The ligandin (non-substrate) binding site of human pi class glutathione transferase is located in...

Article No. jmbi.1999.3029 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 291, 913±926

The Ligandin (Non-substrate) Binding Site of HumanPi Class Glutathione Transferase is Located in theElectrophile Binding Site (H-site)

Aaron J. Oakley1, Mario Lo Bello2, Marzia Nuccetelli2, Anna P. Mazzetti2

and Michael W. Parker1*

1The Ian Potter FoundationProtein CrystallographyLaboratory, St. Vincent'sInstitute of Medical ResearchFitzroy, Victoria 3065, Australia2Department of BiologyUniversity of Rome ``TorVergata'', Via della RicercaScienti®ca, 00133, Rome, Italy

Present address: A. J. Oakley, DePharmacology, The Queen ElizabetNedlands, Western Australia 6009,

Abbreviations used: BS, bromosucibacron blue; CDNB, 1-chloro-2,4-dDNP GSH, 2, 4-dinitrophenyl glutaDTT, 1,4-dithiothreitol; EA, ethacryelectron paramagnetic resonance; Gbinding site; GSH, glutathione; GSTS-transferase; H-site, hydrophobic esite; Hepes, N-(2-hydroxyethyl)pipeethanesulfonic acid; L-site, ligandin2-[N-morpholino]ethanesulfonic acicrystallographic symmetry; SLZ, su

E-mail address of the [email protected]

0022-2836/99/340913±14 $30.00/0

Glutathione S-transferases (GSTs) play a pivotal role in the detoxi®cationof foreign chemicals and toxic metabolites. They were originally termedligandins because of their ability to bind large molecules (molecularmasses >400 Da), possibly for storage and transport roles. The location ofthe ligandin site in mammalian GSTs is still uncertain despite numerousstudies in recent years. Here we show by X-ray crystallography that theligandin binding site in human pi class GST P1-1 occupies part of one ofthe substrate binding sites. This work has been extended to the determi-nation of a number of enzyme complex crystal structures which showthat very large ligands are readily accommodated into this substratebinding site and in all, but one case, causes no signi®cant movement ofprotein side-chains. Some of these molecules make use of a hithertoundescribed binding site located in a surface pocket of the enzyme. Thissite is conserved in most, but not all, classes of GSTs suggesting it mayplay an important functional role.

# 1999 Academic Press

Keywords: detoxi®cation; glutathione transferases; inhibitor complexes;ligandin binding site; X-ray crystallography
*Corresponding author
Introduction

The glutathione S-transferases (GSTs, EC2.5.1.18) are a family of dimeric enzymes thatconjugate a wide variety of carcinogenic,mutagenic, toxic and pharmacologically activecompounds to the cellular nucleophile glutathione(GSH, g-Glu-Cys-Gly). Cytosolic GSTs exist eitheras homo- or heterodimers with a subunit molecular

partment ofh II Medical Centre,Australia.lfophthalein; CB,initrobenzene;

thione;nic acid; EPR,-site, glutathione, glutathionelectrophile bindingrazine-N0-2-binding site; Mes,

d; NCS, non-lfasalazine.ing author:

mass in the range 23-28 kDa and with one activesite per monomer (for a review, see Armstrong,1997). They can be classi®ed into at least sevendistinct families: alpha, beta, kappa, mu, pi, sigma,theta and zeta, based on studies of immunologicalproperties, substrate speci®city and amino acidsequences (Mannervik et al., 1985; Meyer et al.,1991; Buetler & Eaton, 1992; Pemble et al., 1996;Board et al., 1997; Rossjohn et al., 1998). Thesequence identities between any two memberswithin a class is typically greater than 70 %,whereas the ®gure is typically less than 30 %between classes. There are now representative crys-tal structures for six of the cytosolic GST classes.The overall polypeptide fold is very similarbetween the crystal structures but each class exhi-bits unique features, particularly about the activesite and at the C terminus (Wilce & Parker, 1994;Dirr et al., 1994; Armstrong, 1997). The crystalstructures have shown that there are at least twoligand binding sites per monomer: the G-site isvery speci®c for GSH whereas the binding site forthe electrophilic substrate (H-site) is less speci®c inkeeping with the ability of GSTs to react with awide variety of toxic agents.

# 1999 Academic Press

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Figure 1 (legend opposite)

914 Glutathione Transferase Ligand Binding Sites

In addition to their enzymatic roles, GSTs bindto large lipophilic molecules (molecular masses>400 Daltons) leading to the suggestion that GSTsare involved in the storage and rapid transport ofthese molecules in the aqueous phase of the cell(Litwack et al., 1971; Habig et al., 1974a; Tipping &Ketterer, 1981; Caccuri et al., 1990). This ligandinproperty was noted in early work on an abundantprotein from rat liver, later identi®ed as a GST,which showed impressive binding activity(105 ÿ 107 Mÿ1 af®nity constants) towards manylipophilic anion ligands (Litwack et al., 1971; Habiget al., 1974a). These ligands included such mol-ecules as hemin, bilirubin, bile salts, steroids, thyr-oid hormones, fatty acids and drugs (Ketterer et al.,1978). Ligandin binding properties were latershown to be a property of many GST isoformsincluding human pi class GST P1-1 (Caccuri et al.,1990). Ligandin binding is characterised by non-competitive inhibition towards the diagnostic GSTsubstrate, 1-chloro-2,4-dinitrobenzene (CDNB)(Ketley et al., 1975; Mannervik & Danielson, 1988).The kinetic data suggest that the non-substrate orligandin binding site (the L-site) is distinct fromthe G and H-sites. The existence of an additionalbinding site has been supported by the recent ®nd-ings that ligands can bind in the dimer interface ofGSTs from Schistosoma japonica (McTigue et al.,1995) and squid (Ji et al., 1996). However, thedimer interface in human pi class GST is verypolar and appears unlikely to be the location of theL-site. Recently, two laboratorys have suggestedthe L-site in human pi class GST is located at a buf-fer binding site (the site of Hepes or Mes binding)close to Trp28 (Ji et al., 1997; Prade et al., 1997).However, the binding site is 16 AÊ distance fromthe G-site and thus it is dif®cult to explain the non-competitive inhibition kinetics with this model.Furthermore, the area de®ned by the buffer bind-ing site is too small to accommodate the largeligands that are known to bind with high af®nityto the L-site. The location of the L-site in mamma-lian GSTs remains uncertain.

