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Differential effects on enzyme stability and kinetic parameters of mutants related to human triosephosphate isomerase deficiency
BBA - General Subjects 1862 (2018) 1401–1409
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Differential effects on enzyme stability and kinetic parameters of mutants related to human triosephosphate isomerase deficiency
T
Nallely Cabreraa, Alfredo Torres-Lariosa, Itzhel García-Torresb, Sergio Enríquez-Floresb, ⁎ Ruy Perez-Montforta, a
Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Av. Universidad 3000, Coyoacán, 04510, Ciudad de México, Mexico b Laboratorio de Bioquímica-Genética, Instituto Nacional de Pediatría, Insurgentes Sur 3700-C, Col. Insurgentes Cuicuilco, Coyoacán, 04530, Ciudad de México, Mexico
A R T I C LE I N FO
A B S T R A C T
Keywords: Triosephosphate isomerase deficiency Site directed mutagenesis Recombinant enzyme Activity Stability Crystal structure
Human triosephosphate isomerase (TIM) deficiency is a very rare disease, but there are several mutations reported to be causing the illness. In this work, we produced nine recombinant human triosephosphate isomerases which have the mutations reported to produce TIM deficiency. These enzymes were characterized biophysically and biochemically to determine their kinetic and stability parameters, and also to substitute TIM activity in supporting the growth of an Escherichia coli strain lacking the tim gene. Our results allowed us to rate the deleteriousness of the human TIM mutants based on the type and severity of the alterations observed, to classify four “unknown severity mutants” with altered residues in positions 62, 72, 122 and 154 and to explain in structural terms the mutation V231M, the most affected mutant from the kinetic point of view and the only homozygous mutation reported besides E104D.
1. Introduction The fifth enzyme of the glycolytic pathway, triosephosphate isomerase (TIM, E.C. 5.3.1.1), catalyzes the reversible conversion between dihydroxyactetone 3 phosphate (DHAP) and glyceraldehyde 3 phosphate (GAP). After this enzymatic reaction, GAP can be further metabolized by subsequent enzymes in the pathway, continuing the breakdown of glucose, which produces two ATPs per molecule of the carbohydrate [1,2]. Almost all organisms have this enzyme, which, in most cases, is a homodimer with two identical subunits with a molecular mass of 27 kDa composed of approximately 250 amino acids. Both monomers have all catalytic residues but TIM is only active as a dimer. In some archaea TIM has an active and stable homotetrameric quaternary structure, but, in all cases, monomers of this enzyme are inactive and unstable, emphasizing that the enzyme acts as either a dimer or a tetramer [3]. Human triosephosphate isomerase (HsTIM) is formed by two subunits, which have 248 amino acids, that associate to form a 54 kDa homodimer. The enzyme with the wild type (WT) sequence of HsTIM has kinetic parameters that make it a nearly perfect enzyme, because the reaction is a diffusion-limited process. It also has a high thermal
resistance and its monomers interact with high affinity making it a very stable dimer [4,5]. The sequence of TIM has high homology for many species. A few key residues are completely conserved among all known sequences, and in some other positions have relatively little variation. In the sequence of HsTIM, very few mutations occur naturally that allow an individual to survive. The extremely small number of persons who have their HsTIM with an altered sequence suffer from HsTIM deficiency. This condition represents the most severe glycolytic enzyme defect in humans which is almost always lethal in early childhood. Although this disease, and the mutations in HsTIM that produce it, have been extensively reviewed [6–8], biochemical characterization using purified recombinant mutant enzymes has not been investigated. There have been several attempts to predict in silico the effect of mutations in HsTIM as they might reflect in the patients with TIM deficiency. Schneider [6] and Oliver and Timson [9] proposed that mutant proteins associated with pathological phenotypes are less stable than those associated with a less severe disease. Schneider [6], based on structural information, also predicted that catalytic abnormalities might be associated with the severity of the disease. Oliver and Timson [9] could not include kinetic parameters of enzyme function into their bioinformatics investigations. So, both these analyses lack the direct study of the
⁎ Corresponding author at: Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Circuito Exterior S/N, Delegación Coyoacán, 04510, Ciudad de México, Mexico. E-mail address: [email protected] (R. Perez-Montfort).
https://doi.org/10.1016/j.bbagen.2018.03.019 Received 8 February 2018; Received in revised form 14 March 2018; Accepted 19 March 2018 Available online 20 March 2018 0304-4165/ © 2018 Elsevier B.V. All rights reserved.
