Induced Fit Movements and Metal Cofactor Selectivity ofClass II AldolasesSTRUCTURE OF THERMUS AQUATICUS FRUCTOSE-1,6-BISPHOSPHATE ALDOLASE*

Received for publication, October 16, 2003, and in revised form, December 19, 2003Published, JBC Papers in Press, December 29, 2003, DOI 10.1074/jbc.M311375200

Tina Izard‡ and Jurgen Sygusch§¶

From the ‡Department of Hematology-Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38111and the §Department of Biochemistry, Universite de Montreal, Montreal H3C 3J7, Canada

Fructose-1,6-bisphosphate (FBP) aldolase is an essentialglycolytic enzyme that reversibly cleaves its ketohexosesubstrate into triose phosphates. Here we report the crystalstructure of a metallo-dependent or class II FBP aldolasefrom an extreme thermophile, Thermus aquaticus (Taq).The quaternary structure reveals a tetramer composed oftwo dimers related by a 2-fold axis. Taq FBP aldolase sub-units exhibit two distinct conformational states corre-sponding to loop regions that are in either open or closedposition with respect to the active site. Loop closure remod-els the disposition of chelating active site histidine resi-dues. In subunits corresponding to the open conformation,the metal cofactor, Co2�, is sequestered in the active site,whereas for subunits in the closed conformation, the metalcation exchanges between two mutually exclusive bindingloci, corresponding to a site at the active site surface and aninterior site vicinal to the metal-binding site in the openconformation. Cofactor site exchange is mediated by rota-tions of the chelating histidine side chains that are coupledto the prior conformational change of loop closure. Sulfateanions are consistent with the location of the phosphate-binding sites of the FBP substrate and determine not onlythe previously unknown second phosphate-binding site butalso provide a mechanism that regulates loop closure dur-ing catalysis. Modeling of FBP substrate into the active siteis consistent with binding by the acyclic keto form, a minorsolution species, and with the metal cofactor mediatingketo bond polarization. The Taq FBP aldolase structuresuggests a structural basis for different metal cofactor spec-ificity than in Escherichia coli FBP aldolase structures, andwe discuss its potential role during catalysis. Comparisonwith the E. coli structure also indicates a structural basisfor thermostability by Taq FBP aldolase.

Aldolases are essential enzymes that catalyze carbon-carbonbond formation in living organisms. They are ubiquitous andhighly abundant in pathways of intermediate cellular metabo-lism such as gluconeogenesis, the Calvin cycle, and glycolysis,where they reversibly cleave ketohexose sugars. In syntheticchemistry, the action of aldolases is precisely controlled by thestereochemistry of these reactions, and thus these enzymes areoften used as an alternative to conventional chemical methodsin biotransformations and synthetic organic chemistry (1, 2)and especially in the synthesis of novel antibiotics (3, 4).

Aldolases that cleave ketohexose substrates are among themost studied enzymes and, depending on their reaction mech-anism, fall into two distinct groups. The class I enzymes utilizea lysine in Schiff base formation during catalysis and aremainly found in higher order organisms. Determination of thecrystal structures of several class I enzymes (5–12) togetherwith biochemical studies (13–19) have provided mechanisticdetails for ligand recognition and catalysis in class I aldolases.Structurally, these aldolases display an (�/�)8 barrel in a ho-motetrameric arrangement. In contrast, class II enzymes,found in yeast, bacteria, fungi, and blue-green algae, are mostoften homodimeric (�/�)8 barrels (20, 21) and require for catal-ysis a divalent metal cation, typically a transition metal suchas Zn2�. The divalent cation functions as a Lewis acid topolarize the carbonyl bond of the incoming ketoses, therebypromoting cleavage of the adjacent carbon-carbon bond as wellas proton transfer during enamine formation. Class II aldola-ses are activated by monovalent cations, such as NH4

�, aregenerally more stable than their class I counterparts, exhibit awide range of substrate specificity, and are preferred for use inbiotransformation chemistry (22, 23). Their reaction mecha-nisms are diverse. For instance, in one class II enzyme, 2-de-hydro-3-deoxy-galactarate aldolase, a phosphate anion ratherthan an amino acid side chain mediates proton transfer duringenamine formation (24). Chiral discrimination among class IIaldolases is subtle, and in the case of the stereoisomers fruc-tose-1,6-bisphosphate and tagatose-1,6-bisphosphate, recogni-tion and turnover depend on fine details of active site interac-tions made with substrate (25). Class II aldolases alsorepresent potential targets for the development of anti-bacte-rial and anti-fungal drugs because they almost exclusivelybelong to prokaryotes, yeasts, and lower order eukaryotes.

Among class II aldolases, FBP1 aldolase (E.C. 4.1.2) has beenextensively characterized because of its important metabolicrole in intermediate metabolism. The enzyme catalyzes thereversible aldol cleavage of FBP to the triose phosphates, di-hydroxyacetone phosphate and D-glyceraldehyde 3-phosphate.

