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1 Department of Chemistry and 2 Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-2084, USA
Reprint requests to: Robert L. Van Etten, Department of Chemistry, Purdue University, West Lafayette, IN 47907-2084, USA; e-mail: vanetten{at}purdue.edu; fax: (765) 494-5274.
(RECEIVED June 3, 2005; FINAL REVISION July 15, 2005; ACCEPTED July 15, 2005)
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Abbreviations: PTP, protein tyrosine phosphatase • LMW PTP, low-molecular-weight protein tyrosine phosphatase • HMW PTP, high-molecular-weight protein tyrosine phosphatase • DS PTP, dual-specificity protein tyrosine phosphatase • BPTP, bovine protein tyrosine phosphatase • TPTP, Tritrichomonas foetus protein tyrosine phosphatase, DCl, deuterium chloride • NaOD, sodium deuteroxide • D2O, deuterium oxide • DSS, 2,2-dimethyl-2-silapentane-5-sulfonate • kcat, catalytic rate constant or turnover number • Km, Michaelis-Menten constant • NMR, nuclear magnetic resonance • p-NPP, para-nitrophenyl phosphate • Vmax, enzyme velocity at saturating substrate concentration
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051618805.
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Introduction |
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Despite the lack of overall sequence and structural similarity, PTPs exhibit a similar two-step catalytic mechanism, involving the formation and subsequent hydrolysis of a covalent phosphoenzyme intermediate. The catalytic mechanism of the LMW enzymes has been thoroughly characterized and serves as a model for all family members. Both kinetic and computational studies indicate that the first stage of the reaction involves the donation of a proton to the leaving group and a near-concurrent formation of a phosphoenzyme intermediate (Dillet et al. 2000; Asthagiri et al. 2002). Nucleophilic attack by the active site cysteine upon the phosphorous atom of the phosphate substrate produces a phosphoenzyme intermediate (Wo et al. 1992; Cirri et al. 1993; Davis et al. 1994a). Experimental and computational studies of several PTPs, including DS and LMW, have demonstrated that this cysteine exhibits a perturbed pKa and exists in the enzyme as a thiolate anion (Denu and Dixon 1995; Evans et al. 1996; Dillet et al. 2000; Czyryca and Hengge 2001; Kim et al. 2001; Asthagiri et al. 2002; Thomas et al. 2002). A conserved aspartic acid residue serves to protonate the oxygen leaving group of the phosphoester (Zhang and Van Etten 1991a,b). The last, and generally rate-limiting, step of the overall reaction involves hydrolysis of the phosphoenzyme intermediate and release of the final product, inorganic phosphate (Zhang and Van Etten 1991b; Asthagiri et al. 2004).
The similar catalytic mechanism of PTPs is facilitated by the conserved structural conformation of the CX5R motif, often referred to as the phosphate binding loop, or P-loop. Despite a lack of overall structural similarity between PTP families, a comparison of P-loop conformations in known structures reveals that they are highly superimposable and are well suited to binding phosphate (M. Zhang et al. 1995). The P-loop has a distinctive conformation, with the backbone NH groups facing in toward the site of phosphate binding. In HMW PTPs, the motif contains several glycine residues, which readily allow the chain to adopt an optimal conformation for substrate binding. In LMW PTPs, only one glycine is present in the CX5R motif, but a completely conserved active site asparagine residue is present and it exists in a strained, left-handed conformation. This distinctive feature alters the P-loop conformation so that it resembles the glycine-rich P-loops found in HMW PTPs. This strained conformation is maintained through a hydrogen bonding network that depends in part on interactions with residues outside of the P-loop, and allows the backbone NH groups of the active site loop to be oriented toward the substrate phosphate group.
