Understanding protein lids: structural analysis of active hinge mutants in triosephosphate isomerase

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1 UNDERSTANDING PROTEIN LIDS: STRUCTURAL ANALYSIS OF ACTIVE HINGE MUTANTS IN TRIOSEPHOSPHATE ISOMERASE Kursula, I. 1,2 , Salin, M. 1 , Sun, J. 3# , Norledge, B.V. 1* , Haapalainen, A.M. 1 , Sampson, N.S. 3 and Wierenga, R.K. 1 1 University of Oulu, Department of Biochemistry and Biocenter Oulu, P.O. Box 3000, FIN-90014 University of Oulu, Finland, 2 European Molecular Biology Laboratory, Hamburg Outstation, c/o DESY, Notkestrasse 85, Building 25A, D- 22603 Hamburg, Germany, 3 Department of Chemistry, State University of New York, Stony Brook, NY 11794-3400, USA Corresponding author: R.K. Wierenga, University of Oulu, Department of Biochemistry and Biocenter Oulu, P.O. Box 3000, FIN-90014 University of Oulu, Finland, Tel: +358-8-5531199, Fax: +358-8-5531141, E-mail: [email protected] Current addresses: # Pharmaceutical Chemistry, University of California, San Francisco, USA; *The Scripps Research Institute, La Jolla, California 92037, USA Running title: Structural analysis of TIM hinge mutants Edited by Alan Fersht Protein Engineering, Design & Selection © Oxford University Press 2004; all rights reserved Received May 17, 2004; accepted May 19, 2004 PEDS Advance Access published May 27, 2004 by guest on July 16, 2015 http://peds.oxfordjournals.org/ Downloaded from

Transcript of Understanding protein lids: structural analysis of active hinge mutants in triosephosphate isomerase

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UNDERSTANDING PROTEIN LIDS: STRUCTURAL ANALYSIS OFACTIVE HINGE MUTANTS IN TRIOSEPHOSPHATE ISOMERASE

Kursula, I.1,2, Salin, M.1, Sun, J.3#, Norledge, B.V.1*, Haapalainen, A.M.1, Sampson,N.S.3 and Wierenga, R.K.1

1University of Oulu, Department of Biochemistry and Biocenter Oulu, P.O. Box3000, FIN-90014 University of Oulu, Finland, 2European Molecular BiologyLaboratory, Hamburg Outstation, c/o DESY, Notkestrasse 85, Building 25A, D-22603 Hamburg, Germany, 3Department of Chemistry, State University of New York,Stony Brook, NY 11794-3400, USA

Corresponding author: R.K. Wierenga, University of Oulu, Department ofBiochemistry and Biocenter Oulu, P.O. Box 3000, FIN-90014 University of Oulu,Finland, Tel: +358-8-5531199, Fax: +358-8-5531141, E-mail: [email protected]

Current addresses: #Pharmaceutical Chemistry, University of California, SanFrancisco, USA; *The Scripps Research Institute, La Jolla, California 92037, USA

Running title: Structural analysis of TIM hinge mutants

Edited by Alan Fersht

Protein Engineering, Design & Selection © Oxford University Press 2004; all rights reserved

Received May 17, 2004; accepted May 19, 2004

PEDS Advance Access published May 27, 2004 by guest on July 16, 2015

http://peds.oxfordjournals.org/D

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ABSTRACT

The conformational switch from open to closed of the flexible loop-6 oftriosephosphate isomerase (TIM), is essential for the catalytic properties of TIM.Using a directed evolution approach, active variants of chicken TIM with a mutatedC-terminal hinge tripeptide of loop-6 have been generated (Sun and Sampson 1999,Biochemistry 38, 11474-11481). In chicken TIM, the wild type C-terminal hingetripeptide is KTA. Detailed enzymological characterization of six variants showedthat some of these (LWA, NPN, YSL, KTK) have decreased catalytic efficiency,while others (KVA, NSS) are essentially identical to wild type. The structuralcharacterization of these six variants is reported. No significant structural differencescompared to the wild type, are found for KVA, NSS, and LWA, but substantialstructural adaptations are seen for NPN, YSL, and KTK. These structural differencescan be understood from the buried position of the alanine side chain in the C-hingeposition-3 in the open conformation of wild type loop-6. Replacement of this alaninewith a bulky side chain causes the closed conformation to be favored, whichcorrelates with the decreased catalytic efficiency of these variants. The structuralcontext of loop-6 and loop-7 and their sequence conservation in 133 wild typesequences is also discussed.