* This work was supported by research grants from the NaturalSciences and Engineering Research Council of Canada and CanadianInstitutes of Health Research (to J. S.) and in part by Cancer Center(CORE) Grant CA21765 and funds from the American Lebanese SyrianAssociated Charities (to T. I.). This work was also supported in part bythe United States Department of Energy, Division of Materials Sciencesand Division of Chemical Sciences, under Contract DE-AC02-98CH10886. The costs of publication of this article were defrayed inpart by the payment of page charges. This article must therefore behereby marked “advertisement” in accordance with 18 U.S.C. Section1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 1RV8 and 1RVG)have been deposited in the Protein Data Bank, Research Collaboratoryfor Structural Bioinformatics, Rutgers University, New Brunswick, NJ(http://www.rcsb.org/).

¶ To whom correspondence should be addressed: Dept. of Biochemis-try, Universite de Montreal, C.P. 6128, Succ. Centre-Ville, MontrealH3C 3J7, Canada. Tel.: 514-343-2389; Fax: 514-343-6463; E-mail:jurgen.sygusch@umontreal.ca.

1 The abbreviations used are: FBP, D-fructose 1,6-bisphosphate;SeMet, seleno-L-methionine; Taq, Thermus aquaticus.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 12, Issue of March 19, pp. 11825–11833, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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Substrate cleavage occurs during glycolysis, and the reversereaction, aldol condensation, is used during gluconeogenesis orthe Calvin cycle. Binding sites corresponding to both the cata-lytic divalent metal ion as well as the activation site of themonovalent cation were identified in the high resolution FBPaldolase crystal structure from Escherichia coli (21). More re-cently, the crystal structures of E. coli FBP aldolase crystalstructure in complex with a triose phosphate transition stateanalogue suggested structural features involved in substraterecognition and processing (26). One important issue that re-mained unresolved was apparent induced fit movements thatclass II FBP aldolases undergo during the catalytic cycle andtheir relationship with active site binding. To address confor-mational changes during catalysis, the crystal structure ofclass II FBP aldolase was determined to 2.3 Å Bragg spacingfrom the extreme thermophile Thermus aquaticus, successfullycrystallized in the presence of sulfate, a phosphate anion ana-logue. The resultant crystal structure not only defined the roleof the induced conformational changes during the catalyticcycle but also provided an explanation as to the metal cofactoraffinity by Taq FBP aldolase for Co2�, rather than Zn2�, whichis preferred in mesophiles such as E. coli.

MATERIALS AND METHODS

Structure Determination—Native crystals of Taq FBP aldolase weregrown as described (27) by vapor diffusion using sitting drops made upof 8.75 �l of protein at 1.75 mg ml�1, 1 �l of 2 mM sucrose monolaurate,and 5 �l of the precipitant solution containing 1.7 M ammonium sulfate,0.1 M Tris-HCl, pH 7.5, and 10 mM CoCl2 that were then equilibratedagainst 1 ml of the precipitant solution at 295 K. The crystals harvestedwere cryoprotected in mother liquor to which had been added 20%glycerol before freezing in liquid nitrogen. The crystals of the seleno-L-methionine (SeMet) isoform of Taq FBP aldolase were obtained fromhanging drops (27) that were made up of 5 �l of protein solutioncontaining 0.25 mg ml�1 of Taq FBP aldolase and 2 �l of the precipitantsolution made of 0.6 M ammonium sulfate and 20 mM citric acid, pH 4,that was equilibrated against 1 ml of the precipitant solution at 295 K.The crystals were cryoprotected in mother liquor supplemented with15% glycerol before freezing in liquid nitrogen.

X-ray data collection for the SeMet isoform of Taq FBP aldolase andthe native protein in complex with cobalt has been described previously(27) and is summarized in Table I. The structure solution strategyconsisted of determining the structure of the SeMet isoform, crystal-lized in tetragonal space group I41 (a � b � 88.6 Å, c � 164.1 Å), andthen using it to solve by molecular replacement the native structure,crystallized in monoclinic space group P2 (a � 99.5 Å, b � 57.5 Å, c �138.6 Å, � � 90.25°). Multiple anomalous dispersion (MAD) data werescaled together with the CCP4 (28) program SCALEIT. Twelve of thefourteen selenium sites expected in the asymmetric unit were deter-mined using SOLVE (29). The N-terminal SeMet was not found, prob-ably because of positional disorder. Initial phases were calculated withthe program MLPHARE (30) and were improved by 200 cycles of sol-vent flattening and gradual phase extension from 3.65 to 2.8 Å resolu-

tion using the program DM (28). The final R factor for the phaseextension at 2.8 Å resolution was 0.347. The selenium positions allowedunambiguous matching of the electron density to the sequence andconstruction of an atomic model by using the program O (30). Themodest resolution and apparent mobility of the two loop regions (resi-dues 134–152 and 175–190), closing over the active site, made electrondensity tracing challenging.

Structure determination was substantially aided by the higher dif-fracting native enzyme x-ray data. The preliminary SeMet model wassuccessfully used as a search model to determine the native Taq FBPaldolase monoclinic crystal structure by molecular replacement usingthe program AMoRe (28). Structure solution revealed two half-tetram-ers in the asymmetric unit cell of the native enzyme, and except for theloop regions, the subunits within the half-tetramers could be related bynoncrystallographic 2-fold symmetry. The loops could be traced in threeTaq aldolase protomers whose loop regions corresponded to an openconformation, whereas only one loop was fully traced in the remainingTaq aldolase protomer and corresponded to the loop region having aclosed conformation. Electron density could not be associated with loopresidues 140–147 in the closed conformer.