In the presence of inhibitors or substrate analogs, the P-loop is well defined in X-ray structures and appears to be one of the most rigid portions of the LMW enzymes (Su et al. 1994; Zhang et al. 1994, 1997; Wang et al. 2000a,b). This strained conformation is maintained through hydrogen bond interactions between the P-loop cysteine, asparagine, serine, and two other highly conserved serine and histidine residues (S43 and H72 in BPTP). Mutation of either of these two non–P-loop residues results in enzymes with reduced catalytic efficiency and decreased overall protein stability (Chiarugi et al. 1994; Davis et al. 1994b; Evans et al. 1996).
The LMW PTP isolated from the primitive eukaryotic bovine parasite Tritrichomonas foetus contains the highly conserved active site CX5R motif, the catalytically critical aspartic acid, and the conserved serine of the active site hydrogen bonding network. However, the otherwise highly conserved histidine has been replaced by a glutamine residue (Q67). Kinetic studies of the wild-type protein and the Ala, Asn and His mutants of Q67 demonstrated that the glutamine residue is important to the catalytic activity of the enzyme (Thomas et al. 2002). In addition, those experiments revealed that the residue affects the active site electrostatics such that the pKa of the active site cysteine was further depressed in the histidine mutant compared with the wild-type enzyme. To further explore these unique features of the TPTP enzyme and its active site hydrogen bonding network, the three-dimensional solution structure of the wild-type TPTP enzyme is reported here.
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represents the phosphate inhibited, transiently bound form of the enzyme. There are ~134 peaks corresponding to the 138 expected nonproline backbone resonances. For the phosphate-containing sample, nearly complete backbone and side-chain assignments were achieved, excluding only A1, R17, S18, and Y119 (BMRB deposition number 5850
[BMRB]
).4 The most notable peaks in this spectrum belong to several of the P-loop resonances, including L12, G13, N14, I15, and C16. However, these residues were not seen in a phosphate-free sample of wild-type TPTP.5 The appearance of P-loop residues upon the addition of a compound that is known to bind in the active site has also been observed for other enzymes containing a CX5R active site motif, including the bovine LMW PTP, the arsenate reductase proteins from Escherichia coli and Staphylococcus aureus and the cancer-associated PRL enzymes (Logan et al. 1994; Zhou et al. 1994; Ab et al. 1997; Stevens et al. 1999; Jacobs et al. 2001; Messens et al. 2002; Kim et al. 2004; Kozlov et al. 2004; Laurence et al. 2004).
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When compared to tentative assignments made for the phosphate-free sample, a few other peaks appear to have shifted significantly upon the addition of phosphate. Similar results were also observed for the wild-type BPTP protein during a phosphate titration experiment (T. Åkerud, pers. comm.). Interestingly, these include residues V115 and S121 in the loop containing the conserved aspartic acid (N110-D123), which has been shown to fluctuate between different conformations in solution (Åkerud et al. 2002).
Structure determination
The XPLOR-simulated annealing protocol was used to generate structure ensembles. A total of 2494 distance and dihedral angle restraints were used in the structure calculations (Table 1). A superposition of the 20 lowest energy wild-type TPTP structures is shown in Figure 2A. The structures satisfy the distance restraints with no violations > 0.25 Å and show good covalent geometry and nonbonded contacts. The energy-minimized average structure created from this ensemble is shown in Figure 2B (ensemble and average structure deposited as Protein Data Bank [PDB] file f1P8A).
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. Secondary structure elements of the wild-type TPTP enzyme were identified by using the Kabsch-Sander algorithm (Kabsch and Sander 1983). The secondary structure elements include four 
-strands and five 
-helices. The strands are formed by residues A6–V10, L34–A38, V82–A85, and K105–L108. The five helices include residues C21–V29, T51–N60, D76–K79, Q88–M97, and F125–H143. Helix 5, which includes residues between F125 and H143, is considered a single helix with a kink or bend in it between residues K134 and K137.