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KEYWORDS

Archae/evolution/flexible loop/structure/TIM

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INTRODUCTION

Many enzymes use flexible loops that change conformation upon substrate binding toform a catalytically competent active site. One of the most studied of such enzymes istriosephosphate isomerase (TIM; E.C. 5.3.1.1). TIM is a dimeric, glycolytic enzyme,which catalyzes the interconversion of dihydroxyacetonephosphate (DHAP) and D-glyceraldehyde-3-phosphate (DGAP) (Knowles 1991; Figure 1). The flexible loop inTIM is loop-6, which is located after the sixth b-strand of the TIM barrel. In fact, inaddition to loop-6, also loop-7 after b-strand-7 changes conformation upon ligandbinding (Figure 2). In the liganded closed form of the active site, loop-7 interacts withloop-6 via the side chains of its highly conserved YGGS-motif (Sampson andKnowles 1992).

Both NMR data (Williams and McDermott 1995, Rozovsky and McDermott 2001,Rozovsky et al. 2001) and laser induced temperature jump relaxation spectroscopy(Desamero et al. 2003) indicate that loop-6 is opening and closing in both theliganded and the unliganded structures, at about the same timescale, which is similarto the catalytic conversion rate. The closed form is favored in the presence of boundligand, while the open form is favored in the absence of ligand. From X-raycrystallography it is observed that the open conformation, in which loop-6 interactswith loop-5, has characteristically rather high B-factors, while the ligandedconformation, in which loop-6 interacts with the YGGS-motif of loop-7, is welldefined (Kishan et al. 1994). Recent crystallographic data, showing that the closedform can be seen in the absence of ligand (Aparicio et al. 2003) and the open form inthe presence of ligand (Parthasarathy et al. 2002), are in good agreement with theNMR-data indicating that the loop-6 breathing motion exists in both the liganded andunliganded complexes. Such a mechanism will facilitate product release aftercompletion of the reaction cycle (Kursula and Wierenga 2003, Jogl et al. 2003).

Loop-6 consists of 11 residues, which can be divided into a three residue N-terminalhinge, a five residue rigid tip of the loop, and a three residue C-terminal hinge (Figure2). As highlighted in Figures 2 and 3, the N-hinge runs parallel to loop-7 while the C-hinge runs antiparallel to loop-7. The catalytic glutamate 165 is located just before thestretch of 11 residues of loop-6. After these 11 residues, Thr177 and Pro178 are,respectively, the N-cap and N-cap+1 residues of helix-6 (Figures 2, 3). In the open,unliganded conformation loop-6 interacts with loop-5; the conformational switch is amovement of 7Å of the tip of the loop from loop-5 towards loop-7. This opening-closing movement of loop-6 concerns small changes of the phi/psi values in the C-terminal and N-terminal hinges, while the central five residues move as a rigid body(Joseph et al. 1990, Wierenga et al. 1991a). The largest changes in phi/psi values(approximately 50o) are observed for psi(Lys174) and phi(Thr175). These twochanges compensate each other; consequently, only the position of the peptide oxygenof Lys174 is affected. In the closed conformation, O(Lys174) is in a tight van derWaals contact with Cb(Ala169). Only in the closed conformation Cb(Ala169) is nearCg of the strained, planar Pro166 (Figure 3), which subsequently contacts Cz(Tyr164)(Kursula and Wierenga, 2003) . The closure of loop-6 affects the active site geometryin several ways, for example (i) it facilitates the rotation of the side chain of thecatalytic residue Glu165 to a position competent for catalysis; the “swung-in”conformation (Wierenga et al. 1991b), also (ii) the substrate-enzyme interaction isshielded form bulk solvent by the side chain of Ile170 in the closed loop-6

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conformation, and (iii) the N(Gly171) forms a hydrogen bond with the phosphatemoiety of the substrate (Lolis and Petsko 1990).