Crystallographic Refinement—All Taq FBP aldolase structures wererefined with the program CNS (31, 32) using standard protocols. Thefree R value (33) was monitored throughout the refinement. Table IIlists the final parameters obtained for the native model. In the half-tetramer containing both protomer conformations, electron density wasweakest for residues 182–185 and 230 in the closed protomer and

TABLE IICrystallographic refinement statistics

Crystallographic refinement of Taq aldolaseNative

Co2� Y

No. of reflections 68,519 98,502Final model parameters

No. of amino acid residues 1,225 1,226No. of protein atoms 9,192 9,272No. of solvent molecules 933 974Resolution range (Å) 40.4–2.3 40.6–2Last shell (Å) 2.3–2.44 2–2.13R factor (overall)a 0.211 0.225R factor (last shell)a 0.265 0.358Rfree (overall)b 0.252 0.259Rfree (last shell)b 0.303 0.373Average main chain B factor (Å2) 33.25 30.45Average side chain B factor (Å2) 33.77 31.31Average water molecule B factor (Å2) 42.73 39.43

Root mean square deviation from idealgeometry

Covalent bond lengths (Å) 0.006 0.006Bond angles (°) 1.2 1.2a R factor � ¥hkl�Fobs�F calc�/¥hklF abs where N represents the num-

ber of equivalent reflections, and hkl represents the total number ofunique Bragg reflections.

b Rfree represents the R factor calculated for a test data set randomlyselected from the observed reflections prior to refinement. The test dataset contained 5% of the total observed data and was not used through-out refinement.

TABLE ICrystallographic data statistics

The diffraction data were collected at 100 K at beam line X-8C of the National Synchrotron Light Source at Brookhaven National Laboratory.

Data collection Edge Peak RemoteNative

Co2� Y

Wavelength (Å) 0.9791 0.9783 0.93 1.105 1.105Resolution (Å) 2.8 2.8 2.8 2.3 2.0Total data 387,740 390,048 396,141 989,324 912,165Space group I41 I41 I41 P2 P2Unique data 14,208 14,078 14,065 68,760 99,328Redundancy 27.3 27.7 28.2 14.4 9.2Overall completeness 0.912 0.911 0.908 0.977 0.928Completeness (last shell) 0.962 0.960 0.960 0.938 0.929F2 � 3� (F2) 0.859 0.860 0.851 0.793 0.774Rmerge (overall)a 0.092 0.088 0.088 0.075 0.067Rmerge (last shell)a 0.408 0.367 0.411 0.195 0.400�F2/� (F2)� 32.2 32.2 29.1 30.0 27.5

a Rmerge � �unique reflections ��i � 1N Ii � I/�unique reflections��i � 1

N Ii.

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residues 134–151, 180–187, and 230–231 in the open conformer. Thehalf-tetramer having all subunits in their open conformation densitywas weakest for residues 146–148, 182–187, and 229–231 in one sub-unit and 183 and 230 in the other subunit. The temperature factors ofthese residues refined to 40–50 Å2 only with the occupancy set to 0.5.The electron density was also weak for one of the two sulfate anionsbound to the active site of all three protomers in their open conforma-tion, and their occupancy was set to 0.5. Water molecules were initiallyidentified in the Fo � Fc maps and screened for reasonable geometryand refined thermal factor �80 Å2. The tables show the overall crys-tallographic R factor and the free R factor for all observed reflectionswithin the indicated resolution range. A Ramachandran plot analysisby the program PROCHECK (28) indicates that 90.4% of all residues liein most favorable regions, and 9.6% lie in additional allowed regions.The structure analysis also showed that all stereochemical parametersare better than expected at the given resolution. A Luzzati plot indi-cated a 0.33 Å error in the atomic coordinates.

Substrate Modeling—The acyclic conformation of FBP (atomic coor-dinates from Protein Data Bank entry 1FDJ) was modeled into theactive site of the refined structure by superposing the phosphate resi-dues onto the sulfate-binding sites. The substrate was oriented in theactive site such that its C1-phosphate coincided with the fully boundsulfate anion in the closed conformation and keto oxygen orientedtoward the metal cation. In this orientation, the C6-phosphate could bereadily superimposed onto the second sulfate-binding site. 200 cycles ofcoordinate energy minimization were then performed at 300 K usingCNS (version 1.1) without the x-ray term to relax possible bad contactsintroduced by substrate modeling into the active site. The coordinates ofthe protein and the substrate did not deviate significantly after mini-mization compared with the starting coordinates.

RESULTS

Structure of the Taq FBP Aldolase Protomer—The structureof the Taq FBP aldolase protomer (subunit molecular mass of33 kDa) adopts an (�/�)8 barrel fold (Fig. 1). The dimensionsof the protomer are 70 Å in height and 45 Å in width, witha depth of 38 Å. The barrel is closed on its N-terminal end by an�-helix (�0) comprising residues 5–14. The core of the structure

consists of an eight-stranded parallel �-strand assembly(strands labeled �1–�8), and each �-strand is accompanied byan �-helix. In addition to the eight times repeated �-strand-loop-�-helix-loop motif, the structure also contains a �-helixdirectly following a �-strand, in the case of �2 (labeled �-helix�2a), �7 (labeled �-helix �7a), and �8 (labeled �-helix �8a). Fur-thermore, the two C-terminal �-helices, �8a and �8, are anti-parallel to each other and create an arm from the barrel thatmediates oligomerization. Within each protomer, 27 pairs ofresidues are involved in electrostatic interactions.

