1Department of Biochemistry and Biocenter Oulu, University of Oulu, PO Box 3000, FIN-90014 University of Oulu, Finland, 2European Molecular Biology Laboratory, Hamburg Outstation, c/o DESY, Notkestrasse 85, Building 25A, D-22603 Hamburg, Germany and 3Department of Chemistry, State University of New York, Stony Brook, NY 11794-3400, USA 4Present address: Pharmaceutical Chemistry, University of California, San Francisco, CA 94143-2280, USA 5Present address: The Scripps Research Institute, La Jolla, CA 92037, USA
6 To whom correspondence should be addressed. E-mail: rik.wierenga{at}oulu.fi
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Abstract |
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Keywords: Archae/evolution/flexible loop/structure triosephosphate isomerase
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Introduction |
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Loop 6 consists of 11 residues, which can be divided into a three-residue N-terminal hinge, a five-residue rigid tip of the loop and a three-residue C-terminal hinge (Figure 2). As highlighted in Figures 2 and 3, the N-hinge runs parallel to loop 7 whereas the C-hinge runs antiparallel to loop 7. The catalytic glutamate 165 is located just before the stretch 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 and 3). In the open, unliganded conformation loop 6 interacts with loop 5; the conformational switch is a movement of 7 Å of the tip of the loop from loop 5 towards loop 7. This openingclosing movement of loop 6 involves small changes of the phi/psi values in the C-terminal and N-terminal hinges, whereas the central five residues move as a rigid body (Joseph et al., 1990; Wierenga et al., 1991a
). The largest changes in phi/psi values (
50°) are observed for psi(Lys174) and phi(Thr175). These two changes compensate each other; consequently, only the position of the peptide oxygen of Lys174 is affected. In the closed conformation, O(Lys174) is in tight van der Waals contact with Cß(Ala169). Only in the closed conformation Cß(Ala169) is near C
of the strained, planar Pro166 (Figure 3), which subsequently contacts C
(Tyr164) (Kursula and Wierenga, 2003
). The closure of loop 6 affects the active site geometry in several ways; for example, (i) it facilitates the rotation of the side chain of the catalytic residue Glu165 to a position competent for catalysis; the swung-in conformation (Wierenga et al., 1991b
); also (ii) the substrateenzyme interaction is shielded from bulk solvent by the side chain of Ile170 in the closed loop 6 conformation; and (iii) the N(Gly171) forms a hydrogen bond with the phosphate moiety of the substrate (Lolis and Petsko, 1990
).
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Materials and methods |
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The six variants (Table I) derived from chicken TIM were purified as described previously (Sun and Sampson, 1999). While this work was proceeding, it was noted that the sequence of the EGA variant actually was KVA; consequently, this variant was renamed the KVA variant. The initial crystallization conditions were screened using the hanging drop vapor diffusion method with equal volumes of protein solution and mother liquor from a factorial screen (Zeelen et al., 1994
) in a 4 µl drop at room temperature and 4°C (Table I). The six different mutants with or without the bound inhibitor 2-phosphoglycolate (2PG) were crystallized in four different crystal forms (Table I). Crystallographic datasets were collected on the EMBL beamlines X13 and X11 at DESY, Hamburg (LWA-unliganded, NPN-liganded, YSL-liganded, YSL-unliganded, KTK-unliganded) and on a MAR345 image plate mounted on a copper rotating anode X-ray generator (Nonius) (KVA-liganded, NSS-liganded, LWA-liganded). The data sets of KVA-liganded, LWA-liganded and NSS-liganded were collected at room temperature with the crystals mounted in a glass capillary. The other 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 and Minor, 1997
) or XDS (Kabsch, 1993
) (Table II). Further data reduction was done with programs from the CCP4 package (Collaborative Computational Project Number 4, 1994
). The structures were solved with molecular replacement using the program AMoRe (Navaza, 1994
). Refmac (Murshudov et al., 1997
) and Refmac5 with TLS parameters (Schomaker and Trueblood, 1968
; Winn et al., 2001
) were used for the refinement of the structures. Manual rebuilding was done with the help of the program O (Jones et al., 1991
). The quality of the resulting models was evaluated using Procheck (Laskowski et al., 1993
). All models display a good fit to the X-ray data and good geometry (Table II).
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As all structures are derived from chicken TIM, the chicken TIM numbering scheme was adopted. In this numbering scheme, the catalytic residues are Asn11, Lys13, His95 and Glu165, loop 6 involves residues 166176 (PVWAIGTGKTA) and loop 7 residues 208211 (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. In KTK(A), loop 6 is more ordered than in KTK(B); also the structures of the ordered loop 6 regions of KTK(A) and KTK(B) are somewhat different. The KTK crystals were grown in the presence of the inhibitor, but no ligand binding was detected in the active site; consequently, we refer to this structure as the unliganded KTK structure. The absence of bound ligand is probably due to the high pH (9.1; Table I), as it is known that only the form of 2PG protonated at the carboxylic acid group binds to TIM (Campbell et al., 1978). In all structures, except KTK(B), the conformations of the mutated tripeptide regions are well defined in the electron density maps, as illustrated in Figure 5 for LWA-liganded and YSL-unliganded. For the structure comparisons, 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, were used. One subunit (A) of each of the variant structures was superimposed in O (Jones et al., 1991
) on the unliganded and liganded structures of wild-type TIM. For this superposition, the C
atoms of the eight ß-strands were used. The graphical images of the structures, shown in Figures 3, 5 and 6, were made with Dino (Philippsen, 2001
). Sequence alignments were done with ClustalX (Jeanmougin et al., 1998
), using protein sequences from Swiss Prot.
