Department of Biochemistry and Biophysics and Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, CA 94143-0448, USA
1 To whom correspondence should be addressed. Present address: S412C UCSF-GENENTECH Hall, 600 16th Street, San Francisco, CA 94143-2240, USA
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Abstract |
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Keywords: bi-substrate reactions/dUMP/hydrogen bond/5,10-methylenetetrahydrofolate/negative cooperativity
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Introduction |
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An essential role of D169(221) is the initial orientation of the cofactor against the substrate prior to methyl transfer. It is well established that the TS reaction proceeds via a covalent ternary complex intermediate (II in Figure 1) (Carreras and Santi, 1995
). The most definitive evidence that this intermediate is on the TS reaction pathway was the discovery of a mutant of LcTS for which the breakdown of the covalent TSdUMPCH2H4folate complex was slowed to such an extent that the complex could be isolated on SDS gels (Huang and Santi, 1994
). This mutant complex was shown to be chemically and kinetically competent to form the product, 2'-deoxythymidine-5'-monophosphate (Huang and Santi, 1994
). Binding assays and structural studies have shown that for most mutants of D169(221), the enzyme reaction stalls at formation intermediate II (Chiericatti and Santi, 1998
; Sage et al., 1998
). Only four of seven mutants tested were able to form an analog of intermediate II, the covalent complex of TS with FdUMP (5-fluoro-2'-deoxyuridine-5'-monophosphate) and CH2H4folate. This complex differs from II by a single atom (a fluorine in place of a hydrogen that prevents the reaction from proceeding) (Figure 2
). Of the four mutants that formed the analog of II, only D169(221)C, had a KdCH2H4folate comparable to that of the wild-type enzyme (Chiericatti and Santi, 1998
); the remaining three variants had KdCH2H4folates that were at least 106-fold higher than for wild-type TS (Chiericatti and Santi, 1998
).
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The structural impact of the Cys substitution must be defined in order to interpret these effects on enzyme activity, particularly since in this bi-substrate reaction catalytic rate is closely related to optimal alignment of reactants. Therefore, we determined the structure of a complex of D169C with substrate, dUMP and an antifolate, CB3717 (10-propargyl-5,8-dideazafolate). For wild-type EcTS, the orientation of CB3717 in the active site of TSdUMPCB3717 is essentially similar to that of the cofactor in TSFdUMPCH2H4folate, but since CB3717 does not have an N5-methyl moiety (Figure 2), it cannot form a covalent bond to dUMP (Matthews et al., 1990b
; Montfort et al., 1990
; Hyatt et al., 1997
). The structure of D169dUMPCB3717 therefore reveals the orientations of the substrates bound in the active site, unconstrained by the covalent bond between the cofactor methylene group and C5 of dUMP.
Facile formation of TSFdUMPCH2H4folate by D169(221)C suggested that Cys would fill the same role as Asp in stabilizing a productive alignment of TS, dUMP and cofactor analog and that, therefore, factors unrelated to substrate alignment, such as the change in pKa or hydrogen bonding potential of the substituted Cys, accounted for reduction in activity of D169(221)C. A wild-type configuration of active site residues, dUMP and CB3717 in the crystal structure would lend weight to the hypothesis that besides aligning reactants throughout the reaction, D169(221) contributes to catalysis through hydrogen bond stabilization of the enol form of the cofactor. Our results, however, do not clearly distinguish such a role for D169(221). We find instead that small differences in the hydrogen bond geometry of the Cys verses the Asp side chain lead to destabilization of the closed conformation of the ternary complex that could reduce the rate of reaction steps requiring precise alignment of substrates. This result illustrates the strict geometric and hydrogen bonding requirements for the side chain at position 169(221) in the sequence, rather than an effect due to the formal charge on D169.