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function of the mutant HsTIMs having the mutated amino acids described in patients with HsTIM deficiency and the characterization of these enzymes in vitro. In this study, we offer such a comparison of several mutants related to HsTIM deficiency. We rate these mutants in terms of their deleterious effects according to kinetic, thermal and dimerization stability assays, and also bacterial growth inhibition. In addition, using X-ray crystallography, we analyze the molecular basis of the deficiency produced by mutant HsTIMV231M, the only other known homozygous mutant reported besides HsTIME104D.
Table 1 Enzymatic kinetic parameters for WT HsTIM and the mutant enzymes. The mean of three independent experiments is shown. For simplicity, a common color code is followed in all tables and figures to describe the behavior of the enzymes.
2. Materials and methods 2.1. Cloning and purification The DNA sequence X69723.1 (NCBI database) for HsTIM was used to make the single mutants. All mutants were constructed on a modified plasmid pET-3a [5] using the QuikChange protocol (Agilent Technologies, CA). The plasmids containing the different mutants were transformed into the Escherichia coli BL21 Codon Plus (DE3)-RIL strain (Agilent Technologies, CA). Cells were grown at 37 °C in Luria-Bertani medium containing ampicillin and chloramphenicol until an A600 of 0.8 was obtained. At that time, they were induced with 1 mM isopropyl-D-1thiogalactopyranoside following the conditions described in Supplementary Table 1, which were obtained after testing two periods of time for induction (3 and 18 h), and three different temperatures (18, 30 and 37 °C). The cell pellet from 1 L of culture was suspended in 20 mL of buffer A containing 50 mM sodium phosphate pH 8.0, 10 mM imidazole and different concentrations of NaCl (Supplementary Table 1). Cells were lysed by sonication and centrifuged at 20,000 ×g for 20 min. The supernatant was loaded on a 5 mL HisTrap (GE Healthcare) column. A 1 mL column was used, for the HsTIMA62D mutant. The proteins were eluted with a linear gradient of buffer A containing 500 mM imidazole and dialyzed for 2 h against a buffer containing 50 mM Tris pH 8.0, 0.5 mM EDTA and 1 mM dithiothreitol (DTT). The proteins were then cleaved using purified recombinant His tagged tobacco etch virus (TEV) protease expressed using vector pRK508 [10]. The protease was added in a proportion of 1:20 (w/w) and incubated at 30 °C for 18 h. Subsequently, the His-tagged TEV protease was removed using a HisTrap column equilibrated with buffer A. The enzymes were precipitated with ammonium sulfate at 75% saturation and maintained at 4 °C until their use.
Enzyme
Km (mM)
kcat (105 min-1)
kcat/ Km (106 M- 1 s-1)
kcat/ Km relative
WT C41Y A62D G72A E104D G122R V154M I170V V231M F240L
0.46 + 0.07 0.62 + 0.03 nd 3.0 + 0.36 0.57 + 0.08 0.57 + 0.08 0.49 + 0.04 0.03 + 0.004 4.02 + 0.99 0.83 + 0.06
2.73 2.16 nd 4.11 3.31 3.57 2.7 0.11 2.67 3.32
9.8 5.8 nd 2.3 9.6 10.4 9.0 5.8 1.1 6.6
1 0.59 nd 0.2 0.97 1.06 0.91 0.59 0.1 0.6
corresponding mutant enzymes (Table 1). To calculate the kinetic parameters, GAP concentration was varied between 0.05 and 3 mM. The data were adjusted to the Michaelis-Menten model and the values of Km and Vmax were calculated using non-linear regression. Activity was measured in a Cary 60 spectrophotometer (Agilent Technology, CA) with a multi-cell attachment. All assays were performed in triplicate.