Quaternary Structure—Class II Taq FBP aldolase behavesas a homotetramer in solution with a molecular mass of 139kDa (35) consistent with point group 222 (Fig. 2). The mutuallyperpendicular molecular dyads are defined as a right-handedset of axes P, Q, and R, where the P dyad is the crystallographic2-fold axis, whereas the Q and R dyads are the local 2-fold axes.The dimensions of the tetramer are 103 Å in height (along theR axis), 91 Å in width (along the P axis), and 83 Å in depth(along the Q axis). Each protomer is in contact with the otherthree subunits within the tetramer. The intersubunit interac-tions across the Q dyad are more extensive than those acrossthe R dyad (Fig. 2A). As a consequence, the former interfaceburies about three times as large a surface area (1844 Å2

buried per subunit, representing 27% of total surface area) asthe latter (582 Å2 buried per subunit, or 9% of total surfacearea) upon tetramerization, whereas the interactions betweenthe P axis-related subunits are minor (386 Å2 per subunit, 6%of total surface; Fig. 2B). This arrangement results in a dimerof dimers within the homotetramer.

The interdimer interactions across the R dyad involve resi-dues located on �-helices �2, �3, and �4, as well as those in theloop regions following �-helices �3 and �4. Dimer-dimer inter-actions implicate seven residues (Leu89, Arg90, Arg93, Gly95,

FIG. 1. Stereo cartoon drawing of the Taq class II FBP aldolase protomer. �-Helices are depicted by blue helical ribbons, and �-strandsare depicted by yellow arrows. FBP aldolases undergo induced fit movements involving conformational changes of a large loop (located following�-strand �5) and a small loop (located following �-strand �6). All of the figures were produced with Raster3D (43) and either MolScript (44) orBobScript (45).

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Ala121, Val125, and Val127) that participate in hydrophobic in-teractions and four residues (Tyr85, Leu92, Arg93, and Phe96)that promote interdimer hydrogen bonds.

More extensive and predominantly hydrophobic interactionsare formed between Q axis-related subunits. Several secondarystructural elements contribute up to 40 residues that partici-pate in intersubunit (intradimer) van der Waals’ contactsacross the Q dyad. These contacts involve residues located onthe �-helices �1, �2, �2a, �7a, �8a, and �8, as well as thoselocated on the loops following �-helices �1, �6a, and �7a. Thisinterface implicates 27 residues that participate in hydropho-bic interactions, 12 residues (Asn25, Glu28, Tyr55, Gly56, Arg58,

Thr261, Arg265, Pro272, Phe275, Arg278, Tyr280, and Arg285) thatpromote 19 intersubunit hydrogen bonds, and three electro-static interactions between residues Glu70 and Arg58, Glu227

and Lys273, and Asp276 and Arg257. Only one electrostatic in-teraction is observed across the crystallographic related sub-units, between residues Glu7 and Arg72. Finally, some hydro-phobic interactions are also formed across the P axis-relatedsubunits involving residues Met1, Glu7, Glu70, Ala71, andArg72.

Open and Closed Conformations—Taq FBP aldolase crystal-lizes using ammonium sulfate as the precipitating agent atphysiological pH in space group P2 or at pH 4 in space groupI41 (27). The SeMet-substituted protein crystals are tetragonal,with two protomers in the asymmetric unit and diffraction to2.8 Å Bragg spacing. Diffraction to 2.3 Å Bragg spacing wasobserved from native Taq FBP aldolase monoclinic crystalswith four protomers in the asymmetric unit. The two protomersin the tetragonal cell are in the closed conformation, whichcontains one small loop (residues 175–190) and one large loop(residues 134–152) that close over the active site. These twoloop regions show that weak electron density and residues138–149 and 176–188 are disordered in the SeMet model. Incontrast, native monoclinic protein crystals in the presence ofthe cofactor Co2� comprise one protomer in the closed confor-mation shown in Fig. 3B, with continuous electron density forresidues 1–139 and 148–305 and three protomers in the openconformation having electron density for all 305 residuesshown in Fig. 3A. Application of the crystallographic 2-foldsymmetry thus generates one tetramer having all four pro-tomers in the open conformation and one tetramer with twoprotomers in the open conformation and two protomers in theclosed conformation.

Cobalt in the Active Site—The catalytic cobalt in Taq FBPaldolase was identified based on its coordination and peak sizein Fo � Fc electron density maps. During refinement, differenceFourier electron density maps were calculated from modelscomprising all atoms except those subsequently identified ascobalt cations. The three strongest peaks in the differenceelectron density maps were 24–28 times over the noise level.These three peaks are the best candidates for the binding sitesof the catalytic Co2� in the three subunits in their open con-formation within the asymmetric unit. Interestingly, the pro-tomer in the closed conformation has two mutually exclusivebinding sites for cobalt metal cations (Fig. 4A). This dual bind-ing is mediated by a conformational transition involving sidechain rotations that occur following chelation by histidine res-idues 81, 178, and 208 (Fig. 4A). Because these two mutuallyexclusive Co2� cations are only partially occupied, their corre-sponding peaks in the difference electron density maps are onlyhalf the height (14 times the noise level) of that of the singlecobalt ion having full occupancy found in the other three sub-units within the asymmetric unit that display the open confor-mation. Ligands for the two mutually exclusive cobalt cationsin the closed protomer include nitrogen atoms from His81 (2.3and 2.5 Å), His178 (2.1 and 2.4 Å), and His208 (2.2 and 2.4 Å) andone oxygen atom from a water molecule (2.3 and 2.8 Å), respec-tively (Fig. 4A). In contrast, candidate ligands for the singlecobalt cation found in the three open protomers include notonly imidazole rings of His81 (2.2, 2.3, and 2.3 Å), His178 (2.0,2.1, and 2.2 Å), His208 (2.3, 2.3, and 2.4 Å), and one oxygen atomfrom each three water molecules (2.3, 2.4, and 2.4 Å) but alsothe carboxylate of Glu132 (2.2 Å in all three protomers; Fig. 4B).