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Materials and methods |
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The Asp to Cys point mutation at residue 169 in EcTS was made by the method of Kunkel et al. (Kunkel et al., 1987) as described previously (Sage et al., 1998
). Mutant EcTS was expressed in E.coli
2913 (Thy-), which lacks a gene for TS, and purified by the procedure of Maley and Maley (Maley and Maley, 1988
). Co-crystals of purified D169C with CB3717 and dUMP were grown by vapor diffusion at room temperature by the hanging drop method. The crystals were grown from a solution containing 13 mg/ml protein, 10 mM potassium phosphate, 50 µM EDTA, 1.2 M ammonium sulfate, 10 mM dithiothreitol (DTT), 6 mM dUMP and 40 µM CB3717; 5 µl drops were placed over 1 ml reservoirs of 20 mM potassium phosphate, 100 µM EDTA, 10 mM DTT and 2.4 M ammonium sulfate. A pH range from neutral to basic was scanned and the complex was found to crystallize most readily at pH 9.1, although crystals at lower pH values grew over several months. To ensure that the cysteine residues did not oxidize, the concentration of DTT was increased during the crystallizations and crystals were mounted from a DTT bath into capillaries for X-ray data collection.
X-ray data were collected on a Raxis-IIc imaging plate system using monochromatized Cu K radiation (
= 1.54 Å) from a Rigaku rotating anode X-ray generator operating at 50 kV and 300 mA. No significant crystal decay was evident during the period of time (2 days) when data were collected. Data were measured at room temperature, as opposed to being collected from liquid nitrogen-cooled crystals, to match the conditions used for activity assays. Data were reduced to Ihkls using the programs Denzo and Scalepack from the HKL software package (Otwinowski, 1993
).
Structure solution and refinement
The structure was solved by difference Fourier methods starting from the isomorphous structure of wild-type EcTSdUMPCB3717 (Montfort et al., 1990) with ligands and waters removed. Ligands were built into difference density maps using O (Jones et al., 1991
). Manual rebuilding with O was alternated with cycles of simulated annealing and energy minimization using X-PLOR or CNS (Brunger, 1992
; Brunger et al., 1998
). Table I
lists the X-ray data collection and current structure refinement statistics. Coordinates are deposited in the PDB under accession code 1NCE.
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In order to compare structures of wild-type and D169C TS, their structural cores were overlapped using LSQMAN from the DEJAVU suite of programs. Our program NEWDOME, written by Eric Fauman, which uses difference-distance matrices to identify domains of conserved structure in two coordinate sets, was utilized to define the structural cores used for overlapping the structures (Montfort et al., 1990). The errors in interatomic distances were estimated using an empirical approach that takes into account the resolution of the structure and the atomic B-factors of the atoms (Stroud and Fauman, 1995
). Difference-distance plots were generated using the DDMP software from the Center for Structural Biology at Yale University. Ligandprotein interactions were determined using LIGPLOT (Wallace et al., 1995
) and the program DISTANG from the CCP4 suite of programs.
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Results |
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The D169C dimer has an asymmetric structure. One protomer closely resembles wild-type EcTSdUMPCB3717 in conformation, ligand orientation and ligandprotein interactions, while the second protomer is in a more open conformation with the ligands out of the productive alignment seen in wild-type analogs of intermediate II. Each protomer in the D169C complex was separately aligned to the corresponding protomer in the wild-type EcTSdUMPCB3717 structure. The r.m.s. distance between the -carbons (C
s) of the aligned structures is 0.28 Å for one protomer but 0.43 Å for the second protomer. After alignment with each other. the r.m.s. distance between the C
s of the two protomers of the mutant complex is 0.43 Å.