GSTs have been implicated in the developmentof resistance of tumors towards various anti-cancerdrugs (for reviews, see Coles & Ketterer, 1990;Waxman, 1990; Tsuchida & Sato, 1992; Hayes &Pulford, 1995). Sulfasalazine (SLZ, 2-hydroxy-(5-{[4-(2-pyridinylamino) sulfonyl]phenyl}azo) ben-zoic acid or salicylazosulphapyridine) (Figure 1(a))is used as a drug for treating in¯ammatory boweldisorders (Svartz, 1942; Das & Rubin, 1976) andrheumatoid arthritis (Pullar, 1989). It is also aknown inhibitor of alpha, mu and pi class GSTswith concentrations of 28 mM or less required toinhibit 50 % of enzymatic activity (Ahmad et al.,1992). SLZ has been shown to increase the cytotox-icity of the anti-cancer drug, cisplatin, towards twohuman lung cancer cell lines which were shown tooverexpress only the pi class GST (Awasthi et al.,1994) and has been tested in clinical trials withpatients suffering advanced forms of cancer (Guptaet al., 1995). Despite the potential bene®ts of GST

Figure 1. Schematic representation of the inhibitors.(a) Sulfasalazine. The numbering scheme is taken fromvan der Sluis & Spek (1990). (b) S-Nonyl GSH; (c) Ciba-cron blue; (d) bromosulfophthalein; (e) 2,4-dinitrophenylGSH.

Glutathione Transferase Ligand Binding Sites 915

inhibitors and despite the large number of GSTinhibitors that have been synthesized to date, veryfew compounds have proved suitable for clinicaltrials.

We have previously solved the crystal structureof human pi class GST P1-1 in complex with theinhibitors S-hexyl GSH (Reinemer et al., 1992;Oakley et al., 1997a) and g-glutamyl-(S-benzyl)cys-teinyl-D-phenylglycine (Oakley et al., 1997a), thesubstrate GSH (Oakley et al., 1997a), the drug etha-crynic acid (EA) and its GSH conjugate (Oakleyet al., 1997b). Here we present the structures of theenzyme in complex with a variety of very largeligands (molecular mass >400 Da). Firstly, we pre-sent the structure of the SLZ complex. A novelbinding site for hydrophobic ligands has beenidenti®ed and is also utilised by S-nonyl GSH. Ourresults de®ne the key residues in binding SLZ tothe enzyme and our ®ndings form the basis for thedesign of a more potent inhibitor to be used as apotential adjuvant in cancer chemotherapy. Wealso report the structure of the enzyme complexedwith two large dyes, cibacron blue (CB; Figure 1(c))and bromosulfophthalein (BS; Figure 1(d)). CB hasbeen used extensively as a diagnostic GST inhibitor(Tahir et al., 1985; Mannervik & Danielson, 1988)and BS has been used as a model compound forprobing the ligandin binding properties of GSTs(Ketterer et al., 1978; Mannervik & Danielson,1988). Up until now, the location of the bindingsites for either inhibitor have not been known. Thestudies reported here indicate both dyes bind inthe H-site. The structure of the BS complexsuggests the L-site is located in part of the H-site.The basis for the non-competitive binding kineticsseen for BS is explained by the structure of the 2,4-dinitrophenyl (DNP) GSH complex.

Results

Sulfasalazine GSH complex

SLZ binds in the H-site of the enzyme in a verysimilar position to that found for the hexyl moietyof the S-hexyl GSH crystal structure (Reinemeret al., 1992; Oakley et al., 1997a). The inhibitor sitsin a hydrophobic pocket lined with the side-chainsof Tyr7, Phe8, Pro9, Val10, Val35, Ile104 andTyr108 (Figure 2(a)). The salicylic acid moiety ofthe inhibitor is stacked between the aromatic side-chain of Tyr108 and the thiol of the glutathionesubstrate and is nestled against Ile104 (Figure 2(a)).The salicylic carboxylate is within hydrogen bond-ing distance of the Ne atom of Arg13 and the thiolsulphur of GSH. The hydroxyl group of the sal-icylic moiety makes no contacts with the protein.The azo linkage is within hydrogen bonding dis-tance of Tyr108. The phenyl ring is stackedbetween the aromatic side-chain of Phe8 andGly205. An interesting feature of the inhibitorbinding is the location of the pyridinyl ring nestledin a shallow pocket on the surface of the protein.The pocket is quite hydrophobic with major contri-butors being Pro9, Val10, Pro202 and Gly205. Intotal, the inhibitor makes 66 van der Waals con-tacts (<4 AÊ ) and three hydrogen bonds with theprotein. There is only one water-mediated inter-action with the inhibitor that has been identi®ed inthe crystal structure. In one monomer, a watermolecule is within hydrogen bonding distance(2.5 AÊ ) of one of the carboxylate oxygen atoms ofthe salicylic acid moiety. A superposition of theSLZ complex on the wild-type structure, com-plexed with S-hexyl GSH, indicates no signi®cantmovement of side-chains in the active site (ther.m.s. deviation in alpha-carbon positions is 0.32 AÊ

with no movements greater than 0.85 AÊ ). Previouswork by us has demonstrated that the lack of sig-ni®cant differences is unlikely to be due to crystalpacking effects, since complexes of the enzyme inthree different crystal forms all exhibit identicalactive sites (Oakley et al., 1997a).

