European Molecular Biology Laboratory, Meyerhofstrasse 1, Postfach 102209, D-69012 Heidelberg, Germany, 3 Vrije Universiteit Brussel, Instituut voor Moleculaire Biologie, Paardenstraat 65, B-1640 Sint-Genesius-Rode, 4 Research Unit for Tropical Diseases, Christian de Duve Institute of Cellular Pathology and Laboratory of Biochemistry, Catholic University of Louvain, Avenue Hippocrate 74, B-1200 Brussels, 5 Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2620 Wilrijk, Belgium and 6 Department of Biochemistry, University of Oulu, Linnanmaa, FIN-90571 Oulu, Finland
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Abstract |
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Keywords: enzymology/leishmania/stability/structure/triosephosphate isomerase
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Introduction |
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A striking feature of the sequence of leishmania TIM is the presence of a glutamic acid at position 65, whereas in all other known TIM sequences a glutamine is observed at this position, which is at the beginning of loop-3. As discussed previously (Kohl et al., 1994), this glutamine is completely buried inside the dimer interface and is involved in an inter subunit hydrogen bonding network. The nitrogen atom of the side chain acts as a hydrogen bond donor to two buried main chain carbonyl oxygen atoms. One hydrogen bond is to the backbone oxygen of Lys13 in loop-1, a conserved active site residue with `unallowed' dihedral angles in each TIM structure, and the other hydrogen bond is to the backbone oxygen of Ala42 of loop-2. The oxygen atom of the glutamine side chain hydrogen bonds to N(Gly76) of the Thr75Gly76 peptide bond in loop-3 of the other subunit, which are completely conserved residues. This hydrogen bonding network is extended through the interaction of the Thr75Gly76 backbone oxygen with the side chain of the completely conserved Arg98 of loop-4. Therefore, Gln65 is an intricate part of a highly conserved hydrogen bonding network across the dimer interface which may stabilize the strained conformation of the catalytic Lys13.
The glutamate found at position 65 in LmTIM is expected to be a destabilizing feature, yet there is no evidence from the published data that LmTIM is less stable than TbTIM (Kohl et al., 1994). The crystal structure of LmTIM is therefore of interest, because it will show structural rearrangements that might have occurred to accommodate the sequence change. Here we report the crystal structure of leishmania TIM at 1.8 Å resolution and compare its structure with trypanosomal TIM. In addition, we constructed a leishmania E65Q variant and show that this E65Q variant is considerably more thermostable than the wild-type.
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Materials and methods |
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Site-directed mutagenesis for making the E65Q variant was carried out using a three-step PCR method (Mikaelian and Sergeant, 1992) starting from the recombinant LmTIM gene. The method required a forward primer (primer 1), two antisense primers (primer 2 and 3) and the mutagenic oligonucleotide (5'-GTCATCAGCGCTCAGAACGCG-3'; the mutated codon is underlined). Primers 1 and 3 containing the Nde1 and Pst1 restriction sites had already been used for the sequence verification when the gene was cloned (Kohl et al., 1994
). Primer 2 (5'-CAGTCCTGGCGCTCCGAGTGGCCC-3') was located downstream of the site of mutagenesis but upstream of primer 3. Amplification was carried out in a Hybaid thermal reactor using Vent DNA polymerase. The amplified Nde1Pst1 fragment containing the mutation was used to replace the corresponding fragment of the wild-type LmTIM gene in the pET3a vector. The presence of the mutation and correct insertion in the wild-type gene were verified.