The closure of loop-6 is a concerted motion with the loop-7 conformational switch,which allows for hydrogen bonding between the side chains of Tyr208 and Ser211 ofthe conserved YGGS-motif of loop-7 with main chain atoms of loop-6 (Figures 2, 3).The structural switch of loop-7 is also required for making a competent active site: (i)the peptide bond between Gly209-Gly210 rotates 90o, to allow the catalytic glutamateto move to the catalytically competent swung-in conformation (Kursula et al. 2001)and (ii) the peptide flip of the peptide bond between Gly210-Ser211 allows N(Ser211)to hydrogen bond to the phosphate moiety. The flip of the latter peptide moietychanges the phi/psi values of Ser211 from (-80o, 120o) in the open form to (65o, 30o)in the closed form (Noble et al. 1993). The YGGS motif of loop-7 is highlyconserved. As shown in Figure 4, its occurrence is uniquely correlated with thetryptophan in the N-terminal hinge position-3. Figure 4 lists hinge sequences of 133TIM sequences, showing that there are 114 sequences with a tryptophan in N-hingeposition-3, all having the YGGS sequence for loop-7. All N-terminal hinge tripeptideshave a proline at position-1. Consequently, these 114 sequences can be referred to asthe PXW-sequence family (X is isoleucine, leucine or a valine in 112 sequences, or athreonine or a lysine). Of the remaining 19 sequences not having a tryptophan in N-hinge position-3, the YGGS-motif is not conserved. For example in Pyrococcuswoesei TIM, whose structure is known (Walden et al. 2001), the loop-6 sequence isPPELIGTGIPV and the loop-7 sequence is CGAG.

From an analysis of the wild type open and closed structures it can be seen that the C-hinge positions-1 and 2 are solvent exposed, while the C-hinge position-3 pointsinwards to the bulk of the protein (Figure 3), in particular in the open conformation.In wild type chicken TIM, the C-terminal hinge tripeptide is KTA. Directed evolutionapproaches have been carried out to investigate the allowed sequence variability ofthe C-hinge tripeptide of loop-6 of chicken TIM, which belongs to the PXW-sequencefamily. Six families of active variants were identified by in vivo selection (Sun andSampson 1998). A member of each family was chosen for further evaluation, and isreferred to by its three residue sequence. The sequences studied are KVA, NSS,LWA, NPN, YSL and KTK; the sequence motifs LWA, NPN, YSL, and KTK are notobserved in wild type sequences (Sun and Sampson 1998) (Figure 4). Subsequentenzymological characterizations have shown that for some of the selected activevariants (LWA, NPN, YSL, KTK) the values of k

cat/K

m are approximately a factor of

three smaller as compared to wild type, while KVA and NSS have the same kineticparameters as the wild type (Sun and Sampson 1999). Also, NPN and LWA showelevated Km’s for the substrate and elevated Ki’s for the inhibitorphosphoglycolohydroxamate. Here, structures of these six active C-hinge variants arereported and compared with the wild type structures.

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MATERIALS AND METHODS

Crystallographic structure determinations

The six variants (Table I), derived from chicken TIM have been purified as describedbefore (Sun and Sampson 1999). While this work was proceeding, it was noted thatthe sequence of the EGA-variant actually was KVA; consequently this variant wasrenamed as the KVA-variant. The initial crystallization conditions were screenedusing the hanging drop vapor diffusion method with equal volumes of protein solutionand mother liquor from a factorial screen (Zeelen et al. 1994) in a 4 ml drop at room