The electron density maps showed clear densityfor GSH in the G-site of the SLZ complex.Although not added to the crystallization trials, theGSH originated from the enzyme puri®cation pro-cedure where the enzyme is eluted from an af®nitychromatography column using the substrate (LoBello et al., 1993). Superposition of the SLZ com-plex model with other crystal structures of humanpi class GST complexes (Reinemer et al., 1992;Oakley et al., 1997a,b) demonstrates that the boundGSH molecule binds in identical fashion in all thecomplexes. However, unlike all the other com-plexes, it is not conjugated to the electrophilicligand (SLZ) bound in the H-site.

Sulfasalazine kinetics

We found no evidence that SLZ could act as aco-substrate for the conjugation reaction with GSH.

Figure 2 (legend opposite)


However, we found that SLZ acts as a competitiveinhibitor for CDNB in the classical CDNB/GSHreaction with a Ki value of 24(�2) mM. Theseresults are consistent with crystallographic data(see Figure 3) where SLZ was found to bind in theH-site in a similar position to that found for othersubstrates/inhibitors. Furthermore, SLZ occupiessome of the space taken by DNP in the DNP GSHcrystal structure (Figure 3(a)). Because the azo link-age of SLZ is within hydrogen bonding distance of

Tyr108, we also investigated a possible role of thisresidue in the binding of this inhibitor. We havepreviously obtained two active mutants, Y108Fand Y108V, by site-directed mutagenesis (Lo Belloet al., 1997; Nuccetelli et al., 1998). Both thesemutants were inhibited by SLZ with Ki values of13.5(�0.5) mM and 41(�1) mM, respectively. Theseresults suggest that the role of the aromatic ring ofTyr108 or of its hydroxyl group is not a majordeterminant in the binding of SLZ.

Figure 2. Stereo diagrams of ®nal 2Finhibitor ÿ Fc electron density maps of the complexes in the vicinity of the inhibi-tor binding site of human GST P1-1. The maps were calculated from the ®nal model after the inhibitor was taken outand the model subjected to a round of simulated annealing to reduce bias. The maps were calculated using all re¯ec-tions and contoured at the 0.7s level. (a) SLZ; (b) S-nonyl GSH; (c) CB; (d) BS; (e) DNP GSH.


S-Nonyl GSH complex

The ®t of this inhibitor into the electron densitymap is shown in Figure 2(b). The GSH moietybinds in identical fashion to that observed in theGSH-bound enzyme structure (Oakley et al.,1997a). A superposition of this complex with thepreviously published S-hexyl GSH complex(Reinemer et al., 1992; Oakley et al., 1997a) indicatesthat the alkyl tails lie in identical positions andthere are no signi®cant movement of side-chains inthe active site (the r.m.s. deviation in alpha-carbonpositions is 0.17 AÊ with no deviations greater than0.53 AÊ ). In total, the inhibitor makes 87 van derWaals contacts (<4 AÊ ), three salt links and 24hydrogen bonds with the protein. The nonyl tailmakes van der Waals contacts with the side-chainsof Phe8, Val10 and Tyr108 and the main-chain ofGly205. With reference to the S-hexyl complex, theextra three carbon units of the S-nonyl GSH inhibi-tor are found nestled in a shallow hydrophobicpocket on the surface of the protein. This pocket is

constructed from the side-chain atoms of Pro9,Val10 and Pro202 and the main-chain atoms ofGly205.

Cibacron blue complex

In the CB model, the three conjugated rings ofthe dye (Figure 1(c)) occupy the volume of densityin the H-site of each monomer (Figure 2(c)).Because there was no density for rest of the ligand,only the fused ring system has been included inthe ®nal model. The three rings lie in a similarplane as those in other pi class GST complexessuch as EA, EA GSH (Oakley et al., 1997b), g-gluta-myl-(S-benzyl)cysteinyl-D-phenylglycine (Oakleyet al., 1997a) and SLZ. In particular, there is aremarkably close overlap with the fused three-ringed structure of (9R,10R)-9-(S-glutathionyl)-10-hydroxy-9,10-dihydrophenanthrene complexed tothe human pi class enzyme (Ji et al., 1997). Bothring systems occupy almost the same space withan angle of about 30o between the planes of two.

Figure 3. A comparison of the binding locations of GSH conjugates derived from the various crystal structures ofpi class GSTs presented here. The superposition was based on overlaying the coordinates of the respective N-terminaldomains. The ligands are overlayed on a schematic of the N-terminal domain of the human pi class enzyme. (a)Stereo view showing all inhibitors described in the text. The inhibitors are coloured as follows: SLZ, yellow; S-nonylGSH, cyan; CB, dark blue; BS no.1, red; BS no. 2, magenta; DNP GSH, green. The side-chains of Phe8 and Tyr108 areshown. (b) Comparison of the binding locations of DNP GSH (denoted by yellow bonds) and BS no. 1 (denoted byred bonds). These pictures were generated with the program MOLSCRIPT (Kraulis, 1991).