Monovalent Cation Binding—Cations, such as NH4� or K�

are known to activate class II FBP aldolases and in the case ofTaq aldolase, only NH4

� cations activate the enzyme (34).Strong electron density in the difference maps was observed for

FIG. 2. A cartoon drawing of the FBP aldolase oligomer withpoint group 222. The three different molecular dyads comprise aright-handed orthogonal set of axes P, Q, and R as originally defined forthe three 2-fold axes of lactate dehydrogenase (46). In A, the view islooking down the crystallographic dyad (P), while in B the orientation islooking down the molecular dyad (R). The dyads (R and Q in A and Pand Q in B) are indicated by solid lines. Each protomer is shown in adifferent color.

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FIG. 3. Superposition of class II al-dolase structures. Stereo C�-trace su-perposition of the Taq FBP aldolase pro-tomer bound to Co2� in its openconformation (yellow in A), the closed sub-unit of the enzyme when bound to yttrium(yellow in B), and the E. coli FBP aldolasestructure (Protein Data Bank ID code1DOS; red in A), onto the E. coli FBPaldolase structure in complex with thetransition analogue phosphoglycolohy-droxyamate (Protein Data Bank ID code1B57; red in B). The alignment was per-formed pairwise using SwissProt (39).The largest root mean square deviationwas 1.33 Å for 221 C� atoms in commonbetween E. coli aldolase and Taq aldolaseprotomer in closed conformation. Thelarge and small loop regions of Taq aldol-ase protomers are illustrated in gray, andthose of E. coli aldolase are shown in pur-ple. In A are grouped Taq and E. colialdolase subunits whose loop conforma-tions correspond to the open position. Bshows E. coli aldolase and Taq aldolasesubunits with loop conformation in theclosed or bound position. Also shown in Bis substrate FBP that was modeled (usingacyclic FBP coordinates of Protein DataBank entry 1FDJ) into the active site andsubjected to energy minimization. Thebonds of FBP are drawn in green andshown in ball-and-stick representation. C,shows in detail modeling of FBP guidedby two sulfate anions (bonds in gray)whose positions were consistent with thephosphate-binding site of the substrate inthe acyclic form. The bonds of the transi-tion state analogue, phosphoglycolohy-droxymate, are drawn in black.

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catalytic cation sites in all four subunits in the asymmetricunit, with peak heights ranging from 7 to 11 standard devia-tions above the noise level. The putative NH4

� cation sites weremodeled with sodium scattering factors (Fig. 4B). The cation iscoordinated by the nitrogen atoms of the imidazole ring of His78

(with distances ranging from 2.8 to 3 Å in the four subunits)and the oxygen atoms of the side chains of Asp80 (2.8 to 3 Å),Glu130 (2.8 to 2.9 Å), Asn251 (3.2 Å), and a water molecule (3 to3.3 Å).

Novel Cation-binding Site—Taq FBP aldolase crystals weresoaked in the presence of an yttrium chloride salt, which iden-tified an additional metal-binding site (Fig. 4C). This metal-binding site has not been previously reported and is uniquely

found in the protomer in its closed conformation. The metal iscoordinated by two oxygen atoms of the side chain of Asp102

(2.7 Å), one oxygen atom of the carboxylate group of Glu132

(2.9 Å), and the hydroxyl of Ser104 (2.8 Å), Wat545 (2.9 Å) andcontacts a sulfate anion. The metal refined to a temperaturefactor below 40 Å2. The site was modeled using a strontiumscattering factor, which has a scattering factor almost identicalto that of yttrium.

Sulfate Binding—Taq FBP aldolase crystallizes from highconcentrations of ammonium sulfate as the precipitating agent(27). Under these concentrations, sulfate efficiently competeswith phosphate or phosphate-containing compounds for bind-ing to proteins. Two strong peaks of 17–19 times the noise level

FIG. 4. Stereo view of Taq FBP aldolase active site. Final �A weighted Fo � Fc omit electron density map for ligands bound to the enzyme.The contour level of the electron density map is 4 �, and the resolution is 2.3 Å. The bonds of the ligands are drawn in pink, whereas the bondsof the enzyme are shown in light gray. For clarity, water molecules (drawn as spheres) are not labeled. Residues belonging to a 2-fold relatedsubunit are italicized. A, protomer in the closed conformation showing residues in contact with the sulfate anions that coincide with thephosphate-binding sites of FBP, the two mutually exclusive Co2� cofactors (drawn as light blue spheres) and the activating cation (drawn as a blacksphere). B, sulfate and cation binding to the active site as observed in the subunits in their open conformation. Orientation was rotated by 15° withrespect to A to reveal Asn251 that interacts with the monovalent cation. C, FBP modeled into the active site using the sulfate-binding sites of theclosed protomer as phosphate oxyanion templates in the Taq FBP aldolase complex with yttrium. The novel metal-binding site (yttrium) is drawnas a green sphere. The hydroxyls O2 and O3 of the FBP molecule are within close contact of the exterior Co2� site (indicated by dashed lines).