Overexpression and purification of wild-type LmTIM and the E65Q mutant
Overexpression in Escherichia coli strain BL21(DE3)pLysS was exactly as described previously (Kohl et al., 1994). Bacterial cells of 500 ml culture were collected by centrifugation (30 min at 5000 g at 4°C) and frozen at 80°C. The cells were resuspended in 40 ml of lysis buffer [100 mM triethanolamine (TEA), pH 8, 5 mM reduced dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM NaN3, 0.2 mM phenylmethylsulphonyl fluoride (PMSF)] and lysed by sonication. After centrifugation (30 min, 12 000 g, 4°C), most of the overexpressed protein was present in the supernatant. The protein was further purified by ammonium sulphate precipitation at concentrations between 45 and 65%. The 65% pellet was dissolved in 5 ml of 20 mM TEA, pH 8, 1 mM DTT, 1 mM EDTA, 1 mM NaN3 and dialysed overnight against three changes of the same buffer. The protein was loaded on a 25 ml column of CM-Sepharose CL4B, washed with 50 ml of 20 mM TEA, pH 8, 1 mM DTT, 1 mM EDTA, 1 mM NaN3 and eluted with a 100 ml gradient from 0 to 150 mM NaCl in the same buffer. Active fractions were stored at 4°C. The protein appeared as a single band on Coomassie Brilliant Blue-stained 12% SDSPAGE gels. The specific activity of the purified LmTIM was 3700 units/mg; the specific activity of the E65Q mutant was 5600 units/mg.
Enzyme assays
Activity was determined at 25°C by monitoring the absorbance of NADH at 340 nm for 10 min in a SpectraMAX340 Microtiterplate Reader (Molecular Devices, Sunnyvale, CA). Routinely, the assay mixture (200 or 80 µl) contained 0.47 mM GAP, 0.42 mM NADH, 10 mg/ml glycerol-3-phosphate dehydrogenase and ~2 ng/ml TIM in 100 mM TEA, pH 7.6. To determine the catalytic parameters, the GAP concentration was varied between 0.1 and 1.25 mM. In the reverse direction the reaction mixture contained 0.255 mM DHAP, 5 or 10 mM Na2HAsO4, 1 mM NAD, 25 mg/ml glyceraldehyde-3-phosphate dehydrogenase and ~20 ng/ml TIM. For inhibition measurements eight concentrations of the substrate analogue 2-phosphoglycolate (2PG) between 0.025 and 0.4 mM were used and four GAP concentrations (0.15, 0.3, 0.6 and 1.2 mM). The inhibition constant was obtained by fitting the data using Grafit, assuming competitive inhibition.
CD spectroscopy
CD measurements were performed with a Jasco J-715 spectropolarimeter. The loss of secondary structure of LmTIM and the E65Q mutant was observed by recording the CD signal at 222 nm as a function of temperature. During the experiment the temperature of the sample changed at a rate of ~1°C/min between 30 and 90°C. The temperature of the sample was controlled during the measurements by a sensor built into the cuvette holder. The sensor was connected to a Haake N3 circulating bath which adjusted the temperature of the sample. The temperature scale in the final plots represents the temperature actually measured by the sensor in the immediate vicinity of the sample. The protein was always precipitated after the experiments. The apparent melting temperature at the midpoint of the transition (Tm) was obtained by fitting the experimental data points (CD signal versus temperature) with a sigmoidal function.
Mass spectrometry
All mass spectrometric experiments were carried out using an API III triple quadrupole mass spectrometer (Perkin-Elmer Sciex, Thornhill, ON, Canada) equipped with a nanoelectrospray ion source (Wilm and Mann, 1996). For the intact molecular weight measurement the final protein concentration was ~10 µM in 50% methanol40% water10% formic acid. For peptide sequencing, 10 pmol of LmTIM were digested with trypsin (sequencing grade, Boehringer Mannheim) in 10 µl of 50 mM ammonium hydrogencarbonate at an enzyme-to-substrate ratio of 1:50 at 37°C for 4 h. An aliquot of the resulting digestion mixture was diluted with 50% methanol45% water5% formic acid to a final concentration of 1.5 µM and 1 µl was placed in the spraying capillary of the nanoelectrospray ion source. Several of the peptides were fragmented and their sequence determined, including the N-terminal peptide and the peptide containing residue 65 (residues 6070). In order to determine unambigously the presence of a glutamic acid residue at position 65, the peptide mixture was esterified and the sequence of the modified peptide (6070) was determined again. The esterification was carried out essentially as described previously (Hunt et al., 1986
). Briefly, 1 ml of methanol was cooled at -20°C for 1020 min, followed by the addition of 100 µl of acetyl chloride. The reagent was left at room temperature for 10 min before use. A 2 µl volume of the tryptic digestion mixture was dried down in a vacuum centrifuge and reconstituted with 5 µl of the reaction mixture. After incubation for 45 min at room temperature, the sample was dried down in a vacuum centrifuge. The peptide mixture was dissolved in 90% water5% methanol5% formic acid and desalted using a small column (Wilm and Mann, 1996
). The peptides were eluted directly into the spraying capillary of the nanoelectrospray source for mass spectrometric sequencing.
