A 1.8 Å resolution structure of pig muscle 3-phosphoglycerate kinase with bound MgADP and...

doi:10.1006/jmbi.2000.4294 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 306, 499±511

A 1.8 AÊ Resolution Structure of Pig Muscle3-Phosphoglycerate Kinase with Bound MgADPand 3-Phosphoglycerate in Open Conformation:New Insight into the Role of the Nucleotide inDomain Closure

Andrea N. SzilaÂgyi1, Minakshi Ghosh2, Elspeth Garman2 and MaÂria Vas1*

1Institute of EnzymologyBiological Research CenterHungarian Academy ofSciences, P.O. Box 7H-1518, Budapest, Hungary2Laboratory of MolecularBiophysics, Department ofBiochemistry, University ofOxford, South Parks RoadOxford OX1 3QU, UK

E-mail address of correspondingAbbreviations used: PGK, 3-phos

0022-2836/00/030499±13 $35.00/0

3-Phosphoglycerate kinase (PGK) is a typical kinase with two structuraldomains. The domains each bind one of the two substrates, 3-phospho-glycerate (3-PG) and MgATP. For the phospho-transfer reaction to takeplace the substrates must be brought closer by a hinge-bending domainclosure. Open and closed structures of the enzyme with different relativedomain positions have been determined from different species, but acomprehensive description of this conformational transition is yet to beattained.

Crystals of pig muscle PGK in complex with MgADP and 3-phospho-glycerate were grown under the conditions which have previouslyresulted in crystals of the closed, catalytically competent conformation ofTrypanosoma brucei PGK. The X-ray structure of the pig muscle ternarycomplex was determined at 1.8 AÊ and the model was re®ned toR � 20.8 % and Rfree � 24.1 %. Contrary to expectation, however, it rep-resents an essentially open conformation compared to that of T. bruceiPGK. In addition, the b-phosphate group of ADP is mobile in the newstructure, in contrast to its well-de®ned position in T. brucei PGK. Anextensive comparison of the ternary complexes from these remote specieshas been carried out in order to establish general differences between thetwo conformations and is reported here. A second pair of the open andclosed structures was also compared. These analyses have made it poss-ible to de®ne several characteristic changes which accompany the struc-tural transition, in addition to those identi®ed previously: (1) theoperation of a hinge at b-strand L in the inter-domain region whichgreatly affects the relative domain positions; (2) the rearrangement andmovement of helix 8, regulated through the interactions with the nucleo-tide phosphate; and (3) the existence of another hinge between helix 14and the rest of the C-terminal part of the chain, which allows ®ne adjust-ment of the N-domain position.

The main hinge at b-strand L acts in concert with the C-terminal hingeat helix 7 described previously. Simultaneous interactions of the nucleo-tide phosphate groups with the loop that precedes helix 8, b-strand J andthe N terminus of helix 13 are required for propagation of the nucleotideeffect towards the b-strand L molecular hinge. A detailed description ofthe role of nucleotide binding in the hinge operation is presented.

# 2001 Academic Press

Keywords: domain motion; nucleotide-effect; phosphoglycerate kinase;source-speci®city; X-ray structure
*Corresponding author
author: [email protected] kinase; 3-PG, 3-phosphoglycerate.

# 2001 Academic Press

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500 Structural basis of domain movements

Introduction

Relative movement of protein domains has oftenbeen deduced from structural studies (see, forexample, Zhang et al. (1995) and reviews byGerstein & Krebs (1998); Gerstein et al. (1994);Hayward (1999); and Wriggers & Schulten (1997)).Domain ¯exibility seems to be essential for variousreactions catalysed by enzymes; these include sev-eral members of the kinase family, a ubiquitousgroup of enzymes (Matte et al., 1998). However,the structural principles that govern domainmotion are not always understood. Attempts havebeen made to classify the motion at the level ofsupersecondary structure (e.g. by de®ning hingesor rotational axis). However, there are not manydetailed descriptions of these large-scale confor-mational changes in terms of side-chain and back-bone interactions.

3-Phosphoglycerate kinase (PGK) is a mono-meric enzyme (molecular mass � 45 kDa) with twowell-separated structural domains of about equalsize (Banks et al., 1979; Blake & Rice, 1981; Watsonet al., 1982). PGK is an essential enzyme for thebasic metabolism of various living organisms: gly-colysis of aerobes, fermentation of anaerobes andcarbon ®xation of plants. It catalyses the phospho-transfer from 1,3-bisphosphoglycerate to MgADPto form 3-phosphoglycerate (3-PG) and MgATP.Due to the instability of 1,3-bisphosphoglycerate,the reverse reaction is usually investigated. Crys-tallographic studies have shown that 3-PG binds tothe N-terminal, while the nucleotide substrateMgATP, or the product MgADP, binds to theC-terminal domain (Banks et al., 1979; Watson et al.,1982; Harlos et al., 1992; Davies et al., 1994).