temperature and 4oC (Table I). The six different mutants with or without the boundinhibitor 2-phosphoglycolate (2PG) were crystallized in four different crystal forms(Table I). Crystallographic datasets were collected on the EMBL beam lines X13 andX11 at DESY, Hamburg (LWA-unliganded, NPN-liganded, YSL-liganded, YSL-unliganded, KTK-unliganded) and on a MAR345 image plate mounted on a copperrotating anode X-ray generator (Nonius) (KVA-liganded, NSS-liganded, LWA-liganded). The data sets of KVA-liganded, LWA-liganded, and NSS-liganded werecollected at room temperature with the crystals mounted in a glass capillary. Theother crystals were cryo-protected and flash-frozen in a stream of liquid nitrogen(Table I). The data were processed and scaled with Denzo/Scalepack (Otwinovski &Minor 1997) or XDS (Kabsch 1993) (Table II). Further data reduction was done withprograms from the CCP4 package (Collaborative Computational Project Number 41994). The structures were solved with molecular replacement using the programAMoRe (Navaza 1994). Refmac (Murshudov et al. 1997) and Refmac5 with TLSparameters (Schomaker and Trueblood 1968, Winn et al. 2001) were used for therefinement of the structures. Manual rebuilding was done with the help of theprogram O (Jones et al. 1991). The quality of the resulting models was evaluatedusing Procheck (Laskowski et al. 1993). All models display a good fit to the X-raydata and good geometry (Table II).

Structure analysis

As all structures are derived from chicken TIM, the chicken TIM-numbering schemehas been adopted. In this numbering scheme the catalytic residues are Asn11, Lys13,His95 and Glu165, loop-6 concerns residues 166-176 (PVWAIGTGKTA) and loop-7residues 208-211 (YGGS). In all structures, there is a dimer in the asymmetric unit;the structures of the two subunits are essentially the same, except for KTK. InKTK(A), loop-6 is more ordered than in KTK(B), also the structures of the orderedloop-6 regions of KTK(A) and KTK(B) are somewhat different. The KTK crystalswere grown in the presence of the inhibitor, but no ligand binding was detected in theactive site; consequently we refer to this structure as the unliganded KTK-structure.The absence of bound ligand is probably due to the high pH (pH=9.1; Table I), as it isknown that only the form of 2PG protonated at the carboxylic acid group binds toTIM (Campbell et al. 1978). In all structures, except KTK(B), the conformations ofthe mutated tripeptide regions are well defined in the electron density maps, asillustrated in Figure 5 for LWA-liganded and YSL-unliganded. For the structurecomparisons, the unliganded and liganded structures of wild type trypanosomal TIM(5TIM(A); Wierenga et al. 1991b) and leishmania TIM, liganded with 2PG (1N55;Kursula and Wierenga 2003), respectively, have been used. One subunit (A) of eachof the variant structures has been superimposed in O (Jones et al. 1991) on the

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unliganded and liganded structures of wild type TIM. For this superposition, the Caatoms of the 8 b-strands have been used. The graphical images of the structures,shown in Figures 3, 5 and 6, were made with Dino (Philippsen 2001). Sequencealignments were done with ClustalX (Jeanmougin et al. 1998), using proteinsequences from Swiss Prot.

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RESULTS AND DISCUSSION

Altogether eight structures have been determined, at resolutions equal to or better than2.9Å (Table II), and compared with the open and closed structures of wild type TIM.The flexibility of loop-6 of all subunits has been analyzed by making B-factor plots(data not shown), showing that in all liganded structures, loop-6 is well ordered (as inwild type). In the unliganded structures of LWA and YSL loop-6 is also well ordered,while in unliganded KTK loop-6 is substantially more disordered: in the A subunit,residues 171 and 172 and in the B subunit, residues 169-176 of loop-6 are not visiblein the electron density. The conformational flexibility of loops in crystal structures, asvisualized by B-factors, is influenced by crystal contacts, either directly or indirectly;therefore it is difficult to extrapolate such information to dynamical properties insolution. Nevertheless, it is clear that in KTK-unliganded, loop-6 is more disorderedthan in wild type. The structures of KVA, NSS and LWA are essentially the same aswild type. Larger structural differences are seen for the variants NPN, YSL, and KTK(Figure 6). For example, it can be seen that in each of the unliganded structures ofthese variants (YSL-unliganded; KTK-unliganded), the side chain of tryptophan 168(at the N-hinge position-3) has moved towards its wild type closed position. In theliganded structures of YSL and NPN, the main chain tracing of the N-terminal hingepeptide PVW is slightly different from the wild type tracing as seen in the closedform; consequently, also the active site geometry is somewhat different causing smalldifferences in the positions of the ligand and the catalytic glutamate (Figure 6). Fromthe structural analysis it seems clear that the variants can be grouped in two groups:(i) KVA, NSS and LWA: having structures which are essentially identical to the wildtype structures, and (ii) NPN, YSL and KTK: having structural differences withrespect to wild type.