Crystallization, data collection, structure determination and refinement
For the crystallization experiments the leishmania TIM (11 mg/ml) was in a 10 mM tris(hydroxymethyl)aminomethane (Tris)HCl buffer (pH 7.5; 25 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM NaN3) containing also 10 mM of the substrate analogue 2PG. Potential crystallization conditions were checked using screening procedures as described previously (Zeelen et al., 1994). Well diffracting crystals grow at room temperature in a few weeks in hanging drops using 2 µl of the protein solution and 2 µl well solution containing 20% PEG6000, 1 mM EDTA, 1 mM DTT, 1 mM NaN3 in a 100 mM acetic acidNaOH buffer, pH 4.5. Before data collection, the crystal was transferred into a 100 mM citric acidNaOH buffer, pH 5.9 (including 20% PEG6000, 1 mM EDTA, 1 mM DTT, 1 mM NaN3). After a few hours of equilibration in this buffer, the crystal was mounted in a glass X-ray capillary and the data were collected at room temperature with a MAR image plate area detector. The data were processed and merged with the DENZO package (Gewirth et al., 1995
). The data quality is presented in Table I
. The structure was solved in a straightforward manner by molecular replacement using AMORE (Navaza, 1994
). TbTIM was used as the search model. The rotation function and translation function calculations provided straightforward solutions. The correlation coefficient of the correctly oriented and positioned model was 64.9% and the R-factor was 40.3% for data at 2.5 Å resolution. These calculations confirmed that in this crystal form there is only one subunit per asymmetric unit, as the dimer twofold axis of the positioned LmTIM dimer coincides with a crystallograpic twofold axis. Subsequently, loop-6 (Glu167 to Thr179) and loop-7 (Tyr209 to Gln215), known to adopt different conformations in different structures, were removed from the correctly positioned model and electron density maps were calculated, using programs of the CCP4 package (CCP4, 1994
). These maps, interpreted using O (Jones et al., 1991
), showed good density for the substrate analogue 2PG as bound in the active site. These maps also showed clear density for loop-6, indicating that this loop had adopted the closed conformation, in agreement with the presence of 2PG in the active site. Subsequently, the missing loop-6 and loop-7 were rebuilt, the 2PG molecule was added to the model and the structure was refined, initially with X-PLOR (Brünger, 1992
) and finally with TNT (Tronrud, 1992
), using all data and the bulk solvent correction. Water molecules were added to the model during the final refinement steps. The final refinement statistics are shown in Table I
.
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The sequences of LmTIM and TbTIM can be aligned without the introduction of insertions or deletions and therefore the same numbering scheme can be used for both sequences. The active site residues for both enzymes are Lys13, His95 and Glu167. For comparative studies the 1.83 Å structure of trypanosomal TIM [Wierenga et al., 1991 (entry 5TIM in the Brookhaven Protein Data Bank (PDB)] was used. The superpositions, using the C atoms of the framework ß-strands and
-helices, were made with the LSQ option in O (Jones et al., 1991
). The database search for the interactions of aspartate and glutamate side chains was performed with WHAT IF (Vriend, 1990
). For the sequence analysis, 46 TIM sequences were extracted form the SWISS-PROT database.
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Results |
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Since a glutamate and a glutamine are indistinguishable in the electron density maps at this resolution, mass spectrometry was used to verify the sequence at position 65. The mass of intact LmTIM was determined to be 27 047 (±3) Da, corresponding to the calculated value from the sequence (Kohl et al., 1994) without a leading methionine and without any modifications. This was also confirmed by sequencing of the N-terminal peptide. For this the N-terminal tryptic peptide was fragmented and the sequence determined to be SAKPQPIAAANWK, without any modifications. The tryptic peptide containing residue 65 (residues 6070) was also fragmented and the sequence determined to be Y60VISAENAIAK70. Moreover, the glutamic acid residue at position 65 was verified by methylation and sequencing of the modified peptide.