There are several pieces of evidence whichsuggest movement of the two domains during thecatalytic cycle, bringing the two substrates intoclose proximity for catalysis and thus allowing adirect phospho-transfer between them. Early crys-tallographic studies of horse (Banks et al., 1979),yeast (Watson et al., 1982), pig (Harlos et al., 1992;May et al., 1996) and of the bacterial Bacillus stear-othermophilus (Davies et al., 1994) PGKs, however,showed the enzyme in the open conformation. The®rst crystallographic indication of PGK domainmobility came from the 2.0 AÊ resolution structureof a binary complex with 3-PG, which showed adomain rotation of only about 8o (Harlos et al.,1992). Structural evidence for more completedomain closure of PGK has only recently been pro-vided by two independent crystallographic studiesof PGK from two independent species: Trypanoso-ma brucei (Bernstein et al., 1997) and a thermophilicbacterium, Thermotoga maritima (Auerbach et al.,1997). The 2.5 AÊ resolution structure of T. bruceiPGK complexed with 3-PG and MgADP (Bernstein& Hol, 1998), showed the molecule in a fully closedconformation, accomplished by a domain rotationof about 32o. The transferring phospho-groupcould be modelled into the active site, in agree-ment with the assumed direct in-line phospho-

transfer (Bernstein et al., 1997). The 2.0 AÊ resolutionstructure of T. maritima PGK complexed with thesubstrate 3-PG and MgAMP-PNP, an analogue ofthe substrate MgATP, showed the enzyme in asimilar, but not completely closed conformation(Auerbach et al., 1997).

In the above reports, important structuralelements involved in domain motion have beenidenti®ed. From the study of T. brucei PGK, twoindependent hinge points (called C and N-hinges)have been noted at the ends of helix 7 which con-nects the two domains, as has slight bending ofthis helix (Bernstein et al., 1998, 1997). Small shiftsof two other helices in the inter-domain regionhave also been observed: helix 14 relative to theN-terminal domain and helix 13 relative to theC-terminal domain. In the structure of T. maritimaPGK, a reorientation of the loop between helix 14and the connecting b-strand L has been observedand is thought to be an element of the close contactformation of the two domains that completes thecatalytic site (Auerbach et al., 1997).

While these results provide direct structural evi-dence for domain closure, several questions remainunanswered: (1) what is the relationship, if any,between operation of the hinge-points at the endsof helix 7 and the reorientation of the conservedactive site loop between helix 14 and b-strand L;and (2) what structural changes direct such move-ments, which occur far away from the substrate-binding site? In order to answer these questions, adetailed comparison of the open and closed confor-mations is required.

For the pig muscle enzyme, we have previouslydetermined a partially closed structure of a binarycomplex with 3-PG at 2.0 AÊ resolution (Harloset al., 1992), as well as the structure of a pseudo-ternary complex obtained upon diffusion of thenucleotide analogue MnAMP-PNP into the crystalof this binary complex (May et al., 1996). The tern-ary complex was similar to the binary complexexcept for some local conformational changes. Therelative orientation of the two domains remainedunchanged, possibly due to the crystal contactswhich may have prevented the occurrence of anylarge-scale structural changes.

Here we grew crystals of a ternary complex ofthe pig muscle enzyme in the presence of 3-PG andMgADP, using the same conditions that have pre-viously produced crystals of T. brucei PGK ternarycomplex in the closed conformation. The aim wasto make a systematic comparison of the binary(open) and ternary (presumably closed) complexesof pig muscle PGK, i.e. to compare different con-formations of the enzyme from the same source,since sequence differences of up to 50 % existbetween PGKs from different sources. (Watson &Littlechild, 1990; Fleming & Littlechild, 1997). Wealso wished to compare the structures of PGK-tern-ary complexes from these different sources, inorder to identify their species-independentcommon features.

Structural basis of domain movements 501

Results and Discussion

Crystal structure of the new PGK ternarycomplex: open conformation is associatedwith the mobility of the nucleotidebbb-phosphate group

Crystals (space group P21) of pig muscle PGKwere grown in the presence of the substrate 3-PGand the product MgADP. X-ray diffraction datafrom these crystals have led to a re®ned structureof a ternary complex, PGK*3-PG*MgADP, at 1.8 AÊ

resolution with R � 20.8 % and Rfree � 24.1 %.The gross features of the ternary complex mol-

ecule, determined here, are shown in Figure 1(a)together with the arrangement of secondary-structural elements and the positions of the boundsubstrates, which are similar to the known PGKstructures. The sequence as deduced from the pre-sent X-ray data is compared with some otherknown sequences of PGK (Banks et al., 1979;Harlos et al., 1992; May et al., 1996) in Figure 1(b).The most remarkable feature of the new ternaryPGK structure is the open domain conformation,an unexpected characteristic compared to theclosed conformation of T. brucei enzyme (Bernsteinet al., 1997; Figure 2(a) and (b)). However, asexpected, the substrate (3-PG) and the nucleotidebind to the N and C-terminal domains, respect-ively (cf. Figures 1 and 2). The open conformationof the new structure and the binding mode of 3-PGare very similar to the previous structure of pigmuscle PGK pseudo-ternary complex withMnAMP-PNP and 3-PG (May et al., 1996). Forcomparison, another closed structure of the ternarycomplex of T. maritima PGK with MgAMP-PNPand 3-PG (Auerbach et al., 1997) is also shownin Figure 2 and represents an intermediatestate between the pig muscle and T. brucei PGKstructures.

