Crystal Structure of Triosephosphate Isomerase Complexed with 2-Phosphoglycolate at 0.83-Å Resolution*

Inari Kursula and Rik K. WierengaDagger

From the Department of Biochemistry and Biocenter Oulu, University of Oulu, P. O. Box 3000, Oulu FIN-90014, Finland

Received for publication, November 7, 2002, and in revised form, January 8, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The atomic resolution structure of Leishmania mexicana triosephosphate isomerase complexed with 2-phosphoglycolate shows that this transition state analogue is bound in two conformations. Also for the side chain of the catalytic glutamate, Glu167, two conformations are observed. In both conformations, a very short hydrogen bond exists between the carboxylate group of the ligand and the catalytic glutamate. The distance between O11 of PGA and Oepsilon 2 of Glu167 is 2.61 and 2.55 Å for the major and minor conformations, respectively. In either conformation, Oepsilon 1 of Glu167 is hydrogen-bonded to a water network connecting the side chain with bulk solvent. This network also occurs in two mutually exclusive arrangements. Despite the structural disorder in the active site, the C termini of the beta  strands that construct the active site display the least anisotropy compared with the rest of the protein. The loops following these beta  strands display various degrees of anisotropy, with the tip of the dimer interface loop 3 having very low anisotropy and the C-terminal region of the active site loop 6 having the highest anisotropy. The pyrrolidine ring of Pro168 at the N-terminal region of loop 6 is in a strained planar conformation to facilitate loop opening and product release.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Triosephosphate isomerase (TIM,1 EC 5.3.1.1.) is a dimeric enzyme that catalyzes the interconversion of D-glyceraldehyde 3-phosphate (DGAP) and dihydroxyacetone phosphate (DHAP) during glycolysis (1) (see Fig. 1). The active site is located at the C termini of the beta  strands of the classic TIM barrel fold and is shaped by the loops connecting the beta  strands to the following alpha  helices. Of special importance for substrate binding are loops 6, 7, and 8, of which loops 6 and 7 undergo conformational changes as the substrate is bound. In the unliganded state, loop 6 interacts with loop 5, whereas in the liganded state, it interacts with the YGGS motif (residues Tyr210-Gly211-Gly212-Ser213) of loop 7. Oeta of Tyr210 and Ogamma of Ser213 are hydrogen-bonded to the main chain nitrogen atoms of Ala178 and Gly175 in loop 6, respectively. N of Gly173 is hydrogen-bonded to one of the phosphate oxygens of the substrate. In the conformational switch from the open to the closed state, the tip of loop 6 (near Gly175) moves 7 Å as a rigid body (2), whereas in loop 7, the peptide planes after Gly211 and Gly212 rotate 90° and 180°, respectively. The rotation of these peptide planes allows for the tight binding of the phosphate moiety and for the conformational switch of the Glu167 side chain (3). The closure of loop 6 has also been studied by NMR (4-6) and molecular dynamics calculations (7, 8). The calculations indicate that the loop starts to open in the region Lys176-Val177 (7) and that this is initiated by the loosening of the hydrogen bond between Gly175 and Ser213 (8). The region 176-178 (Lys-Val-Ala) is also referred to as the C-terminal hinge of loop 6 (9). The opening/closing motion of loops 6 and 7 appears to be partially rate-limiting for the catalysis of the reaction in both directions (5, 6). It is unclear which structural features are associated with the energy barrier of this conformational switch (8, 10, 11).

The catalytic residues are Glu167, His95, Lys13, as reviewed by Knowles (1), and Asn11 (3, 12). These residues are in loops 6, 4, 1, and 1, respectively. The catalytic mechanism of TIM has been extensively studied by means of x-ray crystallography, NMR, infrared spectroscopy, mutagenesis, and enzymology, including the use of labeled substrates (1). Three planar transition state analogues (see Fig. 1) have been used for experimental studies on the reaction mechanism. In addition, detailed quantum mechanics/molecular mechanics calculations of the TIM reaction have been performed recently (13). Despite the wealth of information, no atomic resolution structures of TIM complexed with ligands have been reported, and not all aspects of the reaction mechanism are understood. For example, how does the enzyme environment allow for the proton abstraction by the glutamate. Also, there are two alternative proposals for the intermediate proton transfer steps, the relative importance of which is not clear (12, 14).