Glu65 is buried at the dimer interface. As is shown in Figure 2, the side chain of Glu65 points towards the active site of its own subunit; it points away from the equivalent Glu65 of the other subunit (the distance between the CD atoms of the equivalent side chains is 17.7 Å). The OE2/OE1 atoms of Glu65 are at a distance of 8 Å from the active site; for example the OE1(Glu65)NZ(Lys13) distance is 8.4 Å and the OE1(Glu65)NE2(His95) distance is 8.1 Å.
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Discussion |
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Changes in pKa of buried residues due to the environment of the protein are known (Yang and Honig, 1992). One example, the V66K variant of staphylococcal nuclease, shows that the pKa of a buried lysine shifts from 10.2 (in the solvent) to below 6.4 (inside the protein) (Stites et al., 1991
). Another example concerns a buried glutamate in the complex of a mutated OMTKY3-inhibitor with a protease which causes the pKa of the buried glutamate to become 8.7 (Qasim et al., 1995
).
Although infrequent, the occurrence of a hydrogen bond between a protonated glutamate and a main chain oxygen atom is also observed in other structures (Table II). In at least one other structure, E.coli 3-isopropylmalate dehydrogenase, the interaction occurs at a buried dimer interface residue, as in LmTIM (Wallon et al., 1997
). Mutagenesis of this residue, Glu256, into a hydrophobic residue (together with a second point mutation) also increases the stability of this enzyme: the apparent Tm for this double mutant, E256I, M259V, is increased by 7°C (Kirino et al., 1994
).
The presence of the protonated Glu65 is expected to be a destabilizing feature of LmTIM for two reasons. First, at a pH above the pKa of free glutamate (in water), there is a free energy penalty for burying a protonated glutamate in the interior of the protein (Yang and Honig, 1992). Second, although the glutamate and glutamine side chains have the same size, the atomic structure near the Glu65 side chain is optimally able to form hydrogen bonding interactions with a glutamine side chain, but not with a protonated glutamate side chain, it remains deficit of one hydrogen bond donor (Figures 3 and 4
). The notion that Glu65 is a destabilizing feature is in fact confirmed by the significant gain in stability of the E65Q variant (Figures 5 and 6
).
Taking TbTIM as the reference structure, one would expect that a change of Gln65 into a glutamate would destabilize the dimer sufficiently so as to favour the formation of monomers. Such destabilization has been shown to occur for the H47N variant of TbTIM (Borchert et al., 1995) and for the R-TIM variant of TbTIM (Schliebs et al., 1997
). In the latter variant the tip of loop-3 is changed by a point mutation (T75R), by which a small, neutral, residue is changed into a bulky, charged residue, thereby interfering with the fit of loop-3 into a pocket near the active site of the partner subunit; consequently, the dimer stability of R-TIM is reduced. In the H47N variant the dimer interface residue His47 is changed into an asparagine; this relatively minor substitution nevertheless reduces the dimer stability and results in a dimermonomer equilibrium (Borchert et al., 1995
). However, LmTIM, with the sequence change Q65E, has the same stability as TbTIM. This is distinctly different from the V66K mutant in staphylococcal nuclease where the stability of the mutant has been compromised (Stites et al., 1991
). Apparently, LmTIM has evolved to overcome this destabilizing feature. Thus, additional compensating stabilizing interactions must be located elsewhere in the structure of LmTIM.
Based on a sequence comparison alone, LmTIM has three more prolines and three fewer glycines than TbTIM. In general, LmTIM has significantly fewer glycines, 16, and more prolines, 10, than the other TIM sequences, which average 21.7 and 8.2, respectively. The ratio of glycines to prolines for LmTIM is 1.60, the second lowest among all TIM sequences with values ranging from 1.55 to 4.80. Considering the denatured state, this difference would give less conformational space in the unfolded state for LmTIM compared with TbTIM favouring the folded state (Shortle, 1996).