All atoms of the bound 3-phosphoglyceratecould be built into the electron density map. At thenucleotide site on the C-domain, no density couldbe seen for the b-phosphate group of ADP. At lowcontour levels (0.6-0.7 s) of the map, there is anindication of a possible O-P bond to this b-phos-phate group, but nothing beyond. AMP could bewell modelled into the observed density with sev-eral bound water molecules around it (Figure 3(a)).However, the presence of ADP in the crystal hasbeen con®rmed by enzymatic determination (cf.Materials and Methods), implying that the b-phos-phate group of ADP must be mobile in this ternarycomplex. This is in marked contrast to the well-de®ned position of the nucleotide phosphategroups in its T. brucei counterpart (Bernstein et al.,1997). The previously determined pig muscle PGKternary complex contained bound MnAMP-PNP in

{ Numbering of residues throughout the text refers tothe pig muscle PGK sequence unless quoted differently,as e.g. Bs (B. stearothermophilus), Tb (T. brucei) or Tm(T. maritima).

the place of MgADP and electron densities couldbe well assigned to all three phosphate groups ofthe nucleotide (May et al., 1996). Apart from this,the positions and the interactions of the adenine,ribose and the a-phosphate group of the twodifferent nucleotides are largely similar in the newand in the previous pig muscle ternary complexes(Figure 3(b) and (d)).

The mobility of the b-phosphate group of ADPis perhaps coupled with the uncertainty in theassignment of the magnesium ion, which is knownto form a complex with the nucleotide phosphategroups. The 1.6 AÊ resolution structure of thebinary complex of B. stearothermophilus PGK withMgADP (Davies et al., 1994) showed that Mg2�

forms a well-de®ned bridge between two phos-phate O atoms of ADP and Asp Bs352 (equivalentto Asp374 of helix 13){. A similar observation hasbeen made of the interactions of Mg2� in theternary complex of T. brucei PGK with MgADPand 3-phosphoglycerate (Bernstein et al., 1997)(Figure 3(c)). In the present structure of pig musclePGK, there is density in the neighbourhood of theAsp374 carboxyl oxygen atom and the nucleotidea-phosphate group, which has been tentativelyassigned to a Mg2� (Figure 3(a) and (b)). No atomscould be found inside the known co-ordinationsphere of this Mg2�, i.e. within 2.0-2.2 AÊ (Harding,1999). The nearest phosphate group oxygen atomof ADP is at 2.99 AÊ . Therefore, this density is morelikely to be that of a water molecule than amagnesium ion.

Thus, although pig muscle PGK and T. bruceiPGK have been crystallised under similar con-ditions, the structures show differences in boththeir overall conformation and in the details of thenucleotide binding. The open conformation is themost surprising aspect, as the simultaneous bind-ing of 3-PG and MgADP has failed to force theenzyme to adopt a closed conformation. It is worthnoting that the pig muscle enzyme has yet to becrystallised in a closed catalytic conformation. Inall the pig muscle PGK structures determined sofar, the distances between the two bound ligandsare larger than those in the closed ternarymolecules (cf. Figure 2(a) and (b)).

The primary sequences of T. brucei andT. maritima PGKs, both observed in their closedconformations, differ by up to about 50 % from themuscle enzymes, although the tertiary structure ofPGK is highly conserved during evolution (Moriet al., 1986; Watson & Littlechild, 1990; Fleming &Littlechild, 1997). The sequence differences mayin¯uence the state of the equilibrium between theopen and closed states, by stabilising one confor-mation over the other through formation of differ-ent protein-protein contacts in the crystal lattice.On the other hand, dynamic light-scattering stu-dies with solubilised pig muscle PGK have shownthat the presence of both substrates stabilises amore compact protein conformation, compatiblewith the assumed closed conformation (Sinev et al.,1989).

Figure 1. X-ray structure-based sequence alignment of PGKs of different origin. (a) Schematic representation of thepresent pig muscle PGK ternary complex with the bound ligands, showing the arrangement and labelling of the varioussecondary structural elements. (b) Aligned PGK sequences derived by superimposing the secondary structural elements(one by one) of the current pig (pig1) ternary complex with pig pseudo-ternary (pig2) complex (May et al., 1996), horsemuscle (Banks et al., 1979), T. brucei ternary complex (Tb; Bernstein & Hol, 1998) and T. maritima ternary complex (Tm;Auerbach et al., 1997). The pig PGK sequences are derived from X-ray structures. The residues in bold are those whichare observed to be different from the known horse PGK sequence and we have underlined those where the electron den-sity maps are fairly conclusive regarding the change from the horse PGK sequence. The asterisks mark the residues con-served in almost all of the known (about 90) PGK sequences (Barton, 1990). Locations of the secondary-structuralelements are labelled above the sequence.


Figure 2. Comparison of the domain positions in thePGK ternary complexes. The Ca traces of four PGK tern-ary complexes: those of 3-PG and MgADP with the cur-rent pig muscle (red, pig1) and of the T. brucei (green;Bernstein & Hol, 1998); as well as those of 3-PG andMn/MgAMP-PNP with pig muscle (orange, pig2; Mayet al., 1996) and of T. maritima (blue; Auerbach et al.,1997) PGKs are shown superimposed according to thebackbone atoms of the core b-strands of (a) the N-domain and (b) the C-domain, respectively. The boundligands are shown as stick models. In (a) the inter-domain helix 7, and in (b) the b-strand L, are high-lighted by ribbons of the respective colours.


However, all PGK species should temporarilyadopt both open and closed conformations in sol-ution during the catalytic cycle. The hinges whichare operational during the domain movementshould therefore be present in all the species, irre-spective of the differences in sequence. Here, wecompare two pig muscle ternary complexes withcorresponding closed structures from remotespecies and our aims are as follows: (1) to locate allthe hinges involved in the transition between theopen and the closed states, (2) to identify differ-ences between the interactions of the substrates

with the enzyme in the two states and (3) if poss-ible, to describe the effect of the substrate inter-actions on the conformational state of the hinge(s).The structures are compared in the following pairs:Pair 1, pig muscle (current work, open) and T. bru-cei (closed) PGKs complexed with MgADP and 3-PG and crystallised in the presence of phosphate;Pair 2, pig muscle (open) and T. maritima (closed)PGKs, another pair of ternary complexes, but withMg (or Mn) AMP-PNP instead of MgADP andcrystallised in the presence of PEG. New featuresof domain closure have emerged from these com-parisons and are outlined below.