The reaction is started with the abstraction of the pro-R proton from the C1 carbon of the substrate, DHAP, by Glu167, leading to a planar enediolate intermediate. In the absence of enzyme, this is energetically a very unfavorable reaction due to the high pKa of the C1 atom of the substrate. In the enzyme environment, several residues contribute to facilitating this step, in particular those residues that stabilize the negative charge on the oxygen atom of the adjacent carbonyl group. These are Nzeta of Lys13 and Nepsilon 2 of His95 if the substrate is DHAP and Nepsilon 2 of His95 and Ndelta 2 of Asn11 if the substrate is DGAP. Alternative mechanisms have been discussed for the steps following the initial proton abstraction (reviewed in Ref. 12), in particular the "classic" and the "criss-cross" mechanism. In the classic mechanism, the proton abstracted by Glu167 is delivered to the adjacent carbon, unless, in the mean time, exchanged with solvent (15). In the criss-cross mechanism, this proton is transferred to O2 (Fig. 1) of the substrate. NMR experiments have indicated that in wild-type TIM, both mechanisms contribute to the reaction (14).


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Fig. 1.   A, the proton shuttling events in the reaction catalyzed by TIM according to the criss-cross mechanism. B, covalent structures of the transition state analogues PGH, PGA, and IPP. The numbering scheme used for PGA is indicated.

The mode of binding of 2-phosphoglycolate (PGA) to TIM has been studied ever since this compound was proposed to be a transition state analogue of the TIM reaction (16). 31P NMR studies indicate that PGA binds to TIM as a trianion (17). It is assumed that the tight binding of PGA is largely due to electrostatic interactions between PGA and the protonated Glu167 and neutral His95 side chains (18). Here, we report on an atomic resolution structure of Leishmania mexicana TIM complexed with PGA and discuss its implications for the reaction mechanism.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Crystallization and Data Collection-- For these experiments, the superstable E65Q variant of L. mexicana TIM was used, in which Glu65 has been mutated to Gln. The kinetic properties of this variant and the wild type enzyme are the same (19, 20). The enzyme was purified and crystallized as described before (3, 19, 20). The crystals grew at room temperature in less than a week from a mother liquor containing 20% polyethylene glycol 6000 in 0.1 M acetic acid/NaOH, pH 4.5, to a size of ~0.8 × 0.4 × 0.2 mm. The crystals belong to the space group C2, and there is one subunit per asymmetric unit. Two complete sets of diffraction data were collected at beamlines I711, MAX-Lab, Lund, Sweden (to 0.90-Å resolution) and BW7B, EMBL/DESY, Hamburg, Germany (to 0.83-Å resolution) on a MAR345 image plate at 100 K. The crystals were flash-frozen in the cryo stream after a brief soak in a cryoprotectant consisting of the mother liquor supplemented with 23% glycerol and additional 2% polyethylene glycol 6000 for cryo protection. The wavelengths used were 0.9600 and 0.8453 Å on I711 and BW7B, respectively. The data collection was carried out, respectively, in two and three passes for high, medium, and low resolution data from a single crystal. The data were processed and scaled with Denzo and Scalepack (21). The data processing statistics for the 0.83-Å structure are summarized in Table I. Truncate (22) was used to calculate the Wilson statistics for the 0.83-Å structure, indicating a Wilson B-factor of 6.9 Å2 (Table I).

                              
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Table I
Data collection statistics of the 0.83-Å structure
Values in parentheses correspond to the highest resolution shell.

Structure Solution and Refinement-- The structure was solved by molecular replacement using the program AMoRe (23). For the 0.90-Å structure, the previously solved 2-Å structure of the E65Q variant of L. mexicana TIM (1IF2 (3)) was used as the search model. The structure was refined first with Refmac (24) using isotropic B-factors and in the later stages anisotropically using SHELX97 (25) to R and Rfree values of 0.1205 and 0.1385, respectively. The resulting model was used as the initial model for the refinement of the 0.83-Å structure.

The refinement of the 0.83-Å structure was carried out using SHELX97 (25) adding the high resolution data gradually as the refinement proceeded. At this stage, the default geometric restraints and weighting scheme were used. Model building was done with O (26). A test set of 2% of all reflections was used to calculate Rfree. During the first six cycles, the model was refined isotropically. At this stage, water molecules were added to the structure, but other solvent molecules or disordered residues were not modeled. The following cycles of refinement were done anisotropically, and a detailed description of the model with disordered side chains and solvent was built. Most hydrogens were clearly visible in the Fo - Fc maps after moving to anisotropic refinement, as can be seen for example in Fig. 8, and were added to the model at the 16th cycle of refinement. For the last cycles, the geometric restraints were released for the protein part and removed for PGA. The refinement was continued until the highest peaks in the Fo - Fc map were below 0.5 e-3; the r.m.s.d. of the final Fo - Fc map is 0.07 e-3. At this stage, the highest peaks were due to disordered solvent or side-chain atoms not located close to the active site. The final crystallographic R and Rfree values were 0.0949 and 0.1085, respectively. The final statistics of the resulting model are presented in Table II. The programs O (26), XtalView (27), PARVATI (28), Ortep (29), Raster3D (30), AnisPlot,2 Molray (31), and DINO3 were used for analyzing the structure and generating the figures. The coordinates and structure factors have been deposited at the Protein Data Bank at the Research Collaboratory for Structural Bioinformatics.