Examination of the structure ensemble reveals that the first five residues of the TPTP enzyme are disordered as observed in other LMW PTP structures (Zhang et al. 1994, 1997; Wang et al. 2000a, b; Rastogi et al. 2002). The region of highest root mean square (RMS) distribution is the loop connecting 4 and 5. This loop contains the catalytic aspartic acid and two conserved tyrosines and corresponds to the region of highest flexibility observed in the BPTP enzyme (Kim et al. 2004).
In order to compare the active site structure when the side chains of Q67 and N14 are restrained within hydrogen bonding distance, a second structure ensemble (referred to as Q67–N14 TPTP) was calculated. These calculations included an additional hydrogen bond (represented by two additional restraints) specified between the HE21 or HE22 side-chain amide protons of Q67 and the side-chain amide oxygen of N14. The 20 lowest energy Q67–N14 structures satisfy the distance restraints with no violations > 0.25 Å and show good covalent geometry and nonbonded contacts. The structural statistics for this ensemble are also shown in Table 1. The energy-minimized average Q67–N14 structure is virtually identical to the wild-type TPTP structure (data not shown).
Steady-state kinetics
Michaelis-Menten kinetic parameters were determined for mutant TPTP enzymes with p-NPP as the substrate (Table 2). The S37A and S37A/Q67A mutants exhibit kcat/Km values that are 9% and 2% that of the wild-type enzyme, respectively. The observed reduction in enzymatic activity is consistent with the anticipated participation of S37 in the active site hydrogen bonding network. They are similar to changes observed for analogous mutations made in the hydrogen bonding network of BPTP (H72A and S43A) (Table 2; Evans et al. 1996).
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As expected, the global fold of the TPTP protein is similar to that observed for other members of the LMW PTP family. The overall architecture of the protein contains two motifs. The -strands form a parallel, highly twisted, four-stranded -sheet that is surrounded by five -helices. The TPTP active site is located in the first motif, at the C-terminal end of the central sheet in an open cleft that is readily substrate accessible (Fig. 2B).
The active site configuration resembles that of other LMW PTPs. The nucleophilic cysteine is located in the center of the active site P-loop (residues C11 R17) across from the proposed catalytic aspartic acid, D117 (see below; Fig. 3A). Biochemical studies of the TPTP enzyme have demonstrated that the active site cysteine exhibits a perturbed pKa of < 4, indicating that it normally exists as a thiolate anion (Thomas et al. 2002). Notwithstanding the low observed pKa of this cysteine residue, it was protonated in all structure calculations, following the procedure used in determination of the BPTP NMR structure (Logan et al. 1994). In its position located across from the nucleophilic cysteine, D117 can facilitate protonation of the substrate leaving group (Fig. 3A). In sequence alignments with other LMW PTP enzymes, D117 appears in the position of the strictly conserved catalytic aspartic acid. Superposition of the wild-type TPTP NMR and BPTP X-ray structure (1PNT) reveals that the backbone position of D117 corresponds to that of the catalytic aspartic acid, D129, of the bovine enzyme.
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). Structural and computational studies of PTPs have shown that optimal phosphate binding is facilitated by multiple P-loop backbone NH groups as well as the arginine side chain (M. Zhang et al. 1995; Z.Y. Zhang et al. 1995; Evans et al. 1996; Dillet et al. 2000). The conserved arginine forms hydrogen bonds between the hydrogens of its positively charged guanidyl group and oxygen atoms of the phosphate. These interactions (in part) are responsible for positioning the phosphate near the nucleophilic cysteine and reducing the net negative charge of the phosphate group (Dillet et al. 2000). In the BPTP structures, interactions with another conserved aspartic acid, D92 in BPTP, hold the arginine side chain in position. In the case of wild-type TPTP, NOEs were observed between residues D87 and R17, supporting the idea that this aspartic acid also helps to position the arginine side chain (Fig. 3A).