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are found in the difference Fourier maps that correspond to twosulfate anions bound to the active site of the protomer in theclosed conformation. One sulfate anion engages in hydrogenbonds to the hydroxyl of Ser211, the amides of Gly179, Asp253,and Thr254, as well as to two water molecules (Fig. 4A). Gly179

resides on the small loop that closes over the active site in thisprotomer. The second sulfate anion is located 10 Å away andmakes an electrostatic interaction with the side chain of Arg278

as well as hydrogen bonding to the hydroxyl of Ser49 and threewater molecules.

The same two sulfate anions are also found in the threesubunits in the asymmetric unit in their open conformation(Fig. 4B). However, because residue Gly179 of the closed pro-tomers is not available for binding to the sulfate anion, thissulfate anion does not bind as tightly as in the protomer inthe closed conformation. Difference electron density for thissulfate anion was weaker (with only a half of the signal tonoise ratio (10) versus to the closed subunit). The occupancywas also reduced to one-half to obtain a refined averagetemperature factor of 46.2 Å2 (compared with 36.2 Å2 in theclosed subunit, where this anion has full occupancy). Bycontrast, the second sulfate anion in the active site binds tothe open subunit in a fashion similar to that in the closedsubunit (Fig. 4B).

Residues Arg116 and His123 at the C-terminal end of �-helix4 bind a third sulfate anion in all four protomers within theasymmetric unit. The site is located at the solvent surface ofeach protomer and is distant from the active site. Again, theelectron density was also weak for this ligand, and the occu-pancy was set to one-half in all subunits to obtain a refinedaverage temperature factor of 43.9 Å2. In the current model,the sulfate ion accepts hydrogen bonds from the side chains ofArg116 (2.7 Å) and His123 (2.8 Å), as well as from two watermolecules (2.7 and 2.9 Å) in each protomer. In the closedconformation, an additional sulfate ion complexes with Arg135

side chain, one water molecule, and the yttrium ion (shown inFig. 4C).

DISCUSSION

Molecular Details of Hyperthermostability—Electrostatic in-teractions are the major determinant in hyperthermostabilityof a protein (35, 36). Taq aldolase, which is stable at 90 °C forseveral hours (37), has 27 intramolecular salt bridges in itstertiary structure. Of these, only 10 similar interactions areobserved in the E. coli FBP aldolase crystal structure. In par-ticular, an intricate network of ion pairs between �-helices �1

and �8 in the Taq FBP aldolase crystal structure is replaced bytwo single salt bridges in the E. coli structure. Glutamateresidues, Glu35 (corresponding to Glu47 in E. coli aldolase),Glu39 (Lys51 in E. coli aldolase), Glu286 (Thr339 in E. coli aldol-ase), and Glu290 (Ala343 in E. coli aldolase) surround lysineresidues Lys289 (Ile342 in E. coli aldolase) and Lys293 (Glu346 inE. coli aldolase) that results in multiple electrostatic interac-tions. In E. coli aldolase, residue substitution, although yield-ing two compensating electrostatic interactions, Lys51–Glu346

and Glu47–Arg335, excludes formation of an ion-pairing net-work. In a sequence alignment of 18 class II FBP aldolasesequences (38), only 10 of 45 residues involved in electrostaticintramolecular interactions observed in the Taq aldolase struc-ture are invariant.

Three intermolecular ion pairs in Taq FBP aldolase areobserved between subunits related by the Q dyad. Only one ofthese (Arg58–Asp70) are found in the analogous E. coli structure(Lys71–His99). In the thermophile structure, Arg58 is involvedin a network of ion pairs involving Glu66 and Arg69 and the2-fold related residues (Arg58, Glu66, Arg69, and Asp70). Addi-tional strong interactions were also found across subunits re-lated by the P dyad. In particular, the side chains of Glu7 andArg72 are within hydrogen bonding distance (2.8 Å) and thesulfur atoms of the 2-fold related Met1 participate in van derWaals’ interactions. No electrostatic interactions were found inthe Taq FBP aldolase structure across the remaining interface.Protomers related by the R dyad involve, however, a consider-able number of hydrophobic contacts involving residues Tyr85,Leu89, Arg90, Leu92, Arg93, Phe96, Ala121, Ala124, Val125, andVal127.

