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

I. Kursula1,2, M. Salin1, J. Sun3,4, B.V. Norledge1,5, A.M. Haapalainen1, N.S. Sampson3 and R.K. Wierenga1,6

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The conformational switch from open to closed of the flexible loop 6 of triosephosphate isomerase (TIM) is essential for the catalytic properties of TIM. Using a directed evolution approach, active variants of chicken TIM with a mutated C-terminal hinge tripeptide of loop 6 have been generated (Sun,J. and Sampson,N.S., Biochemistry, 1999, 38, 11474–11481). In chicken TIM, the wild-type C-terminal hinge tripeptide is KTA. Detailed enzymological characterization of six variants showed that some of these (LWA, NPN, YSL, KTK) have decreased catalytic efficiency, whereas others (KVA, NSS) are essentially identical with wild-type. The structural characterization of these six variants is reported. No significant structural differences compared with the wild-type are found for KVA, NSS and LWA, but substantial structural adaptations are seen for NPN, YSL and KTK. These structural differences can be understood from the buried position of the alanine side chain in the C-hinge position 3 in the open conformation of wild-type loop 6. Replacement of this alanine with a bulky side chain causes the closed conformation to be favored, which correlates with the decreased catalytic efficiency of these variants. The structural context of loop 6 and loop 7 and their sequence conservation in 133 wild-type sequences is also discussed.

Keywords: Archae/evolution/flexible loop/structure triosephosphate isomerase


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Many enzymes use flexible loops that change conformation upon substrate binding to form a catalytically competent active site. One of the most studied of such enzymes is triosephosphate isomerase (TIM) (EC 5.3.1.1). TIM is a dimeric, glycolytic enzyme, which catalyzes the interconversion of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate (DGAP) (Knowles, 1991Go) (Figure 1). The flexible loop in TIM is loop 6, which is located after the sixth ß-strand of the TIM barrel. In fact, in addition to loop 6, also loop 7 after ß-strand 7 changes conformation upon ligand binding (Figure 2). In the liganded closed form of the active site, loop 7 interacts with loop 6 via the side chains of its highly conserved YGGS motif (Sampson and Knowles, 1992Go).



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

 


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Fig. 2. Schematic diagram of the interactions between loop 6, loop 7 and loop 8 in the liganded, closed conformation. The hydrogen bonding interactions are shown by dotted lines. 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 follow immediately after ß6, ß7 and ß8, respectively. The N-hinge tripeptide of loop 6 runs parallel to loop 7 and the C-hinge tripeptide runs antiparallel to loop 7. Loop 7 interacts via side chain–main chain hydrogen bonds with loop 6 and via water-mediated hydrogen bonds with loop 8. The waters are labeled as O1 and O2. The asterisks identify the NH groups and the water molecules, which are hydrogen bonded to the phosphate moiety of the substrate.

 
Both NMR data (Williams and McDermott, 1995Go; Rozovsky and McDermott, 2001Go; Rozovsky et al., 2001Go) and laser-induced temperature jump relaxation spectroscopy (Desamero et al., 2003Go) indicate that loop 6 is opening and closing in both the liganded and the unliganded structures on about the same time-scale, which is similar to the catalytic conversion rate. The closed form is favored in the presence of bound ligand, whereas the open form is favored in the absence of ligand. From X-ray crystallography it is observed that the open conformation, in which loop 6 interacts with loop 5, has characteristically rather high B-factors, whereas the liganded conformation, in which loop 6 interacts with the YGGS motif of loop 7, is well defined (Kishan et al., 1994Go). Recent crystallographic data, showing that the closed form can be seen in the absence of ligand (Aparicio et al., 2003Go) and the open form in the presence of ligand (Parthasarathy et al., 2002Go), are in good agreement with NMR data indicating that the loop 6 breathing motion exists in both the liganded and unliganded complexes. Such a mechanism will facilitate product release after completion of the reaction cycle (Jogl et al., 2003Go; Kursula and Wierenga, 2003Go).

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 opening–closing 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., 1990Go; Wierenga et al., 1991aGo). 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{gamma} of the strained, planar Pro166 (Figure 3), which subsequently contacts C{zeta}(Tyr164) (Kursula and Wierenga, 2003Go). 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., 1991bGo); also (ii) the substrate–enzyme 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, 1990Go).



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

 
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 of the conserved YGGS motif of loop 7 with main chain atoms of loop 6 (Figures 2 and 3). The structural switch of loop 7 is also required for making a competent active site: (i) the Gly209–Gly210 peptide bond rotates 90°, to allow the catalytic glutamate to move to the catalytically competent swung-in conformation (Kursula et al., 2001Go) and (ii) the peptide flip of the Gly210–Ser211 peptide bond allows N(Ser211) to hydrogen bond to the phosphate moiety. The flip of the latter peptide moiety changes the phi/psi values of Ser211 from (–80°, 120°) in the open form to (65°, 30°) in the closed form (Noble et al., 1993Go). The YGGS motif of loop 7 is highly conserved. As shown in Figure 4, its occurrence is uniquely correlated with the tryptophan in the N-terminal hinge position 3. Figure 4 lists hinge sequences of 133 TIM sequences, showing that there are 114 sequences with a tryptophan in N-hinge position 3, all having the YGGS sequence for loop 7. All N-terminal hinge tripeptides have a proline at position 1. Consequently, these 114 sequences can be referred to as the PXW sequence family (X is isoleucine, leucine or a valine in 112 sequences or a threonine or a lysine). In the remaining 19 sequences not having a tryptophan in N-hinge position 3, the YGGS motif is not conserved. For example, in Pyrococcus woesei TIM, whose structure is known (Walden et al., 2001Go), the loop 6 sequence is PPELIGTGIPV and the loop 7 sequence is CGAG.