2.4. Thermal shift assay (differential scanning fluorimetry) We followed the protocol described by Niesen and collaborators [12]. Briefly, the assay system had 0.2 μg of each mutant protein, 8 μl of 100 mM TEA pH 7.4,10 mM EDTA and a 1:100 dilution of SYPRO Orange dye (Invitrogen, CA), in a final volume of 10 μl. The dye was excited at 490 nm and the emitted light intensity was recorded at 575 nm. Data were collected at 1 °C intervals from 25 to 99 °C on a StepOnePlus real time PCR system using a 96-well reaction plate (Applied Biosystems 4346907, MA) and analyzed with the Protein Thermal Shift Software v1.3 from Applied Biosystems to define the thermal melting temperature (Tm). All assays were performed in triplicate.
2.5. Dimer stability All enzymes we analyzed were incubated at the following concentrations: 0.01, 0.05, 0.1, 0.5, 1, 2 and 5 μg of protein/mL for 2 h at 36 °C. At that time, the specific activities of the samples were determined. The amount of enzyme used to measure the specific activity was different for each mutant (Supplementary Table 1). All assays were performed in triplicate. The percentage of residual catalytic activity was plotted against the logarithm of protein concentration and the data were adjusted to a non-linear-fit-three-parameter equation, included in the GraphPad Prism 7.0 software.
2.2. Growth curves of E. coli Δtim- BL21-gold (DE3) cells complemented with WT HsTIM and different mutants We used a genetically manipulated strain BL21-Gold (DE3) without the tim gene (E. coli Δtim-BL21-Gold (DE3)) [Saab et al., unpublished data]. Cells were grown at 37 °C in M9 minimal medium supplemented with glucose (0.2%), casamino acids (0.006%) and ampicillin (100 μg/ mL). The assay was performed in triplicate in 96-well, clear, flatbottom, plates (Corning Costar 3697), in a total volume of 80 μl inoculated either with a single colony of WT bacteria, mutants or cells containing the plasmid without the gene, using a Synergy MX equipment (BioTek, VT). The growth was followed for 16 h and the results were monitored with the software of the equipment (Gen 1.11).
2.6. Crystallization and data collection of HsTIMV231M HsTIMV231M was crystallized via vapor diffusion using the sitting drop method. One microliter of a solution at 35 mg/ of protein mL was mixed with 1 μL of reservoir solution. Crystals were obtained after three weeks of incubation at 20 °C in condition A6 of the Crystal Screen HT kit from Hampton Research (200 mM MgCl2, 100 mM Tris pH 8.5, 30% polyethylene glycol 4000). The crystals were cryoprotected in a solution prepared with the mother liquor supplemented with 20% glycerol; they were immediately frozen in liquid nitrogen. Diffraction data were collected at 100 K using a wavelength of 0.9785 Å at the Life Sciences Collaborative Access Team (LS-CAT) 21-ID-G beamline at the Advanced Photon Source (Argonne National Laboratory, IL). The data were processed with iMOSFLM [13] and reduced with Aimless [14].