A new important hinge at bbb strand L

A superimposition of all four PGK ternary com-plexes optimising the alignment of the backboneatoms of the core b-strands in the N and C-term-inal domains, respectively, is shown in Figure 2(a)and (b). The conformation of each individualdomain, except for a few source-speci®c insertions,is similar in all structures even when the relativedomain positions are widely different. Thus, thedomains move almost as rigid bodies relative toeach other.

Of the two known hinge points at the ends ofhelix 7 suggested previously (Bernstein et al., 1997),only the conformation of the C-hinge is signi®-cantly different in the open and the correspondingclosed ternary structures (Figure 2(a)). This is con-®rmed by the r.m.s. deviation data of the superim-posed helix 7 plus C-domain core b-strands whichare much higher than the respective data obtainedby separate superimposition of these structuralelements (Table 1). Much smaller differences wereobserved in the data when helix 7 and theN-domain core b-strands were chosen for super-imposition, which indicated that in all four ternarycomplexes the N-hinge is similarly closed(cf. Figure 2(a)).

In addition to these hinges at each ends of helix7, we have observed the presence of anotherimportant hinge operating in b-strand L, as illus-trated in Figure 2(b). This strand connects helices13 and 14, and is situated between the twodomains. Helix 13 constitutes an integral part ofthe C-terminal domain, while helix 14 movestogether with the N-terminal domain. These fea-tures are supported by the r.m.s. deviation dataobtained from superposition of the respectivestructural elements (Table 1) which show that therelative movement of helices 13 and 14 is as largeas the relative movement of the C and N-terminaldomains observed when their core b-strands weresuperimposed. A smaller movement of helices 13and 14 relative to the C-terminal and N-terminaldomains, respectively, can be also deduced fromthe r.m.s. data in Table 1, in agreement with pre-vious observations (Bernstein et al., 1998).

The relative movement of helices 13 and 14 isdepicted in Figure 4(a) and (b), which also showthe domain rotations by movements of the respect-

Figure 3. Interactions of the nucleotide with the enzyme in the four ternary complexes. (a) The electron densitymap contoured at 1s and (b) the ball and stick model of the bound nucleotide and its surroundings in the presentpig muscle PGK structure (pig1, red). The hydrogen-bond distances including the bound water molecules are alsoindicated. The water molecule W 36 that connects Asn336 to the N terminus of helix 14 might mimic the position ofthe transferring phospho-group during catalysis. The ball and stick models of nucleotides as bound to the active sitesof the other PGK ternary complexes of (c) Tb (green), (d) pig pseudo-ternary (pig2, orange) and (e) Tm (blue) are alsoshown. Selected distances of the nucleotides from the surrounding conserved side chains and peptide backboneatoms are indicated in AÊ .


ive core b-strands. b-Strand L, which connectsthese two helices, must therefore undergo a largeconformational change. The r.m.s. deviation data(Table 1), support the existence of a signi®canthinge at this location.

In addition, through modelling studies (SzilaÂgyiet al., unpublished results), we have observed thatthe particular backbone conformation of the inter-domain region which is characteristic of the closedPGK molecules, and consists of part of helix 13and the b-strand L, can position the two domainscloser together. However this modelling has alsodemonstrated quite clearly that without operationof the hinges at helix 7, the polypeptide chainwould break. Therefore the new hinge region at b-strand L and the known hinges at helix 7 must actin concert, and both regions have important rolesin the positioning of the two domains.

Rearrangement and motion of helix 8

Superimposition of all four PGK structures tooptimise the alignment of various structuralelements of the two domains and of the inter-

domain region (Figure 4) has led to other newobservations. It is notable that all the relevantparts, highlighted by the ribbons, are well separ-ated into pairs according to the basic confor-mational state of the molecule.

The operation of the new putative hinge point inb-strand L results not only in the movement ofhelix 14, but also of the remaining part of theC-terminal end of the chain (helix 15 and theC-tail). This is clear in (Figure 4(a)), where the mol-ecules are superimposed according to helix 13. TheC-terminal end of the polypeptide chain (residues398-416) including helices 14 and 15 constitutes anintegral part of the N-domain. This feature of thePGK molecule has been known for a long time, butthe role it plays in the folding process (Mas et al.,1995; Ritco-Vonsovici et al., 1995; SzilaÂgyi & Vas,1998) or in the function (Ritco-Vonsovici et al.,1995; Zomer et al., 1998) has not yet been properlyunderstood. This type of structural organisationhas often been observed in proteins and has gener-al importance in enhancing domain-domain inter-actions (Schleif, 1999).