                              
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Table II
Refinement statistics of the final model


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Structural Heterogeneity-- Due to the high resolution of the diffraction data, many of the disordered residues could be modeled with confidence. Altogether, 42 of the 250 residues have been built in more than one conformation. Nevertheless, some of the solvent-exposed side chains had to be considered as highly disordered with no preferred conformations. Those side chains have been included in the model as one or two conformations, depending on the electron density, but sometimes there was residual electron density in the surroundings that could not be explained by the model. Mostly, the alternative conformations occur in the side chains, but in a few cases, also the main chain is disordered. For example, the tip of the flexible loop 6 (residue Gly175) has two distinct conformations. In the major conformation (60% occupancy), the peptide proton of N(Gly175) points to Ogamma of Ser213, providing a reasonable hydrogen-bonding interaction (Ogamma (Ser213)-N(Gly175) distance is 3.29 Å). In the minor conformation, this interaction is weakened and Gly175 is unfavorably close to Ogamma of Ser213; for instance, the distance Calpha (Gly175)-Ogamma (Ser213) is 3.21 Å. This disorder seems to reflect the intrinsic loop-opening properties of loop 6, which is also visualized by the anisotropic B-factors of residues adjacent to Gly175 (Thr174 and Lys176) (Fig. 2).


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Fig. 2.   Structural disorder and anisotropy of loops 6 and 7. For loop 6, only the main chain atoms are depicted, except for Glu167, the side chain of which is pointing toward the inhibitor (PGA). For clarity, only the major conformations of Glu167 and PGA are shown. The main-chain and side-chain atoms of the conserved YGGS motif of loop 7 are shown. As discussed in the text, especially loop 6, the region around disordered Gly175 displays larger anisotropic movement compared with loop 7. The tyrosine and serine side chains of the YGGS motif interact with main-chain atoms of loop 6. The figure was generated using XtalView (27) and Raster3D (30).

In the active site, PGA is bound in two conformations (Fig. 3). In both of them, the O11 atom of PGA is hydrogen-bonded to Oepsilon 2 of Glu167. In the predominant conformation (70% occupancy), O12 of PGA is at a hydrogen-bonding distance to Nzeta of Lys13 and Nepsilon 2 of His95, whereas in the minor conformation (30% occupancy), it points further down and is weakly hydrogen-bonded to Nepsilon 2 of Asn11 (Fig. 3). In the predominant conformation, the Nepsilon 2(Asn11)-O12(PGA) distance is 4.17 Å, whereas in the minor conformation this distance is 3.22 Å. The difference between the two conformations concerns a rotation about the C1-C2 bond of PGA. The torsion angle O1P-C2-C1-O12 is -35° for the major conformation and 0° for the minor conformation. The Glu167 side chain is also 2-fold-disordered. The disorder concerns the position of Oepsilon 1, whereas Oepsilon 2 adopts always the same position, being hydrogen-bonded to O11 of PGA. The structural disorder of the carboxylate moieties of PGA and the Glu167 side chain appears not to be a concerted motion, instead, the active-site geometry (Fig. 3) suggests that these two groups move independently of each other, while keeping the O11(PGA)-Oepsilon 2(Glu167) hydrogen bond intact. In the major conformation of Glu167 (80% occupancy, chi 1 = -52, chi 2 = -169, chi 3 = -66), the Oepsilon 1 atom points to Sgamma of Cys126 at a distance of 4.12 Å, whereas in the minor conformation (chi 1 -50, chi 2 = -161, chi 3 = -53), Oepsilon 1 is rotated away from Sgamma of Cys126 at a distance of 4.69 Å. This structural disorder appears to be correlated with heterogeneity of Sgamma of Cys126, because there exists a minor conformation in which this atom is rotated into the space created by the rotation of Oepsilon 1 of Glu167. In both Glu167 side-chain conformations, the Oepsilon 1 atom is within a hydrogen-bonding distance from O11 of PGA as well as Wat28 (Fig. 3). This water is the first water of a network between Glu167 and bulk solvent. It consists of waters 28, 114, 38A, and 58. Wat58 is also hydrogen-bonded to Ogamma of Ser96 (Fig. 3). There also exists an alternative arrangement for this network, consisting of waters 28, 114, 38B, 107, and 58. 