2.3. Activity assay Enzyme activity was measured at 25 °C following the conversion of glyceraldehyde 3 phosphate (GAP) to dihydroxyacetone phosphate using α-glycerolphosphate dehydrogenase (α-GDH) as a coupling enzyme, as described by [11]. NADH oxidation was monitored at 340 nm. The assay system (1 mL) had 100 mM triethanolamine (TEA) pH 7.4, 10 mM EDTA,1 mM GAP, 0.2 mM NADH and 20 μg/mL of α-GDH. The reaction was initiated by the addition of different quantities of the 1402
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2.7. Structure determination and refinement The structure was solved by the molecular replacement method with the program PHASER [15] using the coordinates of WT HsTIM, at a resolution of 1.7 Å (Protein Data Bank code 2JK2), as the search model. The existence of the V231 M mutation was initially confirmed by difference Fourier maps calculated using the structure of the wild type enzyme. Refinement was alternated with manual building/refinement in COOT [16], PHENIX [17] and the PDB_REDO server [18]. At the start, non-crystallographic symmetry restraints were used for the refinement, using one group that included the four monomers of the asymmetric unit. The restraints were released during the last cycles of refinement, before the addition of water molecules. The electron density of monomer A is very well defined. For monomers B and C, residues 170–177 (in loop 6 or the flexible loop) are less clear in the electron density map. These regions are normally poorly defined in all apo-TIMs. In addition, one of the monomers (D) is somewhat more disordered than the other three (residues 130–240). The regions that comprise the active sites were well defined for all monomers. Five percent of the data were used to validate the refinement. Data collection and refinement statistics are given in Supplementary Table 2. Figures were generated with PyMOL (the Pymol Molecular Graphics System, Version 1.7.4, Schrödinger, LCC). 3. Results 3.1. Cloning and purification All the mutant enzymes and WT HsTIM were cloned and expressed in Escherichia coli strain BL21-CodonPlus (DE3)-RIL. To optimize protein expression, the time and temperature of induction was fine tuned for each mutant, as well as the amount of NaCl used in the lysis buffer (Supplementary Table 1). The enzymes were expressed with a His-tag at the N-terminal position, which was cleaved with TEV protease, as part of the purification process. In general, the yield of protein was between 20 and 200 mg/L of culture. The purity and homogeneity of all the proteins (which was more than 95%) was analyzed by SDS-PAGE and size exclusion chromatography coupled to a multi-angle light scattering instrument, which showed a dimeric behavior for all the enzymes at a concentration of 2 mg/mL (data not shown). HsTIMA62D had a very low yield after purification (5 mg/L of culture) and had a very strong tendency to precipitate. This behavior was due to an inherent low stability of the enzyme (Fig. 1, and growth curves described below). Thus, most of the described assays could not be performed with this mutant, and will be shown as not determined (nd).
Fig. 2. The enzymatic kinetic constants are affected differently for each mutant. A. Km is increased for mutants G72A (6.5 fold) and V231 M (8.7 fold). It decreases 15-fold for mutant I170V. B. kcat is affected for mutant I170V (25 fold). C. The catalytic efficiency (kcat/Km) is only strongly affected for mutants G72A (4.2 fold) and V231 M (9 fold). The assays were performed in triplicate.
3.2. Growth of E. coli Δtim-BL21 gold (DE3) cells complemented with the different enzymes In order to test for major survival impairments due to the mutations of HsTIM, we monitored the growth of bacterial cells lacking their endogenous tim gene and transformed them with the plasmids overexpressing WT HsTIM or the mutant enzymes (Fig. 1). Notably, there is a clear difference in the growth rate of mutant A62D, when compared to all the other mutants and the WT enzyme. No growth at all is seen for HsTIMA62D, when the assay is performed at 42 °C (Supplementary Fig. 1). Fig. 1. Mutant A62D impairs the growth of a Δtim bacterial strain. Growth curves of Escherichia coli strain Δtim BL21-Gold (DE3) transformed with the overexpression plasmid pET-3a (Novagen) encoding WT HsTIM or the mutant enzymes or the empty plasmid. The logarithm of OD600 is plotted against time of incubation in M9 minimal medium supplemented with 0.006% casamino acids and 0.2% glucose at 37 °C. The assay was performed in triplicate on a Synergy MX microplate reader on 96-well plates. No growth is seen for the A62D mutants when the assay is performed at 42 °C (Supplementary Fig. 1).