The protomer that deviates most from the wild-type structure has a more open active site. The distance between the Cs of residues W80 and Y209, on opposite sides of the active site cavity, is 19.9 ± 0.3 Å in this protomer compared with 18.8 ± 0.1 Å in the wild-type complex. Even this 1 Å difference has a major impact on the hydrophobic contacts between cofactor and the protein. The active site closure triggered by binding of cofactor or cofactor analogs involves small shifts of several protein segments towards the ligands (Montfort et al., 1990
). Difference-distance matrices, which contain the intramolecular distances between all pairs of C
s in a protein, are a convenient way of detecting these shifts. Difference-distance matrix plots comparing the D169C dimer with wild-type EcTSdUMPCB3717, which has a closed active site cavity, and apo-EcTS (Perry et al., 1990
), which has an open active site, show that protomer 1 has a typical EcTS ternary complex conformation while the conformation of protomer 2 more closely resembles that of apo-EcTS, except for the position of its C-terminal residues. The asymmetry has functional consequences in that dUMP is covalently attached to the active site thiol of C146 only at the fully closed active site.
Movement of the C-terminus is independent of other segmental shifts upon CB3717 binding in protomer 2
An ~5 Å movement of the C-terminus of TS into the active site, where it contributes to a hydrogen bond network with R21, W83 and the cofactor (Figure 3), is the most dramatic segmental shift that accompanies ternary complex formation (Matthews et al., 1990a
; Montfort et al., 1990
). Both protomers in D169CdUMPCB3717 have their C-termini in this closed conformation. However, the B-factors for the four C-terminal residues are high in both protomers, 65 Å2 on average compared with 28 Å2 for EcTSdUMPCB3717, suggesting that the closed conformation of the C-terminus is less stable in D169C ternary complexes.
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C169 coordinates a hydrogen bond network analogous to that formed in wild-type EcTS
In wild-type TS, D169 participates in a hydrogen bond network, mediated in part by three conserved waters, which connects three of the last four residues of the protein with the cofactor (Figure 3). The network is important for immobilizing and orienting the cofactor prior to catalysis (Perry et al., 1993
). The major features of this network are conserved in protomer 1, where side chain interactions of C169 mimic those of the wild-type D169, but are less conserved in protomer 2 (Figures 4 and 5
). In the first protomer C169 S
accepts hydrogen bonds from two of the three conserved water molecules in the network and from N3 of the quinazoline ring of CB3717 (Figure 4
). In wild-type ternary complexes the two waters, one on the surface of the active site cavity and one in the interior of the protein, are hydrogen bond donors to D169(221) O
2 and D169(221) O
1, respectively (Figure 3
). The hydrogen bond between the C169 thiol and N3 is longer than the hydrogen bond between N3 and D169 O
2 in wild-type EcTSdUMPCB3717 (3.6 ± 0.3 vs 3.2 ± 0.1 Å) but is in the range seen for cysteine sulfhydryl groups in proteins (Petersen et al., 1999
).
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In protomer 2 of the D169C complex, C169 S accepts a shorter (2.9 ± 0.5 Å) hydrogen bond from N3 of CB3717 and only one of the three conserved waters in the network, the internal water discussed in the previous paragraph, is visible in electron density maps (Figure 5
). This water makes the conserved hydrogen bonds to H212 N
1, Y209 NH and C169 S
and also accepts a hydrogen bond from F171 NH.
dUMP is covalently attached to the active site thiol in only one active site
The substrate, dUMP, and cofactor analog, CB3717, are bound at both active sites (Figure 6). In both protomers the substrate, dUMP, makes the same hydrogen bonds with the protein as in the wild-type enzyme. In each protomer the dUMP phosphate moiety is coordinated by a quartet of arginines: R21, R166 and, from the opposite protomer, R126' and R127' [(residues from the second protomer that enter into discussion of the first protomer are indicated with a prime)]. S167 and a conserved water molecule are also hydrogen bonded to the phosphate. The ribose hydroxyl group, O3', is hydrogen bonded to side chains of both H207 and Y209. The 2-exo oxygen on the dUMP pyrimidine accepts a hydrogen bond from the backbone amide of C169. The N177 side chain forms hydrogen bonds to O4 and N3 of the pyrimidine base and H147 may make a long hydrogen bond to O4. Within experimental error the acceptordonor distances are the same as those seen in wild-type EcTSdUMPCB3717.