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Fig. 3.   The mode of binding of PGA in the active site. A, interactions of the carboxylate moiety. The dotted lines highlight the hydrogen-bonding interactions of water molecules (visualized as yellow, pink, or purple spheres) and of the carboxylate moieties of PGA and Glu167. The yellow water molecules occur in both of the alternative networks, the pink one only in the major configuration, and the purple ones only in the minor configuration. B, interactions of the phosphate moiety. Gly173 is in loop 6, Ser213 in loop 7, and Gly234 in loop 8. The figure was generated using DINO.3

Anisotropy-- The large amount of data at this high resolution allowed for expanding the description of the model to anisotropic displacement parameters. The anisotropic description was applied to all atoms, including solvent molecules, which caused a significant drop in both R and Rfree. As seen in other structures at atomic resolution, none of the atoms agree with the traditional isotropic description. As examined by PARVATI (28) and Rastep (30), the mean anisotropy of all protein atoms in the structure is 0.53 (with a sigma  of 0.1) deviating significantly from the value 1, which means perfect isotropy. The anisotropy value refers to the ratio of the shortest and longest principal axis of the thermal ellipsoid describing the anisotropic scattering properties of an atom (28). The mean isotropic B of the protein atoms is 11.8 Å2.

In this crystal form, the dimer 2-fold axis coincides with a crystallographic axis, and consequently, the molecular center is on this 2-fold axis. The analysis of the mean anisotropy of the protein atoms of the dimer as a function of the distance from the dimer center shows the least anisotropy at the center (with a mean anisotropy of 0.9), which increases smoothly to a mean anisotropy of 0.3 at the extremities (25 Å away from the center). A more detailed analysis of the variation of the anisotropy along the polypeptide chain is shown in Fig. 4. Consistently, very low anisotropy is seen at the C termini of the beta  strands. This correlates with the low isotropic B-factors of these regions. The regions with the highest anisotropy are in the TIM-barrel framework alpha  helices, in particular at their C termini.


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Fig. 4.   Variation of the anisotropy and isotropic B (Å2) as a function of residue number. Also shown is the location of secondary structure elements; beta  strands are shown as arrows and alpha  helices as cylinders. The figure was generated with the help of AnisPlot.2

In the active site, the side chain of His95 shows significant anisotropic behavior with an anisotropy value of 0.47 for Nepsilon 2. This anisotropic movement occurs in the plane of the imidazole ring (Fig. 5). The side chains of Lys13 and Asn11 are less anisotropic with anisotropy values of 0.63 for Nzeta of Lys13 and 0.62 for Ndelta 2 of Asn11. Glu167, which is present in two conformations, displays significant anisotropy in the minor conformation with anisotropy values of 0.59, 0.41, 0.37, and 0.54 for Cgamma , Cdelta , Oepsilon 1, and Oepsilon 2, respectively. For the major conformation, the corresponding anisotropy values are 0.66 (Cgamma ), 0.87 (Cdelta ), 0.50 (Oepsilon 1), and 0.43 (Oepsilon 2). The mean anisotropy of PGA is 0.47 and 0.43 for the major and minor conformation, respectively. It is interesting to note the relatively high anisotropy of PGA and the side chain of the catalytic glutamate, despite the fact that the main chain regions defining the active site have relatively low anisotropy as well as low isotropic B-factors (Fig. 4). For example, the anisotropy of the Calpha atoms of Lys13, His95, Asn11, and Glu167 are 0.86, 0.77, 0.84, and 0.69, respectively.


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Fig. 5.   Anisotropy in the active site. A, not much anisotropic movement is observed looking approximately along the plane of His95 and the carboxyl group of PGA. B, viewing 90° degrees away, the directional movement of the His95 ring can be seen. Also the Glu167 side chain and the carboxylate group of PGA are anisotropic. For clarity, only the major conformations of Glu167 and PGA are shown. The figure was generated using ORTEP (29).

The anisotropy of the flexible loop 6 has a direction parallel to the loop opening/closure event. The anisotropy is more pronounced at the C-terminal end of the loop, concerning residues Gly173-Ala178 (Fig. 2). The most anisotropic part concerns residues Thr174 and Gly175 when only the main chain atoms are considered, whereas the side chain of Lys176 has one of the highest anisotropy values in the whole structure (0.10 for the Cbeta atom).

Active-site Geometry-- Fig. 6 highlights important hydrogen bonding interactions in the active site, in particular the hydrogen bonds of the major conformations of PGA and the Glu167 side chain. The phosphate moiety is hydrogen-bonded to main chain N atoms from loops 6, 7, and 8 (Fig. 3), whereas the triose moiety forms hydrogen bonds to side-chain atoms, in particular Lys13, His95, and Glu167 (Fig. 3). The phosphate moiety is also hydrogen-bonded via four water molecules to the protein: via Wat44 to Lys13 and via Wat186, Wat8, and Wat1 to loops 7 and 8. Wat1 is a buried water molecule, not part of a hydrogen bonding network to bulk solvent. Wat1 is buried between the ligand and protein and hydrogen-bonded to N of Gly212, O2P of PGA, and O of Leu232. It is at 3.69 Å distance from C2 of PGA.


