate group of Asp55 at the phosphoruDivalent cations are essential cofactors and probably facilitateon the anions, polarising the P-O bond and by forming a temp1980). The invariant Lys106 and the surrounding hydrogennegative charge that accumulates on the phosphate oxygen ato

lished results). In this structure, N-Spo0A is adimer, which is interesting because Spo0A, unlikeCheY and Spo0F, is believed to dimerise uponphosphorylation in solution (Asayama et al., 1995;our unpublished results). Comparison of the twoN-Spo0A structures suggests an obvious way inwhich changes in the active site can be coupled tochanges in quaternary structure invoking animportant role for Lys106 and Pro107 (unpublishedresults).

Phosphoaspartates have a high standard freeenergy of hydrolysis, a property that allowsresponse regulators to undergo large and energeti-cally favourable conformational changes withoutsacri®cing kinetic control of either their phos-phorylation or their dephosphorylation reactions(Stock et al., 1995). The interactions that stabilisethe aspartate phosphate species in the protein,favouring the net formation of the phosphorylatedregulator from ATP, are pivotal in achieving this.These interactions in N-Spo0A, and by inference inthe wider family of response regulators, are appar-ent from the structure presented here.

Phosphorylated aspartic acid residues are alsoreaction intermediates in enzymes such as the sar-coplasmic Ca2�-ATPase that pumps ions from thecytoplasm of eukaryotic cells. These P-typeATPases belong to the haloacid dehalogenase(HAD) superfamily of enzymes (Aravind et al.,1998). Sequence analysis and comparisons of thecrystal structures of CheY and L-2-haloacid dehalo-genase have led to the suggestion that responseregulators and the P-type ATPases and phospha-tases of the HAD superfamily have a commonactive-site architecture, metal co-ordination sphereand catalytic mechanism (Ridder & Dijkstra, 1999).This conserved active-site structure is obviouslyrobust as well as versatile. Sequence variations out-side this preserved active site shape the reactionkinetics according to the protein's function, whilethe conformational changes resulting from phos-phorylation in¯uence diverse aspects of cellularchemistry and physiology.

Accession codes

Co-ordinates and structure factors have beendeposited at the RCSB with the accession code1qmp.

4 and Lys106, the main-chain amide groups of Ile56, Ile57d to proceed by an in-line displacement mechanism withs atom of the histidine-phosphate on the sensor kinase.phosphoryl transfer variously through shielding charges

late for the trigonal bipyramidal transition state (Knowles,bond donor groups appear well-placed to balance the

ms during the reaction.

14 Phosphorylated Response Regulator Structure

Acknowledgements

This work was supported by The Wellcome Trust, theBBSRC, the Slovak Academy of Sciences and COPERNI-CUS. We thank Guy Dodson for his constant supportand enthusiasm, and Eleanor Dodson, Garib Murshu-dov, Jo Sutherland, George Spiegelman, Jim Hoch andN. Michael Green variously for useful discussions andcomment, and technical assistance.

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Edited by A. R. Fersht

(Received 23 August 1999; received in revised form 4 October 1999; accepted 6 October 1999)