The conjugated rings of CB make van der Waalscontacts with Phe8, Val10, Ile104, Tyr108 andGly205. There are possible hydrogen bondinginteractions between the hydroxyl group of Tyr7and a ring carbonyl group of CB, and a salt bridgebetween the sulfonic acid group of CB and theguanadyl group of Arg13. In total, the inhibitormakes 42 van der Waals contacts (<4 AÊ ) with theprotein. A superposition of the CB complex on thewild-type structure, complexed with either S-hexylGSH or GSH, indicates no signi®cant movement ofside-chains in the active site (the r.m.s. deviation inalpha-carbon positions is 0.35 AÊ with no move-ments greater than 1.0 AÊ ).

Bromosulfophthalein

Analysis of the electron density maps indicatedthe presence of two closely related bindinglocations of BS in the H-site. No density wasobserved for the two benzyl sulfonic acid moieties(Figures 1(d) and 2(d)) in either binding mode, pre-sumably because they are too mobile. Both BS mol-ecules lie in the same plane but one is 5 AÊ closer to

the G-site (see Figure 3(a)). The electron density forone of the two binding modes is much clearer andstronger (which we call BS no.1 and shown inFigure 2(d)) and we have chosen to discuss onlythis mode here because of the uncertainties in theprecise interpretation of the other mode. (BS no.2appears to have a signi®cantly lower occupancythan BS no.1.) The conjugated rings of BS lie in asimilar position to the rings of SLZ with an angleof about 35 � between the plane of the rings of eachligand. Whereas SLZ appears to optimise ringstacking against both Phe8 and Tyr108, BS opti-mises its ring stacking with Phe8 only (Figure 3(a)).The six-membered ring of the BS fused ring system(Figure 1(d)) cannot bind in the same position asthe single rings of other inhibitors such as the ben-zyl groups in the g-glutamyl-(S-benzyl)cysteinyl-D-phenylglycine complex (Oakley et al., 1997a) orthe S-(p-nitrobenzyl) GSH complex (GarcõÂa-SaÂezet al., 1994) because of steric clashes between thebromine substitutents and the side-chains of Tyr7and Val10. The inhibitor makes van der Waals con-tacts with the side-chains of Phe8, Val10, Val35,Trp38, Tyr108 and main-chain of Gly205. In total,


BS makes 14 van der Waals contacts (<4 AÊ ) but nosalt links or hydrogen bonds with the protein. Asuperposition of this complex with the previouslypublished S-hexyl GSH complex (Reinemer et al.,1992; Oakley et al., 1997a) shows there are no sig-ni®cant movement of side-chains in the active site(the r.m.s. deviation in alpha carbon positions is0.23 AÊ ) with one exception. The region aroundVal35 has moved up to 1.3 AÊ away from the activesite in order to accommodate the inhibitor. Thisregion exhibits poor density and high B-factors.Indeed, movement of Val35 is required, since itwould otherwise clash with the bound BS mol-ecule.

2,4-Dinitrophenyl GSH

DNP GSH (Figure 1(e)) binds in an unexpectedfashion to the enzyme in comparison to the struc-tures of other complexes (Figures 2(e) and 3). Pre-vious work has shown that the S-benzyl moietiesof g-glutamyl-(S-benzyl)cysteinyl-D-phenylglycinebound to the human enzyme (Oakley et al., 1997a)and of S-(p-nitrobenzyl) GSH bound to the mousepi class GST (GarcõÂa-SaÂez et al., 1994) stackbetween Phe8, Val10 and Tyr108, making van derWaals contacts with all of these residues. The hexyland nonyl tails in the S-hexyl and S-nonyl-GSHcomplexes also stack against these residues, occu-pying some of the same space as the S-benzylgroups. In the case of DNP GSH presented here,the S-phenyl moiety stacks against only Tyr108(and Ile104) with no contacts to Phe8 or Val10. Thereason for the differences lie in the fact that DNPGSH lacks a bridging methylene group betweenthe aromatic ring and the sulphur atom of GSH.The Ca to Cb bond of the GSH Cys in the complexis rotated by about 90 � with respect to the samebond in the g-glutamyl-(S-benzyl)cysteinyl-D-phe-nylglycine (28) and S-(p-nitrobenzyl) GSH (GarcõÂa-SaÂez et al., 1994) complexes so that the sulphuratom points towards Tyr108. This causes the aro-matic ring of the DNP moiety to move toward theedge of the H-site (Figure 3(a)). The position of thearomatic ring is somewhat reminiscent of the pos-ition found for the aromatic ring in the EA GSHcomplex (Oakley et al., 1997b). The angle betweenthe planes of the aromatic rings is about 20 � andthe ring in the DNP GSH complex is about 2 AÊ clo-ser to Ile104. The nitro groups of the DNP GSHcomplex make no contacts with protein.

The GSH moiety of the complex binds in identi-cal fashion to that observed in the GSH-boundenzyme structure (Oakley et al., 1997a). A superpo-sition of this complex with the previously pub-lished S-hexyl GSH complex (Reinemer et al., 1992;Oakley et al., 1997a) indicates there are no signi®-cant movement of side-chains in the active site (ther.m.s. deviation in alpha carbon positions is 0.21 AÊ

with no deviations greater than 1.0 AÊ ). In total, theinhibitor makes 102 van der Waals contacts (<4 AÊ ),three salt links and 12 hydrogen bonds with theprotein.

Discussion

Location of the L-site

The ®rst pi class GST crystal structure was deter-mined in the absence of an electrophilic substrate(Reinemer et al., 1991). Three possible locations forthe H-site were proposed; one of them was locatedadjacent to the G-site and subsequently found tobe the binding place for the hexyl moiety of theS-hexyl GSH inhibitor that was used to crystallizethe human GST P1-1 enzyme (Reinemer et al.,1992). Subsequent work on other complexes hasreinforced the idea that this site is the binding sitefor electrophilic substrates (Ji et al., 1997; Oakleyet al., 1997a,b; Prade et al., 1997).