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Fig. 6.   Schematic diagram concerning the active-site geometry of the major conformations of PGA and Glu167. Important hydrogen-bonding interactions are highlighted, including all hydrogen-bonding contacts between the ligand and protein atoms. The numbers refer to distances (Å) in the major conformations of the active-site residues and the ligand. Thr75* is in loop 3 of the adjacent subunit. The red dotted lines describe the interactions between PGA and side-chain atoms, the blue lines represent interactions between PGA and water molecules, and the green lines represent interactions between PGA and main-chain atoms. All other hydrogen bonds are shown in black. The indicated protonation states of the His95, Cys126, Glu167, and PGA are discussed in the text.

The side chains of Lys13 and His95 are well hydrogen-bonded to the bulk of the protein. Nzeta of Lys13 is tightly fixed to its position by three hydrogen bonds to Oepsilon 1 of Glu97, O2 of PGA, and Wat45. Nepsilon 2 of His95 is hydrogen-bonded to O12 of PGA (2.74 Å), whereas the side-chain position of His95 is stabilized by a hydrogen bond between Ndelta 1 and N of Glu97 (2.96 Å). The Glu167 side chain is only weakly interacting with the rest of the protein. The Glu167 side-chain carboxylate oxygen atoms span the region between Calpha of Gly211 (at 3.39 Å from Oepsilon 2), O of Leu232 (at 3.60 Å from Oepsilon 2) and Sgamma of Cys126 (at 4.12 Å from Oepsilon 1). The hydrogen atom on Sgamma of Cys126 points toward the main-chain oxygens of 124 and 93 (Figs. 6 and 7), as is also confirmed by the residual positive density in the Fo - Fc map; therefore, the Oepsilon 2(Glu167)-Sgamma (Cys126) is a van der Waals contact. Oepsilon 1 is at a distance of 3.29 Å from Nepsilon 2 of His95, but the geometry does not allow for a good hydrogen bond (Fig. 3). Two other fully conserved side chains (Ile172 in loop 6 and Leu232 at the beginning of loop 8) also contact the Glu167 carboxylate group (Fig. 7). None of these contacting protein atoms allow for good hydrogen bonding. A good hydrogen bond is observed with Wat28 (Figs. 3 and 7). This hydrogen bond exists in both the major and the minor conformation of the Glu167 side chain with distances of 2.71 and 2.80 Å. In both the major and the minor conformation, there exists a short (2.61/2.55 Å) hydrogen bond between Oepsilon 2 of Glu167 and O11 of PGA. The Cdelta -Oepsilon 2 and Cdelta -Oepsilon 1 bond distances in Glu167 are 1.26 and 1.19 Å, respectively, whereas the C1-O11 and C1-O12 bond distances are 1.22 and 1.19 Å, respectively. These distances are in the range of the C-O bond distances of the carboxyl moiety of the fully protonated crystal structure of PGA (33), which are 1.23 Å for the C=O double bond and 1.31 Å for the C-OH single bond. In the trianionic species of PGA, the C-O distances of the carboxylate group are 1.24 and 1.26 Å (33).


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Fig. 7.   Active-site geometry of the TIM·PGA complex. All residues and water molecules contacting the carboxylic acid moieties of PGA and the catalytic glutamate are included. Residues 74*-76* are in loop 3 of the adjacent subunit. Val40 (loop 2, gray) contributes to the hydrophobic cluster near Leu232. The importance of the other regions (11-13, loop 1; 93-97, loop 4; 124-126, loop 5; 165-175, loop 6; 210-213, loop 7, yellow; and 232-235, loop 8) are discussed in the text. The figure was generated using DINO.3