3.3. Enzyme activity Steady state kinetics assays for all enzymes were determined in the direction of glyceraldehyde 3-phosphate to dyhydroxyacetone-phosphate (Table 1 and Fig. 2). All enzymes exhibited a Michaelis-Menten behavior. No activity was detected with HsTIMA62D. 1403
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3.3.1. Km Regarding this parameter, we found mainly two groups of mutant enzymes (depicted in Fig. 2A): 1) those that had a “WT-like” behavior, including HsTIMC41Y, HsTIME104D, HsTIMG122R, HsTIMV154M and HsTIMF240L and 2) severely affected enzymes, including HsTIMG72A and HsTIMV231M, which have a Km 6 and 9 times higher than the WT enzyme, respectively. Interestingly, the Km value of HsTIMI170V is 15 times lower than that of WT HsTIM.
assays (Fig. 5). This is the case for the most affected enzymes in these parameters: HsTIMG72A, HsTIME104D and HsTIMC41Y. There is another group of enzymes that are only slightly affected: HsTIMG122R, HsTIMV154M and HsTIMI170V. HsTIMV231M has a residual activity similar to WT HsTIM, but its Tm value is affected. Together with HsTIMF240L, both mutants represent the borderline between the “WTlike” group and the most affected mutants. 3.6. Overall comparison and rating of the deleteriousness of the mutant enzymes
3.3.2. kcat As shown in Fig. 2B, the only mutant that is strongly affected in this parameter is HsTIMI170V. Its kcat value is 25 times lower as compared to that of the WT enzyme. The rest of the mutants have a similar behavior to the WT enzyme, with a kcat that is 1.5 times higher than the WT enzyme for HsTIMG72A and 1.2 times lower for HsTIMC41Y.
In order to provide an overall and comparative view of this study, the strength of the effects seen in the different measured parameters described in Figs. 1-4, compared with the WT enzyme, is summarized in Table 2. According to these results, we propose a rating of the deleteriousness of the mutants, based on their effects on growth (g), kinetics (k, represented by Km, kcat or kcat/Km) or stability (s, represented by the thermal or dilution stability) (Fig. 6). The critical mutations are those whose three parameters (g,k,s) are affected. This is the case for HsTIMA62D, which was extremely difficult to purify and whose precipitation behavior precluded the measurement of any parameter. This mutant cannot support the growth of the Δtim strain of E. coli. HsTIMG72A is a severe mutant for which two parameters (k and s) are strongly affected. Other important mutations are those that show a strong effect in one parameter. This is the case for HsTIMC41Y, HsTIME104D (with an effect on s) and HsTIMV231M (with an effect on k). HsTIMI170V and HsTIMF240L have moderate effects, since their kinetic or stability parameters are only somewhat affected. Finally, HsTIMG122R and HsTIMV154M do not have any effect on these parameters of enzyme function.
3.3.3. Catalytic efficiency (kcat/Km) Fig. 2C shows that HsTIMG72A and HsTIMV231M are strongly affected in this global parameter, with a respective four- and nine-fold decrease when compared to the WT enzyme. The catalytic efficiency values of HsTIMC41Y, HsTIMI170V and HsTIMF240L decreases by half with respect to the WT enzyme. 3.4. Thermostability We used thermal shift assays to measure the thermal stability of the mutants (Fig. 3 and Supplementary Fig. 2). HsTIMG122R, HsTIMV154M and HsTIMI170V display a melting temperature (Tm) similar to the WT enzyme (60 to 63 °C). HsTIMV231M and HsTIMF240L have a Tm of around 53 °C. HsTIMC41Y, HsTIMG72A and HsTIME104D show a difference of more than 10 °C compared to the WT enzyme, with Tm values ranging between 49.7 and 51.5 °C.