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In the case of D169C, covalent addition of C146 to dUMP at one active site appears to interfere with activation of C146 or dUMP C6 in the second protomer. One possible explanation is the absence of the water-mediated hydrogen bond between E58 and O4 of dUMP that is postulated to facilitate the Michael addition of the catalytic sulfhydryl to dUMP (Huang and Santi, 1997). This hydrogen bond forms when the active site closes during binding of the cofactor or an antifolate. The conserved water in this hydrogen bond is absent or disordered in protomer 2 of D169C.
Alternatively, protomer 2 may better stabilize a dUMP iminium ion postulated to be in equilibrium with covalently bound dUMP in EcTS ternary complexes (Hyatt et al., 1997). Hyatt et al., observing weak electron density at C6 of dUMP in several ternary complex crystal structures, postulated that the covalent bond between C6 of dUMP and the active site sulfhydryl is weakened by geometric strain, and thus is present in only a subset of molecules in their crystals (Hyatt et al., 1997
). They suggested that in the other molecules, dUMP exists as an N1-iminium ion paired with S- from the catalytic cysteine and that this ion pair is stabilized by interactions with polar groups on the protein and cofactor. dUMP N1 is stacked ~0.2 Å more closely against the plane of the aromatic quinazoline ring of CB3717 in protomer 2 of D169C than in protomer 1 and it is conceivable that this interaction may enhance the electronic stabilization of the putative iminium ion, thus favoring non-covalently bound dUMP.
CB3717 is misoriented in protomer 2 which lacks the covalent bond to dUMP
The quinazoline ring is misoriented in the more open protomer 2. Although the hydrogen bond network that anchors CB3717 in the active site is at least partially conserved at both active sites, the quinazoline ring of CB3717 is in the productive orientation seen in wild-type ternary complexes only in protomer 1. This protomer has a closed active site in which several hydrophobic residues are in van der Waals contact with the p-aminobenzoic acid (PABA) moiety or the quinazoline ring. These conserved residues have been shown to provide the main hydrophobic contacts to a range of antifolate inhibitors complexed to various TS species (Montfort et al., 1990; Knighton et al., 1994
; Stout and Stroud, 1996
; Fox et al., 1999
; Anderson et al., 2001
; Phan et al., 2001
; Sayre et al., 2001
). Residues I79, L172 and F176 together make seven contacts less than 3.8 Å to atoms in the PABA moiety in protomer 1, while W83 and L143 make five contacts less than 3.7 Å with atoms C7 and C8 in the quinazoline ring. Although protein contacts with CB3717 in this protomer of the D169C complex are almost as extensive as in the wild-type complex, CB3717 is less well ordered, with average B-factors of 62 Å2 compared with 28 Å2 in wild-type EcTS. Within experimental error, CB3717 in this protomer overlaps the position of this inhibitor in the wild-type EcTS ternary complex.
In protomer 2 the quinazoline ring is still closely stacked on the dUMP but is translated 1.5 ± 0.6 Å out of the wild-type folate-binding site (Figure 7) and dUMP is not covalently bonded to the enzyme. The more open conformation of the active site in this protomer limits the number of contacts the hydrophobic cofactor-binding residues make with CB3717. There are no contacts less than 3.8 Å to the PABA ring and L143 is not in close contact with CB3717. The CB3717 has high thermal mobility with average B-factors of 84 Å2.
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Discussion |
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TS is relatively intolerant toward mutation of D169(221): only the D169C mutant in L.casei or E.coli TS complements Thy- cells in vivo (Michaels et al., 1990; Chiericatti and Santi, 1998
). In L.casei TS four other D169(221) mutants are active but not at a level that supports growth in TS-deficient E.coli. This is not typical of ligand-binding residues in TS. Usually structural rearrangements, including incorporation of new water molecules, compensate for the mutation to some degree (Stroud and Finer-Moore, 1993
).