The active site is at the dimer interface, and the well-defined conformations of Asn11 and Lys13 are stabilized due to the tight interactions with loop 3 of the second subunit; for example, Thr75 at the tip of this loop is hydrogen-bonded to Ndelta 2 of Asn11 and Oepsilon 2 of Glu97, which forms a salt bridge to Nzeta of Lys13 (Figs. 6 and 7). Analysis of the B-factors (Fig. 4) shows that the tip of this loop is very rigid (BCalpha Gly76 = 5.4 Å2) and the anisotropy is very low (ACalpha Gly76 = 0.82).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The reaction catalyzed by TIM is conceptually rather simple, because it only concerns shuttling of protons (Fig. 1). The active site can be seen to consist of two parts: a binding pocket for the phosphate moiety and a catalytic site for the binding of the triose moiety. During the catalytic cycle, the phosphate group of the substrate has to be held in plane with respect to the rest of the molecule to avoid phosphate elimination (18). Therefore, mechanistically, the reaction becomes complicated, also because C-H bonds are very stable (34, 35). For example, the pKa value of methane is estimated to be 40 (36). Consequently, under physiological conditions, a deprotonated carbon atom is very unstable. If the resulting carbanion is stabilized, for example in the presence of an adjacent carbonyl function, then the pKa is much lower, near 20 (37). This is still considerably different from the pKa of a glutamate side chain, which is known to extract the proton from DHAP in the first step of the TIM reaction (1, 15). Nevertheless, the active-site geometry of TIM allows for efficient proton shuttling. The active site is located at the C termini of the TIM-barrel beta  strands and in fact, residues from each of the strands (or subsequent loops) contribute to the active site (Fig. 7). Thr75 from loop 3 of the opposing monomer is hydrogen-bonded to the side chains of Asn11 and Glu97, thus providing essential stabilizing interactions.

In the atomic resolution structure of the current study, the active site of TIM is complexed with the transition state analogue PGA. It is observed that the triose moiety of PGA occurs in two conformations, whereas the phosphate moiety is bound in a well-defined single conformation. The P-O1P (O1P is bonded to C2) distance is 1.62 Å, whereas the other P-O distances are 1.49, 1.50, and 1.55 Å. The corresponding distances in the crystallized trianionic species of PGA are 1.64, 1.51, 1.50, and 1.53 Å, respectively (33). Thus, the phosphate is hydrogen-bonded to main-chain nitrogen atoms from loops 6, 7, and 8 (Fig. 3). The triose moiety is hydrogen-bonded to side chains only (Asn11, Lys13, His95, and Glu167). In addition, it is in van der Waals contact with the side chains of Ile172 in loop 6 and Leu232 in loop 8.

Loop 6 is a loop with conformational flexibility, which is known to be in an open conformation in the unliganded state and in a closed conformation in the liganded state. The conformational differences between the open and closed conformation have been described previously (7), and the loop can be divided into an N-terminal hinge (Pro168, Val169, and Trp170), a rigid tip (Ala171, Ile172, Gly173, Thr174, and Gly175), and a C-terminal hinge (Lys176, Val177, and Ala178) (9). After this, helix 6 starts with Thr179 as the N-cap residue, followed by Pro180 in the N-cap+1 position. The tip of the loop moves as a rigid body, while small changes in the phi  and psi  angles of the N- and C-terminal hinge cause loop opening and closure. The largest changes occur for psi (Lys176) and phi (Val177), which are +57 and -38°, respectively (2). In the closed conformation, as seen in this structure, the Ile172 side chain interacts with the triose moiety of the substrate, as well as with the Glu167 side chain (Fig. 7), whereas N of Gly173 is part of the phosphate binding pocket (Fig. 3). Ile172 is in a very well-defined conformation, and the position of most of its hydrogen atoms can be seen in an Fo - Fc omit map (Fig. 8). In the closed conformation, loop 6 tightly interacts via O of Ala171 with Ogamma of Ser213 (in loop 7) and via N of Gly173 with the phosphate of PGA (Fig. 7). Weaker hydrogen bonding is seen for the C-terminal part, between N of Gly175 and Ogamma of Ser213 and between N of Ala178 and Oeta of Tyr210; also the main chain of this part of the loop shows one of the highest anisotropy properties of the entire structure. It seems, therefore, that the C-terminal part of loop 6 has evolved not to interact too tightly with loop 7, allowing for efficient loop opening, which is a requirement for efficient catalysis (5, 6). These structural features are in good agreement with NMR and computational studies. For example, solid-state NMR studies (4) have shown that the opening of loop 6 is not ligand-gated; instead, also in the liganded state, the loop opens at a rate in the millisecond timescale. Computational studies have also indicated that loop opening is initiated by the loosening of the hydrogen-bonding interactions between the C-terminal region of loop 6 and the YGGS motif of loop 7 (7, 8). The observed interactions between loop 6 and the YGGS motif of loop 7 require small structural rearrangements of the Tyr210 and Ser213 side chains as compared with the open conformation (10). These rearrangements are correlated with peptide rotations of the peptide bonds after Gly211 and Gly212, which generate the phosphate binding pocket as well as are required for the Glu167 side chain to move into the "swing-in" position.