Previous work has suggested that the L-site inhuman pi class GST is close to, but distinct from,the G-site. For example, the af®nity for hemin isincreased in the presence of GSH but hemin canstill bind in the presence of S-methyl GSH (Caccuriet al., 1990). EPR studies indicated hemin is locatedbetween 10 and 14 AÊ from Cys47, a residue locatedon helix a2 which forms a wall of the G-site. Thebinding of either GSH or hemin reduces the reac-tivity of Cys47 in a similar way (Desideri et al.,1991). Previous suggestions for the location of theL-site include in the dimer interface (McTigue et al.,1995; Ji et al., 1996) and at a buffer binding site (Jiet al., 1997; Prade et al., 1997). The dimer interfaceis completely accessible in the crystal form studiedhere. The buffer binding site is involved in crystalcontacts and so is not available for possible bind-ing of ligandin molecules. However, previouswork with CB in a tetragonal space group of theenzyme, where the buffer binding site is free andaccessible, showed no evidence of CB binding atthis location (unpublished results). Furthermore,the distance between Cys47 and the buffer bindingsite is about 21 AÊ , which would appear to rule outthe possibility of it being the L-site based on theEPR measurements described above. In addition,site-directed mutagenesis (Nishihira et al., 1993)and ¯uorescence measurements (Bico et al., 1995)appear to rule out the involvement of Trp28 inligandin binding, which forms part of the bufferbinding site. Typical ligandin molecules are verylarge (molecular mass >400 Da) and are usuallynot substrates for GSTs. We have found, despitetheir large size, SLZ, CB and BS are all readilyaccommodated into the H-site of the enzyme andare not conjugated to GSH.

BS displays non-competitive inhibition for theGST diagnostic substrate CDNB and has been usedas a model ligand to probe the location of theL-site (Bico et al., 1995). If the BS complex is super-imposed onto the DNP GSH complex (Figure 3(b)),there is only one collision between the ligands. Theclosest approach is 1.6 AÊ between a nitro group ofDNP GSH and one of the bromine atoms of BS.The structure of the homologous transition stateanalogue, trinitrobenzyl GSH, complexed to theenzyme has been determined recently (Prade et al.,


1997). This structure suggests there would be noclashes in the transition state complex of CDNBwith GSH. In summary, it appears possible thatthe model ligandin compound BS can bind into theH-site without competitively inhibiting the CDNBconjugation reaction. The possibility of two ligandsbinding in the H-site has previously been observedin a plant GST (Reinemer et al., 1996). Thus, wepropose the L-site is located in the H-site ratherthan at the dimer interface or at the buffer bindingsite. We further suggest that the buffer binding sitepreviously identi®ed as a putative L-site (Ji et al.,1997; Prade et al., 1997) might correspond to thesecond low af®nity binding site that has beencharacterised in a kinetic study of hemin binding(Caccuri et al., 1990). Alternatively, the low af®nitysite might correspond to the minor, second BSbinding site we observed in this study (Figure 3(a)).

Previous work suggests that the GST dimerbinds with high af®nity only one ligandin molecule(Ketley et al., 1975; Caccuri et al., 1990), whereasour work suggests the ligandin molecule can bindto each monomer. This apparent paradox can beexplained by assuming that the crystal lattice can-not distinguish between the bound and unboundmonomers of a dimer complexed with one ligandso electron density for the ligand is averaged outin both binding sites. It follows that the ligandoccupancy of each site in the dimer will be equalto or less than half which ®ts in with the occu-pancy estimates made here (see Materials andMethods). The reason why the ligandin moleculemight only bind in one H-site per dimer could bedue to conformational changes induced in thesecond binding site after binding of ligand to the®rst site. Conformational changes transmittedbetween active sites of the dimer have previouslybeen suggested for the G-site of this enzyme (Ricciet al., 1995; Lo Bello et al., 1998; Oakley et al., 1998).

Novel binding site

The hydrophobic surface pocket that binds thepyridinyl moiety of SLZ and the tip of the alkylchain of S-nonyl GSH is conserved, both insequence and structure, between the known crystalstructures of the pi class GSTs (Reinemer et al.,1991, 1992; GarcõÂa-SaÂez et al., 1994) (Figure 4(a)). Inthe rat mu class GST structure, Val10 (human pi) isreplaced by Val9, Pro9 (human pi) by Asn8,Pro202 (human pi) by Pro206 and Gly205 (humanpi) by Ser209 (Ji et al., 1992) (Figure 4(b)). The con-formation about Gly205 (human pi) is slightlydifferent in the mu class structure which causesSer209 (rat mu) to point away from the pocket.Hence, with the exception of Asn8 (rat mu), thehydrophobicity of the binding pocket is retained inthe mu class enzyme. In the squid sigma class GST(Ji et al., 1995), Pro9 (human pi) is retained, Val10(human pi) is replaced by Leu10 but the confor-mation of the C terminus is quite different(Figure 4(c)). However, nearby are the side-chainsof Phe202 (squid) and the aliphatic portion of

Arg200 (squid sigma). Hence the hydrophobicityof the pocket is also retained in the sigma classenzyme. The situation in the schistosomal enzymeis intriguing (Ji et al., 1996). Pro9 (human pi) isreplaced by the aliphatic portion of Lys10, Val10(human pi ) by Ile10, Pro202 (human pi) by Pro202and Gly205 (human pi) by Gly205 (Figure 4(d)).However, the pocket is ®lled in the schistosomalenzyme by the side-chain of Trp206 which super-poses on top of the pyridinyl ring of SLZ(Figure 4(d)). The plant theta class GST (Reinemeret al., 1996) does have a hydrophobic pocket in thesame region formed by residues Pro9, Val10,Ile122, Ile126 and Tyr178. However, it appearsvery different to the human pi class pocket becauseit is much larger and the only common residue isPro9. There are no recognisable pockets in theinsect theta class (Wilce et al., 1995) or alpha class(Sinning et al., 1993) enzymes. The lack of the pock-et in the latter enzyme, despite its known ligandinbinding properties, indicates that the pocket is notessential for recognition and binding of theseligands.