Table 1. Root-mean-square deviation of various pairs of superimposed elements of the compared open and closedstructures

rms deviation (AÊ /AÊ 2) in pairs of PGK structures

Superimposed part

Sequencenumbering

(muscle PGK)

Number ofsuperimposed

atomspig1 (present) and

T. brucei (Bernstein & Hol, 1998)

pig2, pseudoternary(May et al., 1996) and T. maritima

(Auerbach et al., 1997)

Helix 7 189-197 72 0.243 0.393C-domain 207-212 (G) 144 0.379 0.429(core b strands) 231-236 (H)

277-282 (I)Helix 7 � C-domain 216 1.167 1.18N-domain 17-22 (A) 136 0.414 0.390(core b strands) 57-61 (B)

91-96(C)Helix 7 � N-domain 208 0.578 0.428C-domain � N-domain 280 2.583 1.905

Helix 13 374-379 48 0.218 0.288Helix 14 399-403 40 0.721 0.350Helix 13 � C-domain 192 0.543 0.523Helix 14 � N-domain 176 0.659 0.486Helix 13 � Helix 14 88 1.946 1.386

b-Strand L 388-397 80 0.832 0.898

Helix 8 219-228a 80 1.279 0.959222-228 56 0.821 0.344

Helix 8 � C-domain 200 0.787 0.686Helix 8 � helix 13 104 1.203 0.727Helix 8 � helix 14 96 1.978 1.537

a Not used in further comparisons.


Figure 4(a) shows that helix 13 undergoes a con-formational change at its N terminus, which islocated close to the bound nucleotide phosphategroups, and the implication of this change isdiscussed below. At the same time the whole ofhelix 13 is shifted relative to helix 14 when themolecules are superimposed according to helix 14(Figure 4(b)).

Helix 8 moves considerably relative to theC-domain. Superimposition, optimising the overlapof the core b-strands of the C-domain (Figure 4(c)),clearly indicates that helix 8 moves more or lessindependently of this domain, although it constitu-tes an integral part of the C-domain (cf. also r.m.s.deviation data in Table 1). The pronounced move-ment of helix 8 occurs at a hinge between its N ter-minus and the C terminus of b-strand G, whichconnects helices 7 and 8. The operation of thishinge is directly mediated through an interactionwith the nucleotide, as shown below. The knownC-hinge of helix 7 is situated at the N terminus ofb-strand G and thus may be indirectly affected.

It is also evident from Figure 4(d) that helix 8undergoes substantial conformational changes,especially in its N-terminal part located close to thebound nucleotide. The superimposition, optimisedfor the C-terminal part of helix 8, shows clearlythat the ®rst turn of this helix is better formed inthe closed structures, but that the rest of it is moreordered in the open structures. The hydrogenbonds along this helix are signi®cantly weaker inboth closed structures, resulting in an elongation ofthe helix as a whole, in spite of the fact that its ®rstturn has became more ordered.

Helix 8 also moves relative to helix 14(Figure 4(e)). This motion brings helix 8 of theC-domain into a position more parallel with helix14 (which belongs to the N-domain) and thus theopen channel between the two domains can beclosed by complementary side-chain interactions,as we have already proposed (May et al., 1996).

From the superimposition which optimises theoverlap of helix 14, an additional hinge has beenobserved between this helix and the rest of theC-terminal part. The C-tail, including helix 15,moves together with the core b-strands of theN-domain, while helix 14 does not completelyfollow this motion (Figure 4(e)).

Thus, in addition to the new hinge point atb-strand L, which is mainly responsible fordirecting the relative domain positions, two hingepoints have been described in this section: oneassociated with the loop between b-strand G andhelix 8 and the other located between helix 14 andthe short helix 15 at the C terminus of the mol-ecule. These two hinge points play an importantrole in closing the channel between the domains.

Different interactions of the substrates in theopen and closed states

Understanding the spatial changes of the keysecondary structural elements mentioned above atthe level of side-chain interactions would provideinformation on the molecular mechanism of thedomain closure. We have thus analysed theenzyme-substrate interactions.

Figure 4. Important changes in the secondary structure between the open and closed states. Superimposition of thefour PGK ternary complexes: current structure (pig1, red), pseudo-ternary complex (pig2, orange), Tb (green) and Tm(blue) according to the peptide backbone atoms of (a) helix 13, (b) and (e) helix 14, (c) the core b-strands of the C-domain and (d) the C-terminal half of helix 8. For clarity, only the investigated secondary-structural elements areshown by ribbons of the same colour code and the bound ligands are also shown as stick models.


The interactions of 3-PG with protein side-chainsof the N-domain are very similar in all four com-plexes, in agreement with the observation that theextent of closure at the N-hinge of helix 7 is almostthe same. One noticeable exception in the openstructures is the absence of a 3-PG interaction withthe conserved Lys215 in the C-domain. In theclosed structures Tb219/Tm197 interacts with 3-PGas a consequence of the domain closure and hasbeen reported earlier (Auerbach et al., 1997).

At the nucleotide binding site in the newstructure, we have shown above that the ADPb-phosphate group is mobile. We presume that thismobility of the ADP b-phosphate group is relatedto the present open conformation of the molecule,stabilised by the lattice forces in the crystal. Incomparison between the two pairs of known struc-tures in open and closed conformation, there is aremarkable difference in the hydrogen-bondingnetwork which mediates interactions of the nucleo-tide with the loop preceding helix 8. In the closedPGK structures of T. brucei (Figure 3(c)) and T. mar-itima (Figure 3(e)), the peptide amide N atom ofAla Tb218/Tm196 forms three H-bonds; one withthe O atom of the nucleotide ribose, one with thephosphate group ester oxygen atom linked withthe C50 atom of the ribose and one with the nucleo-tide a-phosphate group. These interactions areeither absent or very weak in both the open pigmuscle PGK structures (Figure 3(b) and (d)), andalso in the open structure of B. stearothermophilus

PGK (Davies et al., 1994; not shown). From the dis-cussion above, helix 8 has been seen to be import-ant in closing the open channels between the twodomains. In the closed structures, therefore, thishydrogen-bond network may play a role inthe movement of helix 8 and consequently in thepositioning of the conserved Lys215 closer to thephosphate group of 3-PG.