The requirement for an aspartic acid at position 169 is especially stringent because each carboxyl oxygen has a distinct and important role: O2 positions the cofactor pterin ring in the active site and O
1 contributes hydrogen bonding and electrostatic interactions to the protein core. Since D169 is located in an
-helix where its carbonyl oxygen makes two hydrogen bonds to main chain atoms, substituted amino acids do not have much backbone flexibility to adjust their side chain positions, in order to compensate for the dual roles of the D169 carboxyl.
D169 O1 forms hydrogen bonds via a conserved water molecule to H212 in an electropositive pocket. H212 in turn donates a hydrogen bond to P261 O (Figure 3
). These interactions are likely to be important for structure since H212 is highly conserved across species and D169(221) variants maintain analogous interactions using the substituted side chains or molecules of solvent. For example, in E.coli D169N, it is clearly the carbonyl and not the amide of the N169 carboxamide that is directed into the H212 pocket (Sage et al., 1998
) where it accepts a hydrogen bond from the conserved water. In the case of L.casei D169(221)A, a phosphate ion replaces the Asp carboxyl group and conserved water in the pocket (unpublished data), suggesting a preference for a negative charge in this pocket.
In L.casei D169(221)C, the cysteine sulfhydryl is oxidized to sulfenic acid, even though great care was taken to crystallize this mutant under reducing conditions (unpublished data). Oxidation of this cysteine is exceptional; for example, none of the cysteines present in the D169(221)C EcTS structure, of which two are solvent accessible, are oxidized. The negatively charged sulfenic acid group in the LcTS mutant replaces the D169(221) carboxyl and the internal water, providing further evidence that a negatively charged group is very stable in this site. One reason that the cysteine mutant is the optimal variant of D169(221) may be that the cysteine sulfhydryl substitutes effectively for a carboxyl group in the conserved pocket. Free cysteine sulfhydryl groups in protein structures tend to be buried, favor interactions with neighboring histidine residues over other amino acids (Petersen et al., 1999), are approximately equivalent in size to a carboxyl group and can act as hydrogen bond acceptors.
C169 S substitutes for D169 O
2 in a hydrogen bond that orients the cofactor
TS binds substrate and cofactor by an ordered, sequential mechanism in which dUMP binds first and CH2H4folate subsequently binds to the TSdUMP complex, triggering closure of the active site. The cofactor-induced conformational change, in turn, promotes opening of the cofactor imidazolidine ring, forming a reactive iminium ion that condenses with dUMP to form covalent, steady-state intermediate II. A hydrogen bond from N3 of the pterin ring of CH2H4folate to D169 O2 is critical for the initial alignment of the cofactor with dUMP during this process as it is the only hydrogen bond the open enzyme makes with the pterin ring (Sotelo-Mundo et al., 1999
; Almog et al., 2001
). All mutants capable of forming the intermediate II analog TSFdUMPCH2H4folate are able to accept a hydrogen bond from N3 except for D169(221)A, which has a KdCH2H4folate more than 107-fold higher than that of wild-type TS (Chiericatti and Santi, 1998
). A hydrogen bond acceptor at residue 169 selects against non-productive cofactor binding modes. For example, when D169 is substituted with the isosteric asparagine, the hydrogen bond-donating carboxamide N
2 causes the pterin ring to bind in a flipped orientation, where its N5-methylene cannot add to dUMP (Sage et al., 1998
).
At both active sites C169 S is positioned to accept a hydrogen bond of good geometry from CB3717 N3, analogous to the hydrogen bond between D169 O
2 and N3 of the cofactor. Cysteine sulfhydryl groups are normally not good hydrogen bond acceptors, but at the pH of crystallization (9.1) cysteine side chains are usually ionized, increasing their propensity to accept hydrogen bonds. The short distance (2.9 Å) between C169 S
and N3 in the more open protomer 2 strongly suggests that C169 is present as a thiolate anion. In protomer 1, the longer (3.6 Å) S
N3 distance may indicate that C169 here is neutral, but more likely it arises because many of the hydrophobic contacts between CB3717 and D169C in protomer 1 can be made only at the expense of a shorter S
N3 hydrogen bond. The different balance between optimal C169 S
hydrogen bond lengths and maximal hydrophobic contacts in the two active sites reflects the inherent binding asymmetry of the EcTS dimer.