Binding modes of aromatic rings in the H-site

Recently, a number of pi class GST crystal struc-tures have been determined in complex with elec-trophilic aromatic ligands. These include mouse piclass GST complexed with S-(p-nitrobenzyl) GSH(GarcõÂa-SaÂez et al., 1994), human pi class GST com-plexed with EA and its GSH conjugate (Oakleyet al., 1997b), g-glutamyl-(S-benzyl)cysteinyl-D-phe-nylglycine (Oakley et al., 1997a) and (9R,10R)-9-(S-glutathionyl)-10-hydroxy-9,10-dihydrophenan-threne (Ji et al., 1997). All these crystal structureshave been superimposed on the SLZ complex. Allthe rings lie in approximately the same plane, withthe exception of the EA GSH conjugate which isdiscussed below. The aromatic rings of both themouse pi class GST inhibitor and the EA complexare positioned so as to optimise ring stackingbetween the aromatic side-chains of Phe8 andTyr108. In the multi-ring structures of SLZ and(9R,10R)-9-(S-glutathionyl)-10-hydroxy-9,10-dihy-drophenanthrene, the inhibitors are positioned sothat one ring optimises its interaction with onlyPhe8 and another ring optimises its stacking inter-action with Tyr108. In summary, it would appearthat single ring inhibitors bind so that they stackbetween the side-chains of Phe8 and Tyr108 tooptimise interactions with both rings whereasmulti-ring structures may bind so that one ringoptimises its stacking interaction with Phe8 andanother against Tyr108 (see Figure 3(a)). A compli-cation to this ``rule'' is that some single ring inhibi-tors can bind in a completely different orientationwhich involves stacking against Tyr108 only.Examples include the GSH conjugate of EA(Oakley et al., 1997b) and DNP GSH (Figure 3).This type of binding has been observed in otherGSTs and has been referred to as the ``out'' bindingmode as distinct from the more prevalent ``in''

Figure 4. Stereo pictures of the novel hydrophobic binding pocket found in a variety of GST crystal structures.(a) Human pi class (Oakley et al., 1997a,b); (b) rat mu class (Ji et al., 1992); (c) squid sigma class (Ji et al., 1995);(d) Schistosoma japonica GST (McTigue et al., 1995).


binding mode (Armstrong, 1997). The small changein Ki observed for SLZ inhibition of the Y108Vmutant over wild-type demonstrates that the aro-matic stacking interactions are only one of manycontributors to ligand binding.

The catalytic mechanism of CDNB conjugationhas been the subject of many studies, since thisreaction is the most commonly used assay for GST

activity (Mannervik & Danielson, 1988). A com-parison of the crystal structures of a rat mu classcomplex with the s-complex intermediate ana-logue, 2,4,6-trinitrocyclohexadienate GSH, and theproduct, DNP GSH, indicates the intermediatebinds in the in mode whilst the product binds inthe out mode (Ji et al., 1993). A structure of 1,3,5-trinitrobenzene GSH bound to the pi class enzyme,


which represents a model for a transition state s-complex, shows it binds in the in mode (Pradeet al., 1997). Thus, like the mu class enzyme, itappears that CDNB binds to GSH in the pi classenzyme in the in mode during the transition statebut converts to the out mode as the product isformed. This conversion may enable more rapidrelease of the product.

Concluding remarks

This work indicates that very large ligands (mol-ecular mass >400 Da) are readily accommodatedinto the H-site of the enzyme. In the case of theSLZ complex, the structure provides the frame-work for the development of new inhibitors, by themethod of structure-based drug design, whichmight prove useful as adjuvants in chemotherapy.In particular, the discovery of the novel bindingsite adjacent to the H-site will provide additionalbinding surface for the design of more potent andspeci®c inhibitors.

Materials and Methods

Crystallization and inhibitor soaking experiments

Wild-type protein was expressed and puri®ed asdescribed (Lo Bello et al., 1995). Crystallization was per-formed by the hanging drop vapour diffusion method asdescribed elsewhere (Oakley et al., 1997a). In all cases,solid powder of each inhibitor was added to a hangingdrop containing preformed crystals. All inhibitors wereof the highest purity available and were obtained fromSigma Chemical Company (Missouri, USA) unless other-wise stated. For the SLZ complex, crystals were soakedfor seven days during which time they gradually tookup the yellow colour of the inhibitor. For the S-nonylGSH complex, crystals were soaked for 20 days. For theCB complex, the crystals were soaked at 4 �C for sixweeks. During this time, the crystals slowly changedfrom clear to a deep blue colour indicating uptake of thecompound. For the BS (Fluka Chemie AG, Buchs, Swit-zerland) complex, the crystals were soaked for sevenweeks. For the DNP GSH (Figure 1(e); synthesisedaccording to the method given by Lo Bello et al., 1993)complex, the crystals were soaked for two weeks duringwhich time they turned yellow.