For the closed structure of T. maritima PGK,strong interactions of the nucleotide g-phosphategroup with NZ of Lys Tm201 (helix 8) and withND2 of Asn Tm317 (b-strand J) have beenobserved (Figure 3(e)). Equivalent interactions aremuch weaker in the corresponding open structureof the pig muscle PGK pseudo-ternary complex(Figure 3(d)).

Other interactions of the nucleotide with thePGK molecule are found to be unaffected by thestate of domain closure. The hydrogen-bonding ofthe b-phosphate group O atoms to Asp374 (Tb377/Tm355) peptide amide N, as well as to the amideN and OH of Thr375 (Ser Tb378/Ser Tm356) donot change during domain movement. Theseconserved residues belong to helix 13. Further,the hydrogen-bond between an O atom of thea-phosphate group and Lys219 (Tb223 and Tm201)from helix 8 is present in all of the known PGK-nucleotide complexes, though it is relatively weak(3.48 AÊ ) in the pig PGK pseudo-ternary complex(Figure 3(d)).


We suggest here that the relative positions ofhelix 8 and helix 13 can be regulated through for-mation of these simultaneous contacts with thenucleotide. During domain closure the N terminiof these two helices not only move closer to eachother by between 1 and 2 AÊ , but are also modi®edby noticeable conformational changes, as shown inFigure 4.

Propagation of the nucleotide effect towardsthe main hinge point

We now investigate the consequences of thenucleotide interactions with the two helices 8 and13 described above, and show how these changesare transmitted towards the new hinge at b-strandL. Valuable insight is provided by comparing theinteractions that exist among the side-chainslocated near the nucleotide phosphate groups(Figure 5) in the open and closed structures.

First, the hydrogen bond between the conservedLys Tb223/Tm201 (NZ) from the N terminus ofhelix 8 and Asn Tbr338/Tm317 (OD1) from a coreb-strand (J) of the C-domain exists only in theclosed structures (Figure 5(a)). Due to the for-mation of this hydrogen bond, the salt bridge ofLys Tbr223/Tm201 with Asp Tbr222/Tm200 is dis-rupted, although it remains intact in the openstructures. Also, new hydrogen bonds are formedin the fully closed structure of T. brucei PGK,between the peptide N atom of Lys Tb223 and thepeptide O atoms of both Lys Tb219 and AspTb222. The distances between the equivalent atomsin the T. maritima PGK are also reduced comparedto the open structures, and are not much longer(only 0.5-0.8 AÊ ) than the length of ordinary hydro-gen bonds. Conversely, the hydrogen bondbetween Lys219 (NZ) and the peptide O atom ofLys215, observed in the new pig ternary structure(Figure 3(a)) does not exist in any of the closedstructures. These changes greatly in¯uence the con-formation of the polypeptide chain at and near theN terminus of helix 8.

As Asn Tb338/Tm317 forms simultaneous con-tacts with both Lys Tb223/Tm201 (NZ) and thenucleotide phosphate group, it moves closer to theN terminus of helix 13 (which is in direct contactwith the nucleotide) and forms a hydrogen bondwith the peptide O atom of Gly Tb374/Tm352(Figure 5(a)), i.e. with one member of a conservedGlyGlyGly triplet. This hydrogen bond is wellformed in the fully closed T. brucei PGK structure,but is relatively weak in the partially closed struc-ture of T. maritima PGK. The conformation of theN-terminal end of helix 13 is dramatically changedas a consequence. The new conformation is furtherstabilised by an additional hydrogen bond betweenGly Tb374/Tm352 (O) with Ser Tb378/Tm 355(OG1), a partially conserved side-chain that isreplaced by a threonine residue in the pigenzymes.

This leads to the disruption of the hydrogenbond between the peptide amide group N atom of

Gly Tb375/Tm353 (another member of the Gly tri-plet) and the conserved Ser Tb401/Tm379 (OG)from helix 14 in the closed structures (Figure 5(b));helix 14 moves away from helix 13, towards b-strand L, by at least 2 AÊ , and allows the formationof a new hydrogen bond in both the closed struc-tures. In T. brucei PGK the new bond is formedbetween Ser Tb401 (OG) of helix 14 and Ser Tb395(OG) of b-strand L, and in T. maritima PGK it isbetween Gly Tm353 (N) of helix 13 and the peptideO atom of Ser373. This results in a new confor-mation of b-strand L at its C terminus in the closedstructures and consequently the connecting hydro-gen-bond system between b-strands K and L, thatprecede and follow helix 13, respectively, becomesmore extended (not shown), as has been noted(Bernstein et al., 1997; Auerbach et al., 1997).

Figure 5(c) provides further insight into the poss-ible connection with the other hinges at helix 7 andwith the other substrate (3-PG) site on the N-domain. In the T. maritima structure, as has beennoted (Auerbach et al., 1997), the multiple hydro-gen-bond interactions of the conserved Glu Tm174of helix 7 with Ser Tm373 (OG), as well as withThr Tm374 (OG1 and N) are disrupted, but they allexist in the other closed structure of T. brucei PGKas well as in both open structures. It seems thatT. maritima PGK represents a unique case amongthe four ternary complexes. This is also re¯ected inthe differing interactions of the conserved ArgTm36. This arginine residue is important withrespect to the connection of the two domains of theenzyme and it directs the carboxylic group of 3-PGfor the reaction (May et al., 1996). The NH group ofthis residue forms a permanent hydrogen bondwith Thr Tm374 (O) (not shown), as observed inthe other three structures too, independent of theiroverall conformations. However, the second hydro-gen bond between Arg Tm36 (NE) with the sameThr Tm374 (O) does not exist in T. maritima PGK.Instead, the NE atom forms a new, weak hydrogenbond with Thr Tm374 (OG1).