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Fig. 8.   The 2mFo - DFc (red) and hydrogen-omit Fo - Fc (blue) map around Ile172. The contour levels are 2.23 e-3 (3sigma ) and 0.21 e-3 (2.2sigma ) for the 2mFo - DFc and Fo - Fc map, respectively. The figure was generated using O (26) and Molray (31).

As seen in Fig. 7, the pyrrolidine ring of Pro168 in the N-terminal region of loop 6 is planar. This is unusual, because the proline ring is known to occur in either of two different conformational states; "up" (chi 1 = -25°) or "down" (chi 1 = +25°) (38, 39), which differ in the position of the Cgamma atom with respect to the other members of the five-atom ring. The properties of the pyrrolidine ring of proline confer a unique structural role for this residue as reviewed previously (40). In this TIM·PGA structure, both puckerings of proline rings are observed, except for Pro168, which has chi 1 of -2°. This conformation is enforced by close van der Waals contacts of the Cgamma atom with the side chains of Tyr166 and Ala171 (Fig. 7). This strained conformation is undoubtedly an important structural feature of the closed form of loop 6. For example, in natural TIM sequences, this proline is fully conserved and random mutagenesis studies have shown that active variants require a proline at this position (9). This strained conformation, not seen in the open loop 6, will destabilize the closed loop, thus facilitating loop opening and product release at the end of the reaction cycle.

In the closed form of loop 6, the side chain of Ile172 folds over the substrate, shielding it and the catalytic glutamate from bulk solvent. Glu167 and the substrate are also contacted by Leu232 (Fig. 7). In this way, the Glu167 side chain in the closed conformation is buried in a pocket formed by the side chains of Ile172 and Leu232 but also lined by Calpha of Gly211, O of Leu232, Sgamma of Cys126, and Wat28. This pocket does not anchor the glutamate side chain via specific hydrogen bonds to the rest of the protein, and the width of the pocket (the distance from Cdelta 1 of Ile172 to Cgamma of Leu232 is 8.35 Å) allows for some conformational flexibility for this side chain. This conformational flexibility correlates with the proton shuttling function of Glu167. The importance of this hydrophobic pocket is highlighted by the full sequence conservation of Leu232 and Ile172. Moreover, a disease-related point mutation Ile172 right-arrow Val has been characterized (41). The patients display severe symptoms due to TIM deficiency (42).

The only (non-ligand) hydrogen-bonding partner for the Glu167 side chain is Wat28. This water is part of a water network, which extends to the bulk solvent (Figs. 3 and 6). The importance of these waters for catalysis has been discussed by Zhang et al. (37). Proton exchange between the catalytic glutamate and bulk solvent via this network might also explain the low yield of tritium transfer from the labeled C1 of DHAP to C2 of DGAP during catalytic turnover (15).

Phosphoglycolohydroxamate (PGH) and 2-(N-formyl-N-hydroxy)aminoethyl phosphonate (IPP) (Fig. 1) are transition state analogues mimicking structural features of the ketone (DHAP) and aldehyde (DGAP) substrate, respectively. The comparison of the mode of binding of these two compounds suggests that the precise details of the active-site geometry concerning substrate binding change as the reaction proceeds from substrate to product (3). This is correlated with the structural differences between an alpha -hydroxy ketone and an alpha -hydroxy aldehyde, because the planar carbonyl function is transferred from the C2 position to the C1 position. In particular, it has been shown that the side chains of Lys13 and His95 create a stabilizing oxyanion hole for the ketone carbonyl group, whereas Asn11 and His95 form such an oxyanion hole for the carbonyl group of the aldehyde substrate (3). In the apo structure, a water molecule is bound in the latter pocket. PGA is a transition state analogue in which the methanol group of the ketone substrate (DHAP) is replaced by a hydroxyl group. The O12 atom mimics the ketone moiety, whereas the O11 atom mimics the C1 atom of DHAP from which Glu167 abstracts a proton. The O11 atom of PGA points to both the Oepsilon 1 and Oepsilon 2 of Glu167, implying that it interacts with the syn orbitals of this carboxylate moiety (Fig. 5), which is the optimal geometry for proton abstraction (18). In the major conformation, O12 of PGA binds to Nzeta of Lys13 and Nepsilon 2 of His95, whereas in the minor conformation the O12 of PGA is bound at the expense of induced strain in the molecule (the C1-C2 dihedral is 0°), with low occupancy in the second oxyanion hole. The disorder of PGA in this structure correlates with the differences in the mode of binding of PGH and IPP, resembling the situation as the reaction proceeds from DHAP to DGAP.