Features of the structural environment of C169 are likely to lower its pKa from the typical value of ~8.5 for cysteine: residue 169 is located at the N-terminus of an -helix, adjacent to H212, with which it could form an ion pair. Structural studies of other D169 mutants suggest that a negatively charged group is highly favored at position 169. Thus, although we have not measured the pKa of C169, we can predict that even at neutral pH, C169 exists predominantly as a thiolate anion that could accept the hydrogen bond from N3 of CB3717 or CH2H4folate. These results are consistent with the observation that for L.casei TS D169(221)C KmCH2H4folate is only ~14-fold higher than for wild-type TS (Chiericatti and Santi, 1998
). They suggest that Cys adopts the role of D169(221) in orienting the pterin ring of cofactor during formation of intermediate II and the reduced activity of D169(221)C is probably due to impairment of subsequent catalytic steps.
Structural role for D169C in catalysis
As a key part of the hydrogen bond network with the pterin ring in the closed conformation of TS (Figure 3), D169 continues to play a role in cofactor orientation after intermediate II is formed. The crystal structure of EcTSdUMPH4folate, an analog of the intermediate that immediately precedes hydride transfer (IV, Figure 1
), shows that this hydrogen bond network serves to align precisely the C6 hydrogen of H4folate for transfer to the methylene substituent of 5-methylene-dUMP (Fritz et al., 2002
). The importance of orienting the cofactor pterin ring against dUMP for catalysis of hydride transfer, which is rate-determining in wild-type EcTS (Spencer et al., 1997
), is demonstrated by mutants of other cofactor-binding residues for which reduced hydride transfer rates correlate with thermally disordered cofactor analogs that are less well aligned with dUMP (Variath et al., 2000
; Fritz et al., 2002
).
The hydrogen bond network not only orients the cofactor directly, through hydrogen bonds to the cofactor pterin ring, but also indirectly, through hydrogen bonds that stabilize the closed enzyme conformation. The energetic cost of maintaining a closed enzyme conformation is precisely balanced by the energetically favorable proteinprotein and proteincofactor interactions made only in this closed state. The crystal structure of D169CdUMPCB3717 shows that mutation of D169(221) to Cys alters this energetic balance. Even though Cys is approximately isosteric with Asp and, as illustrated by the structure of protomer 1, a thiol group (probably anion) is capable of forming the same hydrogen bonds as a carboxyl group, small differences in preferred hydrogen bond geometry between the thiol anion and the carboxyl decrease the stability of the closed conformation of this ternary complex. The B-factors in the crystal structure are high for the ligands and for the protein segments that shift to close the active site, particularly the four C-terminal residues of the protein and the loop containing R21(23). While Cys can substitute for Asp in accepting a hydrogen bond from the pterin ring of the cofactor, the requirements for coordinating a complex hydrogen bond network with ideal geometry are more stringent and are not met by the Cys side chain.
The ability of TS to align the reactants for catalysis depends on its transition to a closed conformation. For example, the ligands at the more open active site are not in the orientations that would be required for either Michael addition (step 1, Figure 1) or hydride transfer (step 4, Figure 1
). It follows that a mutation that decreases the stability of the closed conformations of the ternary complex decreases the rate of reaching the transition state and could have a pronounced effect on kcat. For example, the EcTS mutant I264Am, in which the C-terminal residue of the wild-type protein is removed, has a ternary complex structure with comparable degrees of structural asymmetry, active site disorder and ligand misalignment to those of D169C (unpublished data). These structural effects lead to decreases in activity of the same order as those seen for D169C (33-fold lower kcat, 260-fold lower kcat/KmCH2H4folate) (unpublished data). It is therefore possible that structural perturbations that alter the energetics of the protein conformational change may alone account for the reduction in kcat seen in D169C.