These changes, when considered in conjunctionwith the other differences between the two closedstructures (e.g. the existence of a hydrogen bondbetween Ser Tb401 (OG) and Ser Tb395 (OG) onlyin T. brucei but not in T. maritima PGK) indicatethat this part of the molecule is certainly verymobile during catalysis. The hydrogen bondbetween these two conserved serine residues, aswell as the hydrogen-bond network linking Ser392-Glu192-Thr393-Arg38, are under continuousrearrangement in a concerted way. However, onefeature is clear: in all these ternary complexesArg38 always remains connected to, and movesalong with, b-strand L when the conformation ofthe molecule is changing.

In summary, we present here the ®rst completemolecular description of an important hinge thatoperates at b-strand L of PGK. In the ternary-enzyme-substrate complexes, the conformation ofthis hinge is indirectly affected by the nucleotidesubstrate, through well-de®ned changes in the

Figure 5. Changes in the side-chain interactions leading to the operation of hinge at b-strand L. Two pairs of struc-tures ((pig1 and Tb) and (pig2 and Tm)) were superimposed separately according to the peptide backbone atoms of(a) the b-strand J and ((b) and (c)) the b-strand L, respectively. In (a) the position of the nucleotides is also indicated.Distances between the selected side-chain and backbone atoms are given in AÊ . The colour code remains the same:pig1 (red), pig2 (orange), Tb (green) and Tm (blue).



interactions of conserved side-chains. No suchchanges can be attributed to the previouslydescribed hinges at the ends of helix 7. Therefore,although all the hinges must act in concert, thehinge at b-strand L may play a direct role in deter-mining the relative positions of the two domains ofthis enzyme.

Several questions still remain. For instance: (1)why is the nucleotide substrate alone not able toinduce such a conformational change in the binarycomplex; (2) what is the mode of operation of theN-hinge of helix 7; and (3) is the mobility of the b-phosphate group of ADP, observed here, associ-ated with the transient changes in the interactionof the nucleotide substrate with PGK during thecatalytic cycle? Answers to these questions requirean extension of the comparison of all known PGKstructures to include the binary complexes, as wellas the substrate-free enzyme.

Materials and Methods

Crystallisation

PGK (EC 2.7.2.3) was isolated from pig muscle accord-ing to the method described by Harlos et al. (1992) and

Table 2. Data collection and re®nement statistics

A. Data collectionSpace group P21

Unit cell a � 50.5 AÊ

b � 105.2 AÊ

c � 35.9 AÊ , b � 98.4 �Wavelength (AÊ ) 1.488Resolution range (AÊ ) 25-1.8 (1.88-1.8)a

Total no. of reflections 125,740Unique reflections 34,292Completeness (%) 95.6 (98.1)a

Mean I/s(I) 17.0 (3.0)a

Rmerge(I)(%)b 8.1 (24.4)a

B. RefinementResolution range (AÊ ) 20-1.8No. of reflections 16,447R (%)c 20.8Rfree (%)d 24.1Average B (AÊ 2)

Protein (3037)e 25.8Water (208)e 31.53-PG (11)e 30.2AMP (23)e 26.4

r.m.s.d. bond lengths (AÊ ) 0.011r.m.s.d. bond angles (�) 1.621R.m.s.d. dihedral angles (�) 24.72r.m.s.d. impropers (�) 2.48Ramachandran plot quality% in core allowed regions 91.0% in additional allowed regions 9.0

a Numbers in parentheses denote values in the highestresolution shell,

b Rmerge(I)��hkl jhIhkl iIhkl j/�hkl hIhkl i, where hIhkl i representsthe average intensity of symmetry-equivalent re¯ections,

c R � �hkl j (jFoj ÿ jFcj) j/� (Fo), for a working set comprising95 % of the data,

d Rfree � �hkl j (jFoj ÿ jFcj) j/( (Fo), for a test set comprising5 % of the data selected randomly,

e Numbers in parantheses denote the number of non-hydro-gen atoms.

stored as a microcrystalline suspension in the presenceof ammonium sulphate and 2 mM dithiothreitol. Forcrystal growing, the enzyme was dialysed in 30 mMNaK-phosphate buffer (pH 8.0). Single crystals weregrown at 15 �C within a few weeks in hanging drops(10-15 ml each) initially containing 1.25 M NaK-phos-phate (pH 8.0), 100 mM 3-PG, 25 mM MgCl2, 20 mMADP, 10 mM dithiothreitol, 0.02 % (w/v) NaN3 and15 mg/ml PGK. Reservoir solutions contained 2.4-2.75 M NaK-phosphate (pH 8.0). These conditions wereessentially similar to those described by Bernstein & Hol(1998) for the crystallisation of T. brucei PGK, except thatthe substrate concentrations were chosen to be higher inorder to prevent their partial replacement by phosphateion present in the crystallisation medium. Crystals of theternary complex were obtained. The cell parameters(Table 2) are within 1 % of those of pig muscle PGK crys-tals grown in the presence of ammonium sulphate (A.May et al., personal communication).