The LWA variant

From the enzymological characterization, it is seen that the LWA kinetics aredifferent from wild type. The tryptophan side chain of the LWA-motif points towardsloop-7 in the liganded conformation but no clashes in the structure of the LWA-liganded variant are seen. Nevertheless, a tryptophan in this position changes thekinetic properties in a subtle way, not apparent from the LWA-liganded and theLWA-unliganded structures. Possibly this mutation changes the dynamic properties ofloop-6 sufficiently to interfere with optimal catalysis. In fact, in the known wild typesequences a tryptophan, phenylalanine, or tyrosine is never observed (Figure 4) at theC-hinge position-2. Also in the C-hinge positions-1 or -3 such side chains are neverobserved.

The NPN, YSL and KTK variants

The structural differences of loop-6 seen for NPN, YSL and KTK are substantial(Figure 6). It is interesting that these variants (liganded as well as unliganded) are lessstable than wild type (Sun and Sampson 1999). In these sequences, a bulky residue isseen at the third C-hinge position, an amino acid class never observed in wild typesequences whenever there is a tryptophan in the N-hinge position-3 (Figure 4). In the114 sequences listed in Figure 4, an alanine is most often observed (94 cases) in theC-hinge position-3; the only other observed residues at this position are a serine (10times), a proline (7 times) and a cysteine (3 times). The requirement for a small

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residue at this position can be understood from the structure, as this side chain ispointing inwards into the bulk of the protein in the open state, contacting thetryptophan side chain of the N-hinge position-3. In the closed state, the alanine sidechain of the C-hinge position-3 points further away from this tryptophan (Figure 3).Consequently, a bulky side chain at the C-hinge position-3 would favor the positionof the tryptophan, as seen in its closed conformation. Apparently, such a bulky sidechain shifts the equilibrium of the oscillating motion of loop-6/loop-7 in favor of theclosed conformation, even in the absence of ligand. This is most clearly seen in theunliganded structure of the YSL-variant, where both loop-6 and loop-7 have adoptedthe closed conformation, in both subunits. Also in the liganded NPN and YSLstructures loop-7 has adopted the same closed conformation as in wild type (Figure6). From the liganded NPN and YSL structures it can be seen that the active sitegeometry - in particular of the ligand and the catalytic glutamate - is somewhatdifferent from the wild type active site geometry (Figure 6) and from each other.These small structural differences seen between liganded NPN and liganded YSL forthe ligand and the catalytic glutamate correlate with small rearrangements of thePVW-peptide, which in turn are due to the bulky side chain at the C-hinge position-3.The small structural differences for the ligand and the catalytic glutamate (in ligandedNPN and liganded YSL) are in good agreement with differences in the catalyticprofiles seen for NPN and YSL (Sun and Sampson, 1999). Chemistry is no longer ratelimiting for YSL (also not for KTK). That is, a primary deuterium kinetic isotopeeffect is not observed for the mutant-catalyzed reactions. The lack of isotope effectsis consistent with our observation that the equilibrium between open and closedconformations in the absence of ligand has been shifted to favor the closedconformation. It appears from the structural and kinetic analysis that ligand(substrate) binding has become rate-limiting. Although, the catalytic rate (kcat/Km) ofthese mutants is only decreased 3-fold, a larger decrease in rate of substrate bindinghas occurred to result in substrate binding being rate-determining. For a variant inwhich a closed loop-6 predominates, even in the absence of ligand, the probabilitythat the ligand encounters a protein with an open loop is reduced. This leads to adecreased rate of formation of the Michaelis-Menten complex, as observed for thesevariants. In the case, of NPN, a kinetic isotope effect is still observed and thus therate of both substrate binding and enediol(ate) formation must be reduced with thismutation.