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Results and discussion |
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The LWA variant
From the enzymological characterization, it is seen that the LWA kinetics are different from wild-type. The tryptophan side chain of the LWA motif points towards loop 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 the kinetic properties in a subtle way, not apparent from the LWA-liganded and the LWA-unliganded structures. Possibly this mutation changes the dynamic properties of loop 6 sufficiently to interfere with optimal catalysis. In fact, in the known wild-type sequences a tryptophan, phenylalanine or tyrosine is never observed (Figure 4) at the C-hinge position 2. Also in the C-hinge positions 1 or 3 such side chains are never observed.
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 (both liganded and unliganded) are less stable than wild-type (Sun and Sampson, 1999). In these sequences, a bulky residue is seen at the third C-hinge position, an amino acid class never observed in wild-type sequences whenever there is a tryptophan in the N-hinge position 3 (Figure 4). In the 114 sequences listed in Figure 4, an alanine is most often observed (94 cases) in the C-hinge position 3; the only other observed residues at this position are a serine (10 times), a proline (seven times) and a cysteine (three times). The requirement for a small residue at this position can be understood from the structure, as this side chain is pointing inwards into the bulk of the protein in the open state, contacting the tryptophan side chain of the N-hinge position 3. In the closed state, the alanine side chain 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 position of the tryptophan, as seen in its closed conformation. Apparently, such a bulky side chain shifts the equilibrium of the oscillating motion of loop 6/loop 7 in favor of the closed conformation, even in the absence of ligand. This is most clearly seen in the unliganded structure of the YSL variant, where both loop 6 and loop 7 have adopted the closed conformation, in both subunits. Also in the liganded NPN and YSL structures, loop 7 has adopted the same closed conformation as in wild-type (Figure 6). From the liganded NPN and YSL structures it can be seen that the active site geometryin particular of the ligand and the catalytic glutamateis somewhat different from the wild-type active site geometry (Figure 6) and from each other. These small structural differences seen between liganded NPN and liganded YSL for the ligand and the catalytic glutamate correlate with small rearrangements of the PVW-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 liganded NPN and liganded YSL) are in good agreement with differences in the catalytic profiles seen for NPN and YSL (Sun and Sampson, 1999
). Chemistry is no longer rate limiting for YSL (also not for KTK). That is, a primary deuterium kinetic isotope effect is not observed for the mutant-catalyzed reactions. The lack of isotope effects is consistent with our observation that the equilibrium between open and closed conformations in the absence of ligand has been shifted to favor the closed conformation. It appears from the structural and kinetic analysis that ligand (substrate) binding has become rate limiting. Although the catalytic rate (kcat/Km) of these mutants is only decreased 3-fold, a larger decrease in the rate of substrate binding has occurred, resulting in substrate binding being rate determining. For a variant in which a closed loop 6 predominates, even in the absence of ligand, the probability that the ligand encounters a protein with an open loop is reduced. This leads to a decreased rate of formation of the MichaelisMenten complex, as observed for these variants. In the case of NPN, a kinetic isotope effect is still observed and therefore the rates of both substrate binding and enediolate formation must be reduced with this mutation.
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 the C-hinge position 3, but in this case the tryptophan side chain of the N-terminal hinge PVW 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 der Waals interaction between Trp168 and Ala176. Mutagenesis studies on the additivity of 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 with concerted motions has recently also been described for other enzymes (Gunasekaran and Nussinov, 2004
). In TIM, this interdependence is borne out by the covariation of the N-terminal hinge sequence with the C-terminal hinge sequence and also with the YGGS sequence of loop 7 (Figure 4). Apparently, the evolutionary pressure is so powerful that sub-optimal variants lacking the covariant sequence are eliminated from the wild-type pool as they are not found in the currently available set of 114 wild-type sequences. This elimination is despite their relatively high catalytic activities of
30% of the wild-type catalytic efficiency.
Conclusion
Based on these structural studies, the conservation of a small residue at the C-terminal hinge position 3 can now be understood. Such a small residue is observed in the 114 TIM sequences with the N-terminal PXWN-hinge sequence motif. It is intriguing that through evolutionary selection, another TIM sequence sub-family (the 19 remaining sequences, Figure 4) exists in which the required loop 6/loop 7 dynamics has been generated using another sequence motif. The physico-chemical properties of this TIM sub-family are poorly understood. Interestingly, all these 19 sequences are from TIMs of archaebacteria, whereas all PXW sequence motifs are from TIMs of eukaryotes or eubacteria. Future experiments will address whether these two sequence families represent a different approach to provide the same dynamic loop properties or whether the alternative sequences are a result of selection for catalytic activity with different operating requirements.
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Acknowledgments |
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References |
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Received April 26, 2004; accepted May 19, 2004.
Edited by Alan Fersht