X-ray data collection

The X-ray diffraction data were collected using aMARResearch area detector with CuKa X-rays generatedby a Rigaku RU-200 rotating anode X-ray generator. Thedata were either collected at 288 K or 100 K (see Table 1).For the low temperature data sets, crystals were trans-ferred stepwise into drops of arti®cial mother liquor con-taining up to 25 % (v/v) glycerol and then ¯ash frozen at100 K. The diffraction data were processed and analysedusing programs in the HKL (Otwinowski, 1993) andCCP4 (1994) suites. The relevant data statistics are pre-sented in Table 1.

Model building and refinement

Re®nement began with the model of the GSH complexin the same space group (Oakley et al., 1997a) which hadsubstrate and water molecules removed. Rigid bodyre®nement in X-PLOR (BruÈ nger, 1992) was used to com-pensate for any possible changes in crystal packing. Asthe asymmetric unit of the crystal contained two GSTmonomers, use was made of the non-crystallographicsymmetry (NCS) restraints on all non-hydrogen atomsthroughout the course of the re®nements (turning off therestraints did not result in signi®cant differences betweenmonomers beyond crystal lattice contact points but didresult in an increase in Rfree). Typically, a number ofrounds of positional re®nement were performed fol-lowed by model building and then by rounds of pos-itional and individual NCS-restrained B-factorre®nement. The 2Finhibitor ÿ Fnative electron density mapscalculated in the model building steps were furtherimproved, where necessary, by ten cycles of twofoldNCS averaging using the MAMA (Kleywegt & Jones,1994), RAVE (Kleywegt & Jones, 1994) and CCP4 (1994)program suites. In the ®nal stages of re®nement, a bulksolvent correction was employed using all data. For theSLZ complex, bond lengths and angles were derivedfrom the crystal structure of the free molecule (van derSluis & Spek, 1990). For S-nonyl GSH, bond lengths andangles were derived from the parameters by Engh &Huber (1991). For the CB and BS complexes, bondlengths and angles were derived from a model built andenergy minimised using programs in the BIOSYM soft-ware suite (Molecular Simulations Inc., San Diego, CA,USA). For the DNP GSH complex, bond lengths andangles were taken from the PDB entry 5GST (Ji et al.,1993). In some cases only partial inhibitor binding wasobserved. In these cases the B-factor was set to the aver-age value of the side-chains which contacted the inhibi-tor and the occupancy of the inhibitor re®ned. The ®t ofeach inhibitor into the ®nal (omit) electron density mapsis shown in Figure 2. The ®nal re®nement statistics forall models are presented in Table 2. A stereochemicalanalysis of the re®ned structures with the program PRO-CHECK (Laskowski et al., 1993) gave values either simi-lar or better than expected for structures re®ned atsimilar resolutions.

For the SLZ complex, the ®tting of two SLZ and twoGSH molecules into the electron density maps wasunambiguous (the strongest peaks (>5s) in the ®nal elec-tron density map calculated after omitting the SLZ mol-ecules and a round of re®nement, corresponded to thesulphur atom of each inhibitor molecule). The ambiguityin the rotation of the pyridinyl ring about the C1-N2bond (0 � or 180 � rotation) in the electron density maps(see Figure 2(a)) is resolved by the potential hydrogenbonding interaction between the pyridinyl nitrogen (pre-suming the imide form exists in the enzyme complex asit does in the small molecule structure (van der Sluis &Spek, 1990)) and the carbonyl of Gly205. For the S-nonylGSH complex, there was clear and interpretable densityin the H-site (Figure 2(b)). For the CB complex, the mapsrevealed good density for the protein with the exceptionof the residues around and including helix a2, whichhad poor but interpretable density. A similar situationhas been observed previously with the complex betweenEA and the enzyme (Oakley et al., 1997b) suggesting theregion around helix a2 becomes highly ¯exible in theabsence of ligand in the G-site. This suggestion hasrecently been con®rmed in the structure of the apoen-zyme (Oakley et al., 1998). There was a large volume of

Table 1. Data collection and processing

SLZ S-nonyl GSH CB BS DNP GSH

Temperature (K) 288 100 100 100 100Space group C2 C2 C2 C2 C2Cell dimensionsa (AÊ ) 79.5 77.9 78.2 77.7 78.5b (AÊ ) 91.1 89.4 90.7 90.0 89.6c (AÊ ) 69.6 68.9 68.8 68.8 68.9b (�) 98.4 97.5 97.7 97.5 97.5Maximum resolution (AÊ ) 1.9 2.1 2.45 1.9 1.9No. of crystals 1 1 1 1 1No. of observations 88,372 37,192 49,707 84,660 73,910No. of unique reflections 33,656 25,528 16,729 35,002 35,929Data completeness (%) 87 (62) 93 (91) 96 (94) 93 (87) 95 (92)Completeness >3sI(%) 71 (31) 70 (46) 66 (33) 73 (37) 80 (54)I/sI 13.1 (3.1) 9.6 (4.1) 9.2 (2.7) 14.3 (3.5) 19.1 (5.2)Multiplicity 2.6 1.5 3.0 2.4 2.1Rmerge

a (%) 7.2 (24.1) 6.3 (17.4) 11.8 (39.1) 6.0 (27.3) 4.0 (16.9)

The values in parentheses are for the highest resolution bin (approximate interval of 0.1 AÊ ).a Rmerge � �hkl�ijIi ÿ hIij/jhIij, where Ii is the intensity for the ith measurement of an equivalent re¯ection with indices h,k,l.


unaccounted density in the H-site. Although this featureclearly indicated the binding position of CB, an unam-biguous ®t of the dye to the electron density maps wasnot possible because of the relatively low resolution ofthe data set (2.45 AÊ ), the low occupancy of the dye(despite a six week soaking time) and the size of the fea-tures indicating that a portion of the CB molecule mustbe disordered. Because of the uncertainty, a number ofdifferent conformations were built into the density. Eachwas subjected to positional and temperature factorre®nement and maps were generated from the resultingmodels. One model was selected as the one that bestaccounted for the density in the H-site (Figure 2(c)). This