The correlated sequence conservation of loop-6 and loop-7

A bulkier side chain, in particular a valine, is seen in some wild type sequences at theC-hinge position-3, but in this case, the tryptophan side chain of the N-terminal hingePVW-sequence is replaced by another residue, in particular a glutamate (Figure 4).The structure of one hinge is dependent on the other, primarily because of a van derWaals interaction between Trp168 and Ala176. Mutagenesis studies on the additivityof N-terminal and C-terminal hinge mutations led to the same conclusion (Xiang et al.2001). Sequence conservation of interacting residues in adjacent loops correlated withconcerted motions has recently also been described for other enzymes (Gunasekaranand Nussinov 2004). In TIM, this interdependence is born out by the covariation ofthe N-terminal hinge sequence with the C-terminal hinge sequence as well as with theYGGS-sequence of loop-7 (Figure 4). Apparently, the evolutionary pressure is sopowerful that sub-optimal variants lacking the covariant sequence are eliminated from

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the wild-type pool as they are not found in the currently available set of 114 wild typesequences. This elimination is despite their relatively high catalytic activities ofapproximately 30% of the wild type catalytic efficiency.

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CONCLUDING REMARKS

Based on these structural studies, the conservation of a small residue at the C-terminalhinge position 3 can now be understood. Such a small residue is observed in the 114TIM sequences with the N-terminal PXW-N-hinge sequence motif. It is intriguingthat through evolutionary selection, another TIM sequence sub-family (the 19remaining sequences, Figure 4) exists in which the required loop-6/loop-7 dynamicshas been generated using another sequence motif. The physical-chemical properties ofthis TIM sub-family are poorly understood. Interestingly, all these 19 sequences arefrom TIM’s of archaebacteria, whereas all PXW-sequence motifs are from TIM’s ofeukaryotes or eubacteria. Future experiments will address whether these two sequencefamilies represent a different approach to provide the same dynamic loop properties,or whether the alternate sequences are a result of selection for catalytic activity withdifferent operating requirements.

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ACKNOWLEDGEMENTS

It is a pleasure to thank Ville Ratas for expert help with the crystallization setups. Theskillful support by the staff of the synchrotron beam lines X11 and X13 of theHamburg EMBL outstation at the DESY synchrotron in Hamburg (Germany) isgratefully acknowledged. This work has been supported by the Academy of Finland,the American Chemical Society-Petroleum Research Fund (N.S.), and Sigma Xi (J.S).The coordinates and structure factors of the eight structures have been deposited at theRCSB with the following entry codes: 1SW3, 1SW7, 1SW0, 1SQ7, 1SU5, 1SSG,1SSD, and 1SPQ.

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Edited by Prof. Alan Fersht

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LEGENDS TO FIGURES

Fig. 1. The reaction catalyzed by TIM and the covalent structure of 2-phosphoglycolate (2PG).

Fig. 2. Schematic diagram of the interactions between loop-6, loop-7 and loop-8 inthe liganded, closed conformation. The N-terminal hinge, the rigid tip, and the C-terminal hinge regions of loop-6 are highlighted. Loop-6, loop-7, and loop-8 arefollowing immediately after b6, b7, and b8 respectively. The N-hinge tripeptide ofloop-6 runs parallel to loop-7, while the C-hinge tripeptide runs antiparallel to loop-7.Loop-7 interacts via side chain – main chain hydrogen bonds with loop-6 and viawater mediated hydrogen bonds with loop-8. These hydrogen bonding interactions areshown in dotted lines: main chain – main chain interactions are in red; side chain-main chain interactions are in blue, and water mediated hydrogen bonds are in green.The asterisks identify the NH-groups and the water molecules which are hydrogenbonded to the phosphate moiety of the substrate.