Hydrogen bonding network
Examination of P-loop conformations of virtually all known PTP structures reveals that they are highly superimposable and well-suited to binding phosphate with the aid of the backbone NH groups that are pointed toward the site of phosphate binding (M. Zhang et al. 1995; Zhang et al. 1997). In LMW PTPs, this conformation is uniquely facilitated by a conserved active site asparagine residue, which is found in a strained, left-handed conformation in X-ray structures. This strained conformation is maintained through an extensive hydrogen bonding network between the P-loop cysteine, asparagine, serine, and two other highly conserved serine and histidine residues (S43 and H72 in BPTP) (Zhang et al. 1994; Evans et al. 1996). In the BPTP X-ray structure, the hydrogen bond network contains interactions between the C12 sulfur atom and the S19 hydroxyl proton, the side chain OD1 of N15 and the HE2 proton of H72, and the S43 hydroxyl proton and the N15 HD21 or HD22 protons (Su et al. 1994; Zhang et al. 1994; M. Zhang et al. 1995).
The TPTP enzyme contains a serine (S37) analogous to S43 of BPTP but lacks the otherwise highly conserved histidine residue, which is instead replaced by a glutamine residue, Q67. Kinetic studies of the wild-type TPTP enzyme and its Q67A, Q67N, and Q67H mutants suggested that Q67 participates in the active site hydrogen bonding network and could serve a role analogous to that of H72 in BPTP (Table 2). In addition, 1H NMR studies using the Q67H mutant demonstrated that a histidine residue in this position experiences electrostatic effects similar to those experienced by H72 in BPTP (Thomas et al. 2002).
However, in the average calculated TPTP structure, the side chains of Q67 and N14 are pointed away from one another and are ~8 Å apart (Fig. 3A). The side-chain groups were not constrained in calculations due to a lack of observed NOEs between these two residues. It should be noted that stable hydrogen bonds have been detected via 3hJNC couplings between amide 15N and carbonyl 13C in small, rigid proteins such as ubiquitin (Cordier and Grzesiek 1999, 2002). However, it is not possible to measure such small couplings (between –0.2 and –0.65 Hz) in TPTP due to its size. NOEs were detected between Q67 and other residues near the active site, including P19, consistent with the presence of Q67 at the active site near the P-loop as expected.
In light of this and of the kinetic results that suggest an interaction between Q67 and N14, an alternative structure was calculated in which additional hydrogen bond restraints were specified explicitly between the OD1 of N14 and HE21 or HE22 of Q67. Inclusion of these restraints in the structure calculations forces the side chains of N14 and Q67 to be within hydrogen bonding distance (Fig. 3). Comparison of the wild-type and Q67–N14 structures reveals that several residues have shifted to accommodate this interaction. The side chain of Q67 flips and moves closer to N14 as specified by the hydrogen bond restraint. In addition, E22 also makes a slight rotation, but this does not result in any new contacts. This shows the enzyme can accommodate this interaction without significant alterations or increases in energy or restraint violations.
Comparison of the BPTP X-ray, BPTP NMR, wild-type TPTP, and Q67–N14 structures reveals some potentially significant differences in the P-loop backbone NH orientations (Table 3; Fig. 4). In both the wild-type and Q67–N14 TPTP structures, N14 is found in the strained left-handed conformation, most closely resembling the configuration observed in the BPTP X-ray structure. In the BPTP NMR structure, the asparagine is not found in the strained left-handed conformation, possibly explaining why the backbone NH groups of many of the residues in the P-loop of that structure are not oriented toward the site of phosphate binding. The orientation of the P-loop NH groups facilitates an optimal interaction with the oxygen atoms of the phosphate group. The strained, left-handed conformation observed for N14 in the wild-type and Q67–N14 TPTP structures is a hallmark of the active site hydrogen bonding network.