Table 2. Re®nement statistics

SLZ S-nonyl GSH

Non-hydrogen atomsProtein 3299 3262Substrate/inhibitor 96 58Solvent 182 181Buffer 36 24

Resolution (AÊ ) 1.9 2.1Rconv. (%) 19.3 21.1Rfree (%) 22.2 26.2Reflections used in Rconv. calculations

Number 31,984 24,260Completeness (%) 82.2 88.4

rmsd values from ideal geometryBonds (AÊ ) 0.010 0.007Angles (�) 1.4 1.2Dihedrals (�) 22.6 24.0Impropers (�) 0.9 0.7Bonded B (AÊ 2) 2.9 2.8

Mean B (protein) (AÊ 2) 24.9 10.7Main-chain 23.6 10.0Side-chain 26.2 11.4

Mean B (solvent) (AÊ 2) 34.7 12.6Mean B (inhibitor) (AÊ 2) 36.2 26.9Occupancy of inhibitors

A monomer 0.56 1.0B monomer 0.60 1.0

Residues in most favoured regionsof Ramachandran plot (%) 93.6 94.7

model also exhibited the biggest drop in Rfree. In thismodel, only density for the fused ring system wasobserved, presumably the rest of the molecule is toomobile to be seen. For the BS complex, ®tting of thelarge volume of unassigned electron density in the H-sitewas greatly aided by the location of strong features cor-responding to the bromine atoms (Figure 2(d)). Place-ment of water molecules at these sites left residualstrong density in difference maps con®rming theirinterpretation as the bromine substituents of BS. Carefulanalysis showed there were two BS molecules in theH-site and they had overlapping densities. Each mol-ecule was assigned an occupancy of 0.5. Despite the long

CB BS DNP GSH

3262 3262 326244 90 64101 315 55724 24 24

2.45 1.9 1.922.9 21.2 18.128.0 24.9 23.8

15,910 32,933 32,23490.9 88.8 90.6

0.009 0.008 0.0071.3 1.2 1.223.7 22.9 23.10.7 0.7 0.72.0 2.4 2.422.9 18.6 14.823.0 17.6 13.822.8 19.8 15.922.4 26.9 28.346.1 70.6 26.8

1.0 0.50 1.01.0 0.50 1.0

90.5 92.7 94.1


soak time, the density for the non-bromine atoms wasweak indicating that each molecule was probably pre-sent at even lower occupancy. In addition, weak densityfor GSH was observed and was also assigned a partialoccupancy of 0.5. For the DNP GSH complex, the ®ttingof the inhibitor was unambiguous (Figure 2(e)).

Kinetics of SLZ binding

Expression plasmids, site-directed mutagenesis, pro-tein expression and puri®cation of wild-type, Y108F andY108V mutant enzymes were carried out as reported (LoBello et al., 1997; Nuccetelli et al., 1998). Inhibition exper-iments were performed using cuvettes of 0.5 cm opticalpathlength in 0.5 ml (®nal volume) of 0.1 M potassiumphosphate buffer (pH 6.5) containing 1 mM EDTA,1 mM GSH, CDNB (from 0.1 to 1 mM), suitableamounts, respectively, of wild-type, Y108F, Y108Venzymes and in the presence of ®xed inhibitor concen-trations ranging from 0 to 0.12 mM. The enzymaticactivities were determined spectrophotometrically at37 �C by following the increase in absorbance at 340 nmwhere the conjugate of CDNB with GSH absorbs(e � 9600 Mÿ1 cm-1) (Habig et al., 1974b). The enzymeswere preincubated with the inhibitor for ®ve minutesprior to the addition of the substrates. Spectrophoto-metric measurements were performed in a double-beamUvicon 940 spectrophotometer (Kontron Instruments)equipped with a thermostated cuvette compartment.Kinetic parameters were determined at a ®xed GSH con-centration by ®tting the collected data to the Michaelis-Menten equation by non-linear regression analysis usingthe Graph PAD Prism (Graph PAD Software, San Diego,CA) computer programs. The apparent Km values thusobtained were replotted against the correspondinginhibitor concentration (where Ki is the intercept on theX-axis of the latter plot).

Protein Data Bank accession numbers

The crystallographic data have been deposited in theBrookhaven Protein Data Bank under the ®lenames:13GS for the SLZ complex, 12GS for the S-nonyl GSHcomplex, 20GS for the CB complex, 19GS for the BS com-plex and 18GS for the DNP-GSH complex.

Acknowledgements

We thank Dr Matthew Wilce for his work on the earlystages of this project, Dan Nurco (University of Califor-nia at Davis) for alerting us to the existence of the SLZcoordinates and one of the referees for their insightfuland helpful comments about the early studies of ligan-dins. This work was supported in part by an AustralianResearch Council Project Grant and an AustralianResearch Council Senior Research Fellowship (toM.W.P.), a National Research Council of Italy Grant(Target Project on Biotechnology) (to M.L.B.), a NationalHealth and Medical Research Council PostgraduateResearch Scholarship and an International Centre for Dif-fraction Data Crystallography Scholarship (to A.J.O.).

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Edited by R. Huber

(Received 25 March 1999; received in revised form 7 July 1999; accepted 7 July 1999)

The ligandin (non-substrate) binding site of human pi class glutathione transferase is located in...

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Transcript of The ligandin (non-substrate) binding site of human pi class glutathione transferase is located in...