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Fig. 4. Sequence variation of loop 6 and loop 7; 133 TIM sequences of the SWISSPROT database have been aligned. Only full-length sequences have been included; 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 entry with a unique N-hinge (residues 166–168 in chicken TIM)–C-hinge (174–176 in chicken TIM) sequence combination is listed separately. The numbers in parentheses refer to the number of found sequences. In the case of multiple observations, only one example species is mentioned in the first column. At the bottom of the figure, the eight sequences are listed which do not have a tryptophan in the N-hinge position 3. In these, the YGGS motif does not occur either and is replaced by other sequences. Such sequences are found only in archaebacteria.

 
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, whereas the C-hinge position 3 points inwards 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 evolution approaches have been carried out to investigate the allowed sequence variability of the C-hinge tripeptide of loop 6 of chicken TIM, which belongs to the PXW sequence family. Six families of active variants were identified by in vivo selection (Sun and Sampson, 1998Go). A member of each family was chosen for further evaluation and is referred 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 not observed in wild-type sequences (Sun and Sampson, 1998Go) (Figure 4). Subsequent enzymological characterizations have shown that for some of the selected active variants (LWA, NPN, YSL, KTK) the values of kcat/Km are approximately a factor of three smaller as compared with wild-type, whereas KVA and NSS have the same kinetic parameters as the wild-type (Sun and Sampson, 1999Go). Also, NPN and LWA show elevated Kms for the substrate and elevated Kis for the inhibitor phosphoglycolo-hydroxamate. Here, structures of these six active C-hinge variants are reported and compared with the wild-type structures.


    Materials and methods
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 Abstract
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 Materials and methods
 Results and discussion
 References
 
Crystallographic structure determinations

The six variants (Table I) derived from chicken TIM were purified as described previously (Sun and Sampson, 1999Go). 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., 1994Go) 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, 1997Go) or XDS (Kabsch, 1993Go) (Table II). Further data reduction was done with programs from the CCP4 package (Collaborative Computational Project Number 4, 1994Go). The structures were solved with molecular replacement using the program AMoRe (Navaza, 1994Go). Refmac (Murshudov et al., 1997Go) and Refmac5 with TLS parameters (Schomaker and Trueblood, 1968Go; Winn et al., 2001Go) were used for the refinement of the structures. Manual rebuilding was done with the help of the program O (Jones et al., 1991Go). The quality of the resulting models was evaluated using Procheck (Laskowski et al., 1993Go). All models display a good fit to the X-ray data and good geometry (Table II).


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Table I. Crystal forms of the various TIM variants

 

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Table II. Data processing and refinement statistics

 
Structure analysis

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 166–176 (PVWAIGTGKTA) and loop 7 residues 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. 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., 1978Go). 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., 1991bGo] and leishmania TIM, liganded with 2PG (1N55; Kursula and Wierenga, 2003Go), respectively, were used. One subunit (A) of each of the variant structures was superimposed in O (Jones et al., 1991Go) on the unliganded and liganded structures of wild-type TIM. For this superposition, the C{alpha} 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, 2001Go). Sequence alignments were done with ClustalX (Jeanmougin et al., 1998Go), using protein sequences from Swiss Prot.



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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 1{sigma} (2mFo – DFc, blue) and 2.5{sigma} (mFo – DFc, orange).

 


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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 with wild-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 all images, the wild-type protein and 2PG are yellow and orange, respectively.

 

    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Altogether eight structures were determined, at resolutions equal to or better than 2.9 Å (Table II), and compared with the open and closed structures of wild-type TIM. The flexibility of loop 6 of all subunits was analyzed by making B-factor plots (data not shown), showing that in all liganded structures loop 6 is well ordered (as in wild-type). In the unliganded structures of LWA and YSL, loop 6 is also well ordered, whereas 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 visible in the electron density. The conformational flexibility of loops in crystal structures, as visualized by B-factors, is influenced by crystal contacts, either directly or indirectly; therefore, it is difficult to extrapolate such information to dynamic properties in solution. Nevertheless, it is clear that in KTK-unliganded, loop 6 is more disordered than in wild-type. The structures of KVA, NSS and LWA are essentially the same as wild-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 of these 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 the liganded structures of YSL and NPN, the main chain tracing of the N-terminal hinge peptide PVW is slightly different from the wild-type tracing as seen in the closed form; consequently, also the active site geometry is somewhat different, causing small differences in the positions of the ligand and the catalytic glutamate (Figure 6). From the 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 with the wild-type structures, and (ii) NPN, YSL and KTK, having structural differences with respect to wild-type.

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, 1999Go). 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 geometry—in particular of the ligand and the catalytic glutamate—is 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, 1999Go). 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 Michaelis–Menten 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., 2001Go). Sequence conservation of interacting residues in adjacent loops correlated with concerted motions has recently also been described for other enzymes (Gunasekaran and Nussinov, 2004Go). 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 PXW–N-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.


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


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 Introduction
 Materials and methods
 Results and discussion
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Received April 26, 2004; accepted May 19, 2004.

Edited by Alan Fersht