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). The difference could be due to the inherent flexibility of the protein in solution, a feature almost uniquely revealed by NMR spectroscopy (Gao et al. 2004). In the BPTP NMR structure, H72 is located on a long loop between helices 2 and 3 that shows some structural dispersion among members of the ensemble (Logan et al. 1994). Severe exchange broadening was also observed during backbone assignment for the corresponding loop in HCPTPA (residues 67–80), indicative of its flexible nature in solution (Rastogi et al. 2002). In the wild-type and Q67–N14 TPTP structure ensembles, the loop between helices 2 and 3 also shows some structural dispersion among members of the ensemble (Fig. 2A), further suggesting that this hydrogen bond interaction is not present at all times in solution. These observations bring forth the idea that Q67 (or H72 in BPTP) is found in a structurally disperse loop, accounting, at least in part, for its apparent lack of participation in the active site hydrogen bonding network in the NMR structures. However, in the BPTP crystal structure, the atoms of this histidine have an average B factor of ~15, indicating that it is very well-ordered (Zhang et al. 1994). The X-ray structure, on the other hand, may show only one form of the protein that was trapped in the crystal, while the NMR structure shows an ensemble of structures present in solution.
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Phosphate ion concentration significantly affects the solution dynamics of LMW PTPs. For BPTP, protein dynamics data have been collected in the presence of 200 mM phosphate, a competitive inhibitor of the LMW PTPs. Those studies established that the loop containing H72 is very stable, as indicated by low order parameters, and that the P-loop residues undergo minimal movement on the micro- to millisecond timescale (Åkerud et al. 2002). Thus, in the presence of 200 mM phosphate, residues in the active site hydrogen bonding network appear to be undergoing little motion. The sample used in the BPTP NMR structure determination contained 100 mM phosphate, which also facilitated the observation of resonances in and around the active site (Logan et al. 1994; Zhou et al. 1994). However, despite the presence of a phosphate ion bound at the active site, the active site configuration was different than that observed in the BPTP X-ray structure (Fig. 5). Severe exchange broadening was also evident for residues of the P-loop and the loop containing H72 of the human isoenzyme HCPTPA when measurements were made in the presence of 50 mM phosphate (Rastogi et al. 2002). The sample of TPTP with 20 mM phosphate contains the lowest concentration of phosphate used for structure determination of a LMW PTP. Based on the observations of apparent increasing protein dynamics in the presence of decreasing amounts of phosphate, it is not surprising that the wild-type TPTP structure shows higher dispersion among members of its ensemble. We propose the existence of a transient hydrogen bond between Q67 and N14 as a result of the high flexibility of the TPTP enzyme in the presence of low phosphate concentrations. Such a transient hydrogen bond would explain the observed strained conformation of N14 in the TPTP structure and the previously observed kinetic data (Thomas et al. 2002).
While the hydrogen bond between BPTP H72 and N15 is important for enzyme stability and substrate binding, examination of the BPTP X-Ray structure reveals that the strained conformation of the active site asparagine is also stabilized through interactions with the hydroxyl proton of S43 (Fig. 5). The S43A mutant of BPTP showed Vmax activity that was 59% that of the wild-type enzyme (Table 2; Evans et al. 1996). Similarly, the S37A mutant of TPTP showed a Vmax that was 60% that of the wild-type enzyme. The reduction in activity of the S37A mutant is similar to that observed for the Q67A TPTP mutant (Table 2). These results are consistent with the participation of S37, located at the C-terminal end of 2, in the hydrogen bonding network of TPTP. The position of S37 in the wild-type and Q67–N14 TPTP structures is almost identical (Fig. 5). The distance between the side-chain oxygen of S37 and the side-chain nitrogen of N14 is ~5.2 Å in the wild-type structure and ~4 Å in the Q67–N14 structure. Although these residues are not within hydrogen bonding distance, they are much closer to each other than the side chains of N14 and Q67. These distances are similar to those observed for S43 and N15 in the BPTP NMR structure, where the H72–N15 hydrogen bond does not appear to be present. This may indicate that the interactions of S37 and N14 in TPTP are less transient than are those of Q67 and N14.