Fig. 3. Comparison of the loop-6/loop-7 interactions in wild type TIM. The loop-6region (residues 166-176) and the beginning of helix-6 are shown; of loop-7, only theYGGS-motif (residues 208 -211) is shown. The unliganded, open structure(5TIM(A)) is in blue, the liganded, closed structure (1N55) is shown in yellow. Theligand 2PG is also shown. A) standard view, B) top view. The numbers refer to thechicken TIM numbering scheme.

Fig. 4. Sequence variation of loop-6 and loop-7. 133 TIM sequences of theSWISSPROT database have been aligned. Only full-length sequences have beenincluded; each of these sequences has the catalytic residues in loop-1 (asparagine,lysine), loop-4 (histidine), as well as a glutamate just before loop-6. Each loop-6 entrywith a unique N-hinge (green) - C-hinge (red) sequence combination is listedseparately. The numbers in parentheses refer to the number of found sequences. Incase of multiple observations, only one example species is mentioned in the firstcolumn. At the bottom of the figure, the eight sequences are listed which do not havea tryptophan in the N-hinge position-3. In these, the YGGS motif does not occureither and is replaced by other sequences. Such sequences are found only inarchaebacteria.

Fig. 5. Electron density maps of the mutated regions of LWA-liganded (A) and YSL-unliganded (B) after omit refinement. The maps are contoured at approximately 1sigma (2mFo-DFc, blue) and 2.5 sigma (mFo-DFc, orange).

Fig. 6. Comparison of the structures of a) NPN-liganded with wild type TIM(liganded), b) YSL-liganded with wild type TIM (liganded), c) YSL-unliganded withwild type TIM (liganded), and d) KTK-unliganded with wildtype (unliganded).Included are residues 164-178 (loop-6), 208-211 (loop-7), and 2PG (if present). In allimages, the wild type protein and 2PG are colored yellow and orange, respectively.

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Table I Crystal forms of the various TIM variants.

Variant Crystallization

conditions

Cryoconditions Space

group

Cell dimensions (Å,

°)

KVA

(liganded)

25 % PEG6000, 0.1 M

ADA pH 7.5, 0.2 M

Li2SO4, 10 mM 2PG

(+22°C)

Data collection at room

temperature

P212121 57.44, 73,85, 136.43,

90, 90, 90

NSS

(liganded)

2.6 M (NH4)2SO4, 0.1 M

citrate pH 5.5, 0.2 M

NaCl, 10 mM 2PG

(+22°C)

Data collection at room

temperature

P41212 87.22, 87.22, 162.94,

90, 90 ,90

LWA

(liganded)

1.4 M Na-citrate, 0.1 M

TEA pH 7.5, 10 mM

2PG (+22°C)

Data collection at room

temperature

P212121 57.60, 73.38, 135.61,

90, 90, 90

LWA (no

ligand)

19.0 % PEG6000, 0.1 M

citrate pH 5.2, 3 % t-

butanol (+22°C)

0.1 M citrate pH 5.2, 40%

PEG400, 3 % t-butanol,

1.6% PEG6000

P6122 61.67, 61.67,

500.89, 90, 90, 120

NPN

(liganded)

2.0 M (NH4)2SO4, 0.1 M

citrate pH 5.5, 0.2 M

NaCl, 10 mM 2PG

(+22°C)

2.0 M (NH4)2SO4, 0.1 M

citrate pH 5.5, 0.2 M

NaCl, 10 mM 2PG, 20 %

glycerol

P41212 86.76, 86.76, 163.38,

90, 90, 90

YSL

(liganded)

2.5 M (NH4)2SO4, 0.1 M

citrate pH 5.5, 0.2 M

NaCl, 10 mM 2PG

(+22°C)

2.5 M (NH4)2SO4, 0.1 M

citrate pH 5.5, 0.2 M

NaCl, 10 mM 2PG, 20 %

glycerol

P41212 88.33, 88.33,

163.87, 90, 90, 90

YSL (no

ligand)

2.4 M (NH4)2SO4, 0.1 M

citrate pH 5.5, 0.2 M

NaCl (+22°C)

2.4 M (NH4)2SO4, 0.1 M

citrate pH 5.5, 0.2 M

NaCl, 40% glycerol

P41212 88.42, 88.42,

161.87, 90, 90, 90

KTK (no

ligand)