FIG. 4—continued

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Additional structural features that contribute to thermosta-bility are short loops and close packing as exemplified by fewercavities (36). Pairwise superposition of the crystal structures ofclass II FBP aldolase from E. coli and T. aquaticus withSwissProt (39) is shown in Fig. 3; the largest root mean squaredeviation was 1.33 Å for 221 C� atoms in common betweenE. coli aldolase and Taq aldolase protomer in closed conforma-tion. The superpositions demonstrate that the Taq aldolasestructure has shorter loops and a higher degree of secondarystructure, creating a more compact structure that is consistentwith structural attributes found in thermostable proteins.Overall, loop residues make up 158 amino acids in the crystalstructure of the E. coli enzyme, whereas this amount is reducedto 125 residues in Taq aldolase. For instance, the loop connect-ing �-helices �2a and �2 in the E. coli structure consists of 10residues (residues 70–79), whereas only three residues (resi-dues 56–58) are engaged in a tight turn on the thermophilicstructure. In addition, the loop following �-helix �3 in theE. coli structure involves 11 additional residues (residues 127–137) not present in Taq aldolase. The E. coli structure has also10 additional residues without secondary structure at its Nterminus compared with the Taq structure. By contrast, �-he-lix �7a and nine additional residues comprising the sequence215PELVERFRASGGEIGEAA232 in Taq aldolase are not pres-ent in the E. coli structure. Hydrophobic amino acids from thissequence pack against �-helices �8a and �8 that are antiparal-lel to each other and create an arm that protrudes from thebarrel (Fig. 1). The arm appears to mediate oligomerizationbecause of the large number of hydrophobic amino acids mak-ing intersubunit van der Waals’ interactions with the Q-relatedprotomer (Fig. 2A). The arrangement results in residues on�-helix �8a of the Q-related protomer interacting with residuesresiding on �-helix �7a. The inserted sequence of 17 residuesthus not only enhances subunit stability but also promotesdimer formation and is consistent with the enhanced thermo-stability of Taq aldolase.

Induced Fit Movements in Class II FBP Aldolases—Class IIFBP aldolases undergo induced fit movements during cataly-sis. C� backbone traces for Taq aldolase protomers in open andclosed conformation (shown in Fig. 3) suggest that the smallloop (residues 175–190) and a larger loop (residues 134–152)undergo conformational changes upon active site ligand bind-ing. Comparison of the crystal structure of E. coli class II FBPaldolase determined to 1.6 Å (21) and 2.5 Å (24) as well as incomplex with phosphoglycolohydroxamate to 2.7 Å (26), whichresembles the ene-diolate transition state of the dihydroxyac-etone phosphate substrate, corroborates a lid closure mecha-nism mediating ligand binding. In the unbound E. coli struc-tures, these flexible loops exhibited positional disorder tovarious extents, and both small and large loops display an openconformation, shown in Fig. 3A. In the case of the large loop, itsconformation, although open, has shifted somewhat toward aclosed position compared with the more open conformationobserved in the thermostable enzyme. In the bound E. colistructure, the small loop closes over the active site (shown inFig. 3B) and adopts a conformation observed in the Taq en-zyme. Furthermore, as in the Taq aldolase closed protomerconformation, a number of residues of the equivalent large loopregion of the E. coli enzyme could not be traced in the ligandcomplex, supporting enhanced flexibility by residues in thelarge loop and originating most likely from fewer positionalconstraints as a result of the conformational change by theadjacent small loop.

What is the mechanism that is responsible for lid closure?The small loop conformations point to occupancy of the sul-fate-binding site as a means by which to influence the stabil-

ity of the small loop in its closed conformation and hence lidclosure. The three protomers in their open conformation bindonly weakly to the sulfate anion that interacted with thesmall loop in the closed position as indicated by partial occu-pancy and consistent with a high millimolar Kd (1.7 M am-monium sulfate concentration used in crystallization condi-tions). In contrast, the protomer in its closed conformationexhibits full occupancy sulfate binding because of lid closure,indicating tighter sulfate ion binding. The protomer in theclosed conformation cannot open its lid because of sterichindrance of Ile139 with the symmetry-related Val143. Simi-larly, the lid position in the three subunits having an openconformation is also stabilized by crystal contacts. In partic-ular, Val143 interacts with the symmetry-related Phe108, andthe carbonyl of Ala144 is hydrogen-bonded to the side chain ofthe symmetry-related Arg164. The crystal contacts by stabi-lizing distinct loop conformations clearly show that lid clo-sure enhances sulfate anion affinity. Conversely, the twodistinct conformational states are not inconsistent with aninduced fit mechanism whereby lid closure is promoted byligand attachment.

The sulfate anion-binding sites have enabled the identifica-tion of the FBP binding mode in the Taq aldolase active site.The position of the C1-phosphate moiety in the structure of theE. coli enzyme in complex with the transition state analogue,phosphoglycolohydroxamate, coincides with that of a sulfateion present in all protomers of our structure (Fig. 3C) and thatmediates lid closure. Furthermore, mutagenesis of Arg331 inE. coli aldolase perturbs FBP C6-phosphate binding (40), sug-gesting that the sulfate anion interacting with the equivalentArg278 in all Taq aldolase protomers delineates the C6-phos-phate binding locus. Superposition of the FBP C1- and C6-phosphate moieties with the appropriate sulfate anion-bindingsites is consistent with binding by the acyclic keto form of FBPin both open and closed conformations. The FBP orientation,shown in Fig. 4C, is free of steric conflicts, and loop closureindeed traps the C1-phosphate, whereas the C6-phosphate isable to interact with Arg278. Additionally, FBP C3 and C4

hydroxyls hydrogen bond with Asp80 and Asp253, respectively.The equivalent aspartate residues in the E. coli structure,Asp109 and Asp288, respectively, when mutagenized compro-mise FBP as well as dihydroxyacetone phosphate binding (41)validating the docking of the acyclic keto form of FBP in thisorientation. The docked FBP conformation corresponding tothe nascent dihydroxyacetone phosphate portion of FBP alsomimics the binding observed for the transition state analoguein the active site of E. coli aldolase, shown in Fig. 3C (26).Unique to the closed conformation, FBP docking allows inter-action by the C2 keto with the Co2� cation bound furthest fromGlu132, i.e. the solvent-accessible hence exterior binding site,consistent with a reaction mechanism where the cation is ableto polarize the keto moiety. Cyclic forms of FBP did not allowsuperposition of phosphates moieties with the binding sites forthe sulfate oxyanions.