Determination of the ADP content

A small volume of MgADP was taken from the crys-tallisation mother liquor and the concentration wasdetermined with a coupled enzymatic assay using pyru-vate kinase and lactate dehydrogenase (Tompa et al.,1986).

Data collection and refinement

Crystals were ¯ash-frozen (Garman & Schneider,1997) into a gaseous nitrogen stream held at 100 K. Priorto cooling, the crystal was soaked for two minutes in asolution containing mother liquor and 15 % (v/v) glycer-ol to provide cryoprotection. X-ray data were collectedfrom a single crystal on beam-line 7.2 (l � 1.488 mm) atSRS, Daresbury, UK. A MarResearch imaging platedetector of diameter 300 mm was used, positioned130 mm from the crystal, and the resolution at the edgeof the detector was 1.79 AÊ . An oscillation angle of 1 � perimage was used. The data were processed using theHKL package (Otwinowski & Minor, 1996) and were96 % complete to 1.8 AÊ resolution. Table 2 summarisesthe data collection and model re®nement statistics.

The starting model was chosen as the structure of theMnADP binary complex of pig muscle PGK crystallisedin the presence of ammonium sulphate (A. May et al.,personal communication). Re®nement was carried out incycles of the XPLOR package (BruÈ nger, 1992) togetherwith manual rebuilding of the model at the end of eachcycle using O (Jones et al., 1991). The primary sequenceof horse muscle PGK was used as in all previous modelsof pig muscle PGK. A random set of re¯ections (5 %)was excluded from the re®nement and was used to cal-culate Rfree. Initially, the two domains (residues 1-188and 202-392) were re®ned as rigid objects in order todetermine their relative orientation (R � 37 %, Rfree�37 %). The 2FoÿFc and FoÿFc maps calculated at thisstage clearly showed the positions of the bound sub-strates and the open conformation of the enzyme. Thiswas followed by cycles of positional re®nement, simu-lated annealing and restrained isotropic temperature fac-tor (B) re®nement. After each cycle of B-factorre®nement, the model was inspected and adjusted to theelectron density maps. In general, the maps showedgood density for most parts of the molecule and for thesubstrates, except for a few loop regions, the N terminusand the b-phosphate group of bound ADP. After the


®rst cycle of re®nement, substrate molecules 3-PG andAMP (in place of ADP) were included in the model.Water molecules were added to the structure in stereo-chemically acceptable positions using Waterpick (inXPLOR), but those which subsequently re®ned withB-factors above 50 AÊ 2 were deleted from the model. Bulksolvent corrections were applied at a later stage in there®nement and the lower resolution limit of dataincluded in the re®nement was extended from 8 AÊ to20 AÊ . In total, 91 % of the non-glycine and non-prolineresidues were in the most favourable region of theRamachandran plot and the rest were in the additionalallowed region (PROCHECK (Laskowski et al., 1993)).

There were several deviations observed from theknown horse muscle sequence and these are highlightedin Figure 1(b). Among them the following are notable,since a larger residue was identi®ed in place of a smallerone: Ser69 (Gly), Lys147 (Glu), Lys182 (Gln), Ala186(Gly), Lys262 (Ala), Gln294 (His), Ile297 (Thr), Pro317-Lys318 (Thr-Glu), Ser324 (Ala) and Leu413 (Ala);the residues in parentheses are those present in the horsestructure. We believe that these represent real differencesin sequence between the pig and horse muscle PGKs andthey are underlined in Figure 1(b). Some of the residueswhich were treated as alanine in the previous pig struc-ture (May et al., 1996) could also be identi®ed correctly.While most of these were found to be identical with thehorse sequence, there were exceptions, such as Gly68(Val), and Gln349 (Arg). For some residues, the sequencein the pig (May et al., 1996) was found to be the same asthat of the horse muscle enzyme (Banks et al., 1979).However, in our structure the density corresponded toAla, both in the low-resolution and the 1.8 AÊ map. Theseresidues were mostly on the surface and they have beenretained as Ala in the ®nal structure (e.g. 29-31 (Lys-Asn-Asn), 102 (Glu), 155 (Lys), 198-200 (Lys-Ala-Leu),260 (Glu), 299 (Gln) and 321 (Lys)).

For molecular graphics, the Insight II 95.0 software(Biosym/MSI, San Diego, CA) was used. X-rayco-ordinates of PGK from pig muscle in complex with3-PG and MnAMP-PNP (May et al., 1996), from T. brucei(Bernstein & Hol, 1998) and from the bacteriumT. maritima (Auerbach et al., 1997) were kindly providedby the authors before publication.

Protein Data Bank accession numbers

The co-ordinates have been deposited in the ProteinData Bank 1hdi with accession number.

Acknowledgements

The authors are grateful to Professor L.N. Johnson(Laboratory of Molecular Biophysics, University ofOxford) for her continuous interest and support through-out the work. They thank Dr C.C.F. Blake (former mem-ber of the LMB, University of Oxford) for critical readingof the manuscript, Steve Lea for photographic assistanceand the staff at beamline 7.2, SRS Daresbury, U.K. Finan-cial support from the Hungarian National ResearchFund (OTKA T 029208) and from NATO (TravellingGrant LG 960126) is gratefully acknowledged.

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Edited by R. Huber

(Received 20 June 2000; received in revised form 26 October 2000; accepted 26 October 2000)

A 1.8 Å resolution structure of pig muscle 3-phosphoglycerate kinase with bound MgADP and...

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Transcript of A 1.8 Å resolution structure of pig muscle 3-phosphoglycerate kinase with bound MgADP and...