21 % PEG6000, 0.1 M

TRIS pH 9.1, 10 mM

2PG (+4°C)

0.1 M TRIS pH 9.1, 40%

PEG400, 4% PEG6000, 5

mM 2PG

C2 80.71, 57.33, 106.15,

90, 92.89, 90

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Table II Data processing and refinement statistics.KVA

(liganded)

NSS

(liganded)

LWA

(liganded)

LWA

(no ligand)

NPN

(liganded)

YSL

(liganded)

YSL

(no ligand)

KTK

(no ligand)

Data collection statistics

No of observed reflections 102 115 276 315 68 825 101 348 173 113 85 130 109 427 87 857

No of unique reflections 37 515 31 858 40 543 15 311 19 906 16 663 20 217 25 268

Redundancy 2.72 8.67 1.70 6.62 8.70 5.11 5.41 3.48

Resolution range (Å)(*) 40-2.0

(2.1-2.0)

20-2.2

(2.3-2.2)

40-1.7

(1.73-1.70)

30-2.8

(2.9-2.8)

100-2.6

(2.7-2.6)

20-2.8

(2.9-2.8)

19.7-2.9

(3.0-2.9)

40-2.1

(2.2-2.1)

Completeness (%) (*) 93.6 (42.8) 100.0 (100.0) 62.6 (23.4) 94.3 (82.4) 99.0 (97.5) 99.7 (97.4) 98.8 (99.4) 97.8 (78.8)

Rmerge (%) (*) 8.9 (32.2) 11.3 (39.8) 8.9 (25.4) 8.3 (11.7) 10.9 (39.2) 8.9 (60.0) 10.5 (29.8) 5.7 (14.5)

I/sI (*) 8.9 (2.1) 21.5 (6.1) 8.6 (1.9) 10.4 (7.0) 16.5 (5.2) 25.0 (3.4) 15.1 (7.1) 22.0 (7.4)

Refinement statistics

Resolution range (Å) 69.0-2.0 76.7-2.2 13.2-1.7 28.9-2.9 17.8-2.7 20.0-2.9 19.7-2.9 38.3-2.1

Number of reflections 35 610 30 185 47 599 13 977 16 795 13 881 13 996 23 998

Working set 33 722 28 582 45 069 13 279 15 892 13 154 13 232 22 728

Free set 1888 1603 2530 698 903 727 764 1270

R-factor (%) 14.3 19.6 19.6 20.2 18.5 20.5 15.6 16.0

Free R-factor (%) 17.5 23.7 22.8 25.2 23.6 24.4 19.3 19.8

Protein atoms 3734 3671 3756 3 746 3 738 3 758 3 744 3664

Ligand atoms 18 18 18 - 18 18 - -

Solvent atoms 324 289 382 211 283 131 108 373

Geometry

Rmsd bond distances (Å) 0.013 0.015 0.013 0.010 0.012 0.012 0.010 0.012

Rmsd bond angles (°) 1.4 1.5 1.5 1.1 1.2 1.3 1.2 1.2

Rmsd B-factors (main

chain) (Å2)

0.65 0.44 0.53 0.07 0.22 0.23 0.20 0.66

Rmsd B-factors (side

chain) (Å2)

2.10 1.40 1.60 0.21 0.68 0.65 0.71 0.72

NCS-information

Rmsd for C_-atoms of

subunits A and B (Å)

0.13 0.20 0.15 0.13 0.13 0.15 0.22 0.18

Ramachandran plot

Most favored (%) 93.1 93.1 94.3 93.4 92.1 92.4 91.5 94.6

Additionally allowed (%) 6.9 6.9 5.7 6.2 7.9 6.9 8.3 5.4

Generously allowed (%) 0 0 0 0.5 0 0.2 0.2 0

Disallowed (%) 0 0 0 0 0 0.5 0 0

(*) highest resolution range

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FIGURE 1

FIGURE 2

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FIGURE 3

FIGURE 4

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FIGURE 5

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FIGURE 6

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