The loop formed by residues 134–152 has undergone, in theclosed subunit conformation shown in Fig. 3B, a significantconformational change compared with the open position shownin Fig. 3A that flips it toward the active site. Leu136 in the opensubunit conformation is repositioned 9.4 Å, based on C� coor-dinates, closer to the active site and whose side chain, pointingtoward the active site interior, would provoke a steric clashwith the His178 side chain, located on the small loop, were itpositioned as in the open conformation in Fig. 3A. Lid closurethus requires coordinate movement of both small and largeloops. The displacement by the His178 side chain (5.2 Å usingC� coordinates) into the closed conformation remodels the

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Co2�-binding site with respect to the binding site observed inprotomers in the open conformation; the Co2� cation at theinterior site (closest to Glu132) now interacts only indirectlywith Glu132 through an intervening water molecule, Wat545.As a result, Glu132 and Wat545 are able to promote synergisticbinding of the yttrium cation with the sulfate anion, implyingthat cations in presence of oxyanions could activate class IIaldolases by enhancing the stability of the closedconformation.

Exchange by the transition metal cation between the twomutually exclusive binding sites is not sterically hindered,involving merely small side chain rotations by the chelatinghistidine residues. The emerging picture of the catalytic cyclein class II aldolases is therefore that of a two-stage processwhereby the C1-phosphate stabilizes a large conformationalchange, as exemplified by lid closure, that remodels the activesite. The active site rearrangement allows the Co2� cation toexchange between two overlapping sites (1.85 Å apart in Taqaldolase). Occupancy of the exterior site renders the Co2� cat-ion competent to act as a Lewis acid by polarizing the ketooxygen during the catalytic cycle. It may be speculated that theconformational change and/or active site cation exchange gatecatalytic events.

Metal Cation Preference—One outstanding question is the ba-sis for metal cation preference by class II FBP aldolases. In thethermophilic enzyme, the largest activation occurs with metalcofactors Co2� and Fe2�, whereas in the mesophilic enzyme,Zn2� displays highest activation. Inspection of the active sitesshows that the Co2� cation is hexa-coordinated in the open con-formation in Taq aldolase, whereas at the equivalent interior sitein the E. coli aldolase structure, the Zn2� cation is tetra-coordi-nated. Both cations coordinate the same number of nitrogenatoms from histidine residues but differ as to the number ofcoordinating oxygen atoms. The Co2� cation coordinates an ad-ditional water molecule and interacts in a bidentate manner withthe Glu132 carboxylate side chain rather than binding in a mo-nodentate manner as observed in the mesophilic enzyme. Thecomposition of the coordination sphere of two cations thus entailsa greater preference for oxygen lone pairs by Co2� cation that isconsistent with greater hardness of the Co2� cation as a Lewisacid compared with Zn2� (42). Residues comprising the activesite and coordinating the Zn2� and Co2� cations are identical inthe mesophilic and thermophilic class II aldolases, and superpo-sition of the active sites reveals no significant structural differ-ences. The cation binding preference thus appears to be deter-mined by hardness of the metal cation as Lewis acid and possiblyby subtle structural differences between the two enzymes.Greater active site coordination by the Co2� cation in the ther-mophilic enzyme compared with Zn2� suggests enhanced activesite integrity at higher temperatures. A similar considerationwould also apply to the reaction trajectory. The Zn2� cationin E. coli aldolase complexes the transition state analogue,phosphoglycolohydroxamate, by interaction through the C2

and C3 oxygens as well as the active site histidine residues(26). Stability of a similar transition state architecture in thethermophilic enzyme would be enhanced at higher tempera-tures by interaction with the Co2� cation. Enhanced activesite integrity is supported by the 15-fold reduction in activ-ity using Zn2� as a metal cofactor in the thermophilic enzyme(35). Furthermore, the higher activation by Fe2� comparedwith Co2� in Taq aldolase (35) concurs with the even greaterhardness of the Fe2� cation, supporting the hypothesis thathardness of the metal cofactor enhances active site integrityat high temperatures.

Acknowledgments—We thank Veronique Sauve for growing Taq al-dolase crystals and Dr. John Cleveland (St. Jude’s) for helpful com-ments on the manuscript. The assistance of Dr. Tae-Sung Yoon in datacollection and reduction is gratefully acknowledged. Assistance by X8-Cbeam-line personnel is gratefully appreciated.

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Tina Izard and Jurgen SyguschALDOLASE

STRUCTURE OF THERMUS AQUATICUS FRUCTOSE-1,6-BISPHOSPHATE Induced Fit Movements and Metal Cofactor Selectivity of Class II Aldolases:

doi: 10.1074/jbc.M311375200 originally published online December 29, 20032004, 279:11825-11833.J. Biol. Chem. 

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