Although the H72A BPTP enzyme shows an activity that is 15% that of the wild-type enzyme (Evans et al. 1996), the corresponding glutamine to alanine mutant of TPTP shows activity that is 76% that of the wild-type enzyme (Thomas et al. 2002). Kinetic measurements were carried out in a phosphate-free buffer, creating conditions in which we have observed high protein flexibility. Our interpretation of these results is that a transient hydrogen bond between Q67 and N14 is formed under these conditions. Formation of this bond is more difficult in the Q67N mutant and impossible in the Q67A mutant, consistent with the observation of reduced enzymatic activity for these mutants. In BPTP, the H72A mutation has a much larger effect on the enzyme activity than does the S43A mutation. This is not observed in TPTP, where the Q67A and S37A TPTP mutants show comparable reductions in activity relative to that of the wild-type enzyme. In light of these structural and kinetic observations, it appears that in TPTP the interactions of S37 and Q67 with N14 contribute equally to stabilizing the strained conformation of N14 within the P-loop. This would be a further unique feature of the TPTP enzyme.
Conclusions
The exceptional similarity of the P-loop structures from HMW and LMW PTPs demonstrates the importance of providing a specific conformation for optimal binding of the phosphate and transition state. In HMW PTPs, adoption of this conformation is made possible by the glycine-rich nature of the P-loop. However, in LMW PTPs, this conformation is stabilized through the interactions of the active site hydrogen binding network.
Biochemical data clearly suggest that the roles of Q67 and S37 in the hydrogen bonding network of TPTP are similar to that of the conserved histidine and serine found in all other eukaryotic LMW PTPs. However, in the wild-type TPTP NMR structure measured at low phosphate concentration, Q67, S37, and N14 do not appear to be within hydrogen bonding distance. Despite this, the active site asparagine is found in the expected strained left-handed conformation, a hallmark of the hydrogen bonding network, and the P-loop is nearly superimposable with that of other PTPs. The observation of N14 in the strained conformation may be due to the presence of transient hydrogen bonds within the network due to protein dynamics in solution and the presence of low phosphate concentrations. Transient hydrogen bonds would give the active site the flexibility needed to adopt the optimal conformations needed for phosphate binding and release. A flexiblephosphate binding loop and activesitewouldallow the accommodation of a variety of phosphorylated substrates, which seems necessary given the wide variety of enzymes containing the CX5R active site motif.
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Mutagenesis and expression of recombinant TPTP
The S37A and S37A/Q67A mutations were prepared according to the protocol given in Thomas et al. (2002). All proteins were expressed and purified by using the two-step ion exchange and size exclusion procedure outlined in Thomas et al. (2002).
Preparation of NMR samples
The production of 15N and 15N/13C uniformly labeled protein samples was achieved by growing E. coli BL21 (DE3) cells harboring the wild-type TPTP plasmid in M9 minimal media. Phosphate-free NMR samples containing 1–2 mM wild-type TPTP were prepared in 100 mM sodium acetate and 0.1 mM DSS dissolved in 10% D2O/90% H2O. Samples containing phosphate were prepared in 130 mM NaCl, 20 mM NaH2PO4, and 0.1 mM DSS dissolved in 10% D2O/90% H2O. Sodium azide was added to each sample to a final concentration of 0.003 M NaN3, and the pH was adjusted to 5.2 by the addition of NaOD or DCl. Shigemi tubes were used for all NMR experiments.