In terms of structure, this sequence difference can be compared with TbTIM, which is the most closely related TIM in the database. Within a sphere of 6 Å from CD(Glu65) there are no charged side chain atoms (of aspartates, glutamates, histidines, lysines or arginines), either in LmTIM or in TbTIM; there is only one sequence difference within this sphere, which concerns the residue at position 43. This is a serine in TbTIM and a proline in LmTIM. This sequence difference could to some extent compensate for the energetic costs of the glutamine to glutamate change at position 65. First, the O(Ala42) of the Ala42Pro43 peptide bond is hydrogen bonded in both structures to N(Trp12) of loop-1. The carbonyl oxygen in an X-Pro peptide bond has been suggested to make stronger hydrogen bonds owing to increased electronegativity at the carbonyl caused by the three CN bonds at the proline nitrogen (Williamson, 1994). Second, the side chain of the proline is better suited for a rather hydrophobic pocket when compared with serine. Also, this serine cannot form satisfactory hydrogen bonds in TbTIM. Finally, the serine-to-proline change is not expected to induce strain in the main chain because the phi/psi values at this position are (92, 171) in each structure, in good agreement with preferred values for prolines (Karplus, 1996
).
Other sequence differences between LmTIM and TbTIM may also compensate for the destabilizing Q65E sequence difference. These are mapped on to the C trace in Figure 7
. There are three more sequence differences close to the main chain atoms of Glu65. In leishmania TIM there are Ser63, Val91, Ile92, whereas in trypanosomal TIM these residues are alanine, isoleucine and valine, respectively. The last two sequence differences cause minor packing rearrangements. The first sequence difference could contribute to the stability of LmTIM because the extra side chain atom of Ser96 makes a good (buried) hydrogen bond to O(Ala64) and is not involved in short contacts with neighbouring atoms. As can be seen in Figure 7
, the residues making up the hydrophobic core are mostly unchanged and there are few differences at the dimer interface. In fact, most of the differences are located away from the active site and are solvent exposed (Figure 7
).
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Despite the large gain in thermal stability, the values of the kinetic parameters of the E65Q variant of LmTIM and also of the eightfold mutant of TLPste and the sixfold mutant of barnase have not changed. This is in contradiction to what has been observed for `wild-type' superstable enzymes which have lower catalytic rates at mesophilic temperatures (Wrba et al., 1990; Jaenicke et al, 1996
). It has been suggested that these lower catalytic rates could be due to superstable enzymes being more rigid at mesophilic temperatures (Wrba et al., 1990
; Jaenicke et al, 1996
). Apparently, evolutionary pressure to optimize catalytic performance at thermophilic temperature results in a loss of catalytic activity at mesophilic temperature. However, the point mutation studies of LmTIM, TLP-ste and barnase show that mesophilic enzymes can be converted into superstable enzymes without affecting the catalytic properties at mesophilic temperatures. It is therefore predicted that rational design of superstability, without losing catalytic power, can be achieved for many enzymes after a careful analysis of their structures. Such an analysis can suggest generally applicable mutations (which stabilize the folded state with respect to the unfolded state) such as Gly to Ala, Xaa to Pro or the introduction of SS bridges (Van den Burg et al., 1998
) or mutations which stabilize helices (Villegas et al., 1995
; Mainfroid et al., 1996
) or loops (Zhou et al., 1996
; Blanco et al., 1998
) or fill up cavities (Lee and Vasmatzis, 1997
). Such an analysis can also locate specific destabilizing structural features of the wild-type structure which can be corrected by suitable mutagenesis, as is shown here for leishmania TIM.
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Acknowledgments |
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Notes |
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2 Present address: Max Planck Institut für Biophysik,Heinrich Hoffmann Str. 7, D-60528 Frankfurt, Germany
7 To whom correspondence should be addressed. E-mail: rik.wierenga{at}oulu.fi
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Received July 23, 1998; revised December 7, 1998; accepted December 11, 1998.