A key feature of the active site is a very short hydrogen bond between the carboxylate groups of PGA and Glu167. The O-O distance for the usual hydrogen bond between two oxygen atoms, with the hydrogen being firmly attached to one of them, is around 2.8 Å (43), in good agreement with the O-O distances highlighted in Fig. 6. However, the distance between O11 of PGA and Oepsilon 2 of Glu167 is 2.61 and 2.55 Å, when considering the major and minor conformations, respectively. This is significantly shorter than a normal hydrogen bond and similar to the O-O distance expected for a low barrier hydrogen bond (LBHB) (43). An LBHB concerns a short and very strong hydrogen bonding interaction, in which the hydrogen is more diffusely distributed between the two atoms. The possible role of LBHBs in the mechanism of TIM as well as other enzymes has been discussed since 1993 (43-45). NMR studies have shown that, in the complex of TIM with PGH, there exists an LBHB between the NOH hydrogen and the catalytic glutamate (46). Clearly, the glutamate side chain and ligand geometry can flip between different conformations allowing for strong interactions between the glutamate and different atoms of the substrate. This apparently allows the side chain to shuttle protons directly between the two carbons of the substrate (as in the classic mechanism) as well as via the oxygen atoms (as in the criss-cross mechanism). The former, which explains the tritium incorporation from DHAP to DGAP (15), requires that the proton transfer between the oxygens is mediated by the His95 side chain (32, 47). In this TIM·PGA complex, the Nepsilon 2 of His95 is hydrogen-bonded to O12 of PGA (2.74 Å) and interacts with O11 at a distance of 3.21 Å (Fig. 6). The electron density suggests that Nepsilon 2 is protonated and Ndelta 1 is unprotonated. As seen in Fig. 5, the anisotropic B-factors of His95 indicate that the histidine side-chain motion is a concerted motion in the plane of the imidazole ring. When examining the mode of binding of IPP and PGH (3), it can be seen that such dynamic properties would facilitate proton transfer between the two substrate oxygen atoms via Nepsilon 2 of His95, as has been observed to occur (32).

The atomic resolution structure discussed here provides precise structural information on the TIM·PGA complex. The observed dynamic properties of the side chains of Glu167 and His95 agree with the known proton-shuttling roles of these residues. PGA is a true transition state analogue, because it has the same planarity and charge as the intermediate formed from the substrate after proton abstraction by the catalytic glutamate. This structure appears to mimic the active-site geometry of the enzyme·substrate complex, trapped after the very first proton abstraction step by Glu167 has been initiated. The Glu167 side chain shares a proton with the ligand donor atom, and no further proton shuttling has occurred. Additional proton transfer via the His95 side chain seems also not to have happened. The true substrate differs from PGA such that the O11(H) group is the C1(H)-OH moiety in the ketone substrate (DHAP). The LBHB-like hydrogen bond between O11 of PGA and the catalytic glutamate indicates that the active-site geometry favors transition state stabilization required for proton transfer from the C1(H)-OH moiety of DHAP to Glu167 and thereby facilitates efficient catalysis of the proton-shuttling steps. The observed structural heterogeneity in the active site is an important feature of the mode of binding of PGA, because it correlates with conformational changes taking place during the reaction. Atomic resolution structures of TIM complexed with other transition state analogues will be required to obtain a complete understanding of the structural rearrangements occurring in the active site during the proton transfer steps of the catalytic cycle.

    ACKNOWLEDGEMENTS

The excellent support by Dr. Xiao-Dong Su and Dr. Yngve Cerenius on I711 (MAX-Lab, Lund) and Dr. Paul Tucker on BW7B (EMBL/DESY, Hamburg) is gratefully acknowledged. We thank Dr. Petri Kursula for helpful discussions on the manuscript and helping with the figures. The technical assistance of Ville Ratas with protein purification and crystallization is highly appreciated.

    FOOTNOTES

* This work was funded by the Academy of Finland (grant number 44198).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and the structure factors (code 1N55) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

Dagger To whom correspondence should be addressed. Tel.: 358-8-553-1199; Fax: 358-8-553-1141; E-mail: rik.wierenga@oulu.fi.

Published, JBC Papers in Press, January 9, 2003, DOI 10.1074/jbc.M211389200

2 P. Kursula, unpublished.

3 A. Philippsen (2001) www.bioz.unibas.ch/~xray/dino.

    ABBREVIATIONS

The abbreviations used are: TIM, triosephosphate isomerase; DGAP, D-glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; E65Q, the L. mexicana TIM variant, in which Glu65 has been replaced by Gln; IPP, 2-(N-formyl-N-hydroxy)aminoethyl phosphonate; LBHB, low barrier hydrogen bond; PGA, 2-phosphoglycolate; PGH, phosphoglycolohydroxamate; YGGS, the region Tyr210-Gly211-Gly212-Ser213 of loop 7 in TIM; r.m.s.d., root mean square deviation.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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