NMR spectroscopy
NMR spectra were collected on a 600-MHz Varian INOVA spectrometer at 30 ± 1°C or 25 ± 1°C for the phosphate-free and phosphate-containing samples, respectively. Backbone assignment was achieved with the HSQC, HNCACB, CBCA(CO)NH, and HNCO spectra (Cavanagh et al. 1996). Side-chain assignments were achieved with the CCONH, HCCONH, 15N-TOCSY-HSQC, HBCBCGCDHD, HBCBC GCDCEHE, and HCCH-TOCSY experiments (Gao et al. 2004). Distance information was obtained by using the 15N-NOESY-HSQC (150-msec mixing time) and 13C-NOESY-HSQC (150-msec mixing time) spectra (Gao et al. 2004). An HNHA spectrum was acquired on the phosphate-containing sample for generation of dihedral angle restraints based on the 3JHNH coupling constant values (Gao et al. 2004). For the phosphate-containing sample, a 15N-HSQC spectrum was taken between each three-dimensional experiment in order to assess the quality and stability of the protein over time. This was not done for the phosphate-free sample because it was not known to be necessary at the time of data acquisition. Indirect chemical shift referencing was performed using the frequency ratios of 1H, 15N, and 13C according to the protocol of Wishart et al. (1995). Multidimensional NMR spectra were processed by using NMRPipe (Delaglio et al. 1995). Spectra were assigned using the graphical interface SPARKY (T.D. Goddard and D.G. Kneller, University of California, San Francisco).
Structure determination
NOE distance restraints were generated from NOESY spectra using the average dN(i, i + 3) NOE peak height and corresponding known distance in helical conformations for calibration of distances. Peak heights involving nonstereospecifically assigned methylene protons, methyl groups, and H
and H
protons of phenylalanines were corrected by dividing the peak height by the number of degenerate protons prior to distance restraint generation. These corrections were made in conjunction with the 1/r6 averaging method in XPLOR (Fletcher et al. 1996). To account for the narrower line width of the methyl resonance, an additional 0.5 Å was applied to each upper bound for NOEs involving methyl groups. If two methyl groups were involved, a distance of 1.0 Å was applied to the upper bound (Wagner et al. 1987). Using the three-dimensional HNHA experiment 3JHNH coupling constants were calculated and 43 
dihedral angle restraints were generated (Karplus 1959; Kuboniwa et al. 1994). Dihedral angle restraints (58 and 56
) for residues located in secondary structure elements were generated by using TALOS, and each error bound generated by the program was doubled in all structure calculations (Cornilescu et al. 1999; Amezcua et al. 2002). Seventy-four hydrogen bond restraints (two per hydrogen bond) were generated for residues predicted to be in secondary structure elements. For the Q67–N14 TPTP structure, two additional hydrogen bond restraints were specified between OD1 of N14 and HE21 or HE22 of Q67.
Calculations were performed by using the simulated annealing protocol of XPLOR version 3.851 with the 1/r6 distance averaging method (Brunger 1992). One hundred structures were calculated for both the wild-type and Q67–N14 ensembles. Accepted structures did not contain violations > 0.25 Å and 5° in the user-defined distance and dihedral angle restraints, respectively. The average structure of each ensemble was generated from the 20 lowest energy structures and was refined against the structure restraints using the Powell minimization protocol. The quality of the structures was assessed using PROCHECK-NMR (Laskowski et al. 1996).
Steady-state kinetics
Michaelis-Menten kinetic parameters were determined for the S37A and S37A/Q67A TPTP enzymes by using p-NPP as the substrate (Thomas et al. 2002).
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4 HN assignment of S18 was possible only through assignment of the 13C-NOESY-HSQC peaks of P19. In BPTP, the corresponding serine residue S19 is visible in the HSQC at an amide proton frequency 10.18 ppm (Zhou et al. 1994). In the 13C-NOESY-HSQC strips of P19, a peak at 10.4 ppm was visible and on the basis of homology was assigned to S18. This peak is not visible in the 15N HSQC of wild-type TPTP.
5 The 15N-HSQC spectrum of a phosphate-free TPTP sample had ~128 peaks corresponding to the 138 expected nonproline backbone resonances. Unassigned residues included A1, L12–S18, S41, Q47, S48, Q54–K58, Q67, R68, I90–S92, S100–C102, N110, N113, Y119, Y120, F125–F129, F139, and L140. Importantly, the residues in the phosphate binding loop (L12–S18) and Q67 could not be assigned, which prevented any further study of the active site hydrogen bonding network in the absence of phosphate.
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Acknowledgments |
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References |
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