A caution regarding our interpretation of the crystal structure is that the D169CdUMPCB3717 complex was crystallized at a higher pH than the wild-type ternary complex with dUMP and CB3717 (9.1 compared with 8.0). It is unlikely that this difference in pH per se is responsible for the more open conformation of protomer 2. The wild-type EcTS ternary complex with dUMP and tetrahydrofolate, for example, does not show this type of asymmetry although it was crystallized at a higher pH of 8.5 (Fritz et al., 2002). However, the pH determines the ionization states of C169 and its hydrogen-bonding partner, H212, which in turn determines hydrogen bonds formed in the network with the C-terminal residues and CB3717, and these influence the conformation of the enzyme. Because of the environment of D169 we expect that the pKa of C169 would be lowered, such that the thiolate form is probably responsible for the measured activity of D169C even at neutral pH. Our structure is a snapshot of the protein complex at pH 9.1 where, by analogy with the catalytic Cys/His pair in papain, the Cys would likely be in thiolate form and the His would be predominantly neutral (Pinitglang et al., 1997
). One reason why the complex crystallized more readily at pH 9.1 than at pH 8.0 may be that the protein is more heterogeneous at the lower pH, with the H212/C169 pair in the His:/Cys-SH or His-H+/Cys-S- ionization states some of the time. A structural basis for impairment of enzyme activity at lower pH would pertain in this case also, in that the mutation would still result in a broader distribution of conformations for reaction intermediate II, only a subset of which are likely to reach the transition state.
Even though our results show that D169 has a critical role in orienting the cofactor for catalysis, they do not rule out a second role in transition state stabilization. Since C169 S accepts a hydrogen bond from N3 of CB3717, it can in principle still play the postulated role of D169 in catalyzing proton transfers to and from the pterin ring during breakdown of intermediate II (Figure 8
), although less efficiently.
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The asymmetry of the D169C ternary complex dimer is not a feature introduced by the mutation. On the contrary, asymmetric dimers have been seen in other TS ternary complexes with dUMP and antifolate inhibitors (Stout and Stroud, 1996; Anderson et al., 1999
; Sotelo-Mundo et al., 1999
) and this dimer asymmetry is consistent with solution studies that show asymmetric binding of CH2H4folate or antifolate inhibitors, to both wild-type and mutant TSs (Danenberg and Danenberg 1979
; Dev et al., 1994
; Chen et al., 1996
; Spencer et al., 1997
). Rather, the structural asymmetry has been unmasked in the crystal structure by an overall decrease in stability of the closed conformation of the ternary complex. Crystallization conditions for EcTS ternary complexes bias the structures towards fully closed dimers with symmetrically bound, productively oriented ligands at both active sites, but even in crystal structures of wild-type complexes, asymmetry is revealed by higher average B-factors for one protomer than for the other (Montfort et al., 1990
). In general, when mutations abolish some of the proteincofactor interactions that balance the entropic cost of forming a compact, well-ordered active site, the crystal structures of the mutant ternary complexes are asymmetric, with a more open active site and no covalent bond between dUMP C6 and the protein at one protomer, (presumably the more weakly binding one) (Fritz et al., 2002
; unpublished results).
Because TS generally crystallizes as a symmetric dimer, most crystallographic studies of TS have provided little insight into the asymmetric binding detected in solution. An exception is the crystal structure of Pneumocystis carinii TS (PcTS) in a complex with dUMP and CB3717 (Anderson et al., 1999). This structure was an asymmetric dimer with CB3717 bound only to one active site. The structure showed that small structural changes transmitted through residues 173176 and 196199 in the dimer interface (146149 and 163166 in EcTS) had a dramatic (all or none) effect on ligand binding that resulted in loss of a factor of ~106 in binding energy for CB3717 at one active site versus the other (Anderson et al., 1999
). These results show the extremely high requirements for an enzyme site to bind a ligand.
As one of the few EcTS crystal structures with ligands bound differently at the two active sites of the dimer, D169CdUMPCB3717 sheds light on the structural basis for negative cooperativity in ligand binding. In order to understand how binding to one active site of D169C alters the binding affinity at a second, distant, active site, we inspected the dimer interface, which has perfect two-fold symmetry in the apo-enzyme (Perry et al., 1990), for asymmetric contacts. The active site cavity in TS is located at the dimer interface with residues from both protomers contributing to each active site, hence the most direct mechanism for relaying structural effects of binding from one active site to the other is through the interface, as was the case for PcTS.
In the D169C ternary complex, some of the same changes in residues 146149 and 163166 of the dimer interface are seen as were present in PcTS. However, in this case the changes are much smaller, the orientation of dUMP is not affected and both ligands are bound at each site. The asymmetry in the two active sites that persists is subtle and is related to the degree of closure of one active site relative to the other: the more open conformation is associated with ligand misalignment and fewer proteinCB3717 contacts, consistent with weaker binding affinity. These differences can be traced to different packing interactions at the edges of the ß-sheet. In particular, packing of two loops, one from each protomer, that come together at the dimer interface to form the binding site for the phosphate moiety of dUMP is different at the two active sites (Table II).
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Many proteins have a clearly defined mechanism for negative cooperativity in ligand binding which has a functional role, such as regulation of activity or product release (Pettigrew et al., 1990; Bystrom et al., 1999
; Stock et al., 2000
). In TS, conformational change in response to ligand binding occurs by small shifts of several protein segments and there is no obvious conserved route for relaying these changes from one active site to impair binding and catalysis at the other, hence there may be no physiological reason for half-the-sites activity; evolution may have simply optimized one active site to the detriment of the other. However, if part of the basis for half-of-the-sites activity in TS involves opening the active site of the inactive protomer when the active protomer is tightly closed, then negative cooperativity in ligand binding may aid product release.
Conclusion
We have determined the structure of the most conservative EcTS variant, in the functional sense, of the conserved residue D169(221) in a complex with dUMP and a cofactor analog in order to shed light on the role of D169(221) in catalysis. We have previously shown that a hydrogen bond between D169(221) O2 and N3 on the pterin ring of the cofactor contributes to the mechanism for cofactor binding and almost all residues that cannot form this hydrogen bond do not form an analog of the first covalent steady-state intermediate in the reaction. As expected, D169(221)C, which readily forms the analog intermediate, forms the analogous hydrogen bond to N3 of CB3717.
However, several features of the structure indicate that intermediate II, and presumably other ternary complex intermediates in the reaction, are a mixture of fully closed structures and more open structures in which the reactants are not aligned for catalysis. As a consequence, these intermediates have a lower probability of reaching a transition state structure than in the wild-type enzyme. Evidence from I264Am, another cofactor-binding mutation (unpublished data), suggests that this heterogeneity in ground-state structures alone can account for the diminished activity, particularly since TS is a bi-substrate enzyme that derives much of its catalytic power through optimizing the alignment of the reactants prior to the chemical steps of the reaction. However, we do not rule out the possibility that the sulfhydryl group of Cys is less effective than the Asp carboxyl in stabilizing the transition state during breakdown of the covalent ternary complex, as previously suggested (Matthews et al., 1990b; Chiericatti and Santi, 1998
) and that this may also contribute to a reduction in kcat. The asymmetry of the D169(221)C ternary complex structure further shows that cofactor binding affinity is tightly coupled to extent of partitioning to the closed enzyme conformation required for optimal alignment of reactants and this links negative cooperativity in cofactor binding (Dev et al., 1994
) to the reduced or absent enzyme activity measured for one protomer of the enzyme (Pookanjanatavip et al., 1992
; Maley et al., 1995
; Variath et al., 2000
). These results again emphasize the absolute requirement for structure in interpreting results of mutagenesis.
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Received August 22, 2002; revised December 10, 2002; accepted December 19, 2002.