3.7. Structural basis for the kinetic effects of mutation HsTIMV231M
3.5. Dimer stability
HsTIMV231M is the most affected mutant from the kinetic point of view, considering the catalytic efficiency parameter. In addition, this enzyme is, together with E104D, the only homozygous mutation reported to date. We therefore solved the crystal structure of this mutant in the apo conformation at 2.3 Å resolution (Fig. 7, Supplementary Table 2 and Supplementary Fig. 8). The inspection of the four molecules that constitute the asymmetric unit revealed the structural basis of TIM deficiency for this mutant (Fig. 7). Comparing it with the available crystal structures of the WT enzyme, both in the apo conformation (PDB ID: 2JK2) and in complex with the substrate 2-phosphoglycerate (2PG or PGA), allowed us to define the conformational changes that occur as a consequence of the V231 M mutation (Figs. 7A, B). The most important changes are found just around residue 231 (Fig. 7C), which is located very close to the active site (Figs. 7A, B). These changes are
Stability to dilution experiments measure the reduced activity of the TIM enzymes related to monomerization events [19–21]. We incubated the HsTIM mutants at different decreasing concentrations for 2 h and measured the remaining activity (Fig. 4 and Supplementary Fig. 3). At the lowest enzyme concentration assayed (1 ng/mL), we noticed that the residual activity showed an important gap for the enzymes that are still active (between 31 and 57%) and another group of enzymes: HsTIMC41Y, HsTIMG72A and HsTIME104D, which are strongly affected by the dilution effect and had between 2.6 and 7.7% activity of their original activity. Remarkably, we found a strong correlation for some of the mutants between thermostability data and the results of the dimer stability
Fig. 3. Mutants C41Y, G72A and E104D are strongly affected in thermal stability. The Thermal Shift Assay (TSA) was used to measure the thermal stability of the WT and the mutant enzymes. Each assay had 0.2 μg of protein in 100 mM TEA pH 7.4 and SYPRO Orange dye in a final volume of 10 μl. The experiment was performed using a 96-well plate in a real time PCR system. The assay was performed in triplicate. The thermal melting temperature was calculated using the peak of the first derivative plot shown in Supplementary Fig. 2.
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Fig. 4. Mutants C41Y, G72A and E104D are strongly affected in dimeric stability. The enzymes were incubated at different protein concentrations (see Supplementary Fig. 3) in 100 mM TEA pH 7.4 and 10 mM EDTA for 2 h at 36 °C. After that time, their residual catalytic activity was determined. This residual catalytic activity was plotted against the logarithm of protein concentration. The apparent dissociation constants (Kd app) were determined for the WT enzyme and each mutant. The values of each enzyme were adjusted using the Three-parameter Logistic equation of Prism 7.
Fig. 6. Deleteriousness rating of the HsTIM mutants. The rating is based upon the severity of the effects on kinetic parameters (k), stability (s) and growth (g), also shown in Table 2. Critical: implicates that all these parameters are affected. Severe: both k and s are affected. Important: either k or s are affected. Moderate: a mild effect on k or s.
Fig. 5. Correlation between thermal and dimeric stability of mutants C41Y, G72A, E104D and F240L. Double plot of the residual activity at 1 ng/ml (depicted in Fig. 4) and the Tm temperatures (depicted in Fig. 3). The order of the proteins in the x-axis follows the residual activity values, from highest to lowest. In general, a mild effect is seen for both parameters in mutants G122R, V154 M and I170V, and a strong effect in mutants G72A, E104D and C41Y. Mutants V231 M and F240 L represent a borderline between these two extremes.
substrate. This effect is correlated with the change in the kinetic parameter Km, described in this work.
represented by movements in the main chain around residues 212–219 and 231–234, which show a maximal displacement of 1.1 and 2.1 Å, respectively, when compared to both conformations of the WT enzyme (apo and 2PG, Fig. 7). In particular, the movement of residues 231–234 would approach the main chain of the protein to 2PGA by 0.5 Å (Fig. 7C), which is now too close, and hinders the binding of the
4. Discussion In this work, we performed an experimental analysis of most of the reported mutants related to human triosephosphate isomerase deficiency. Other attempts have been made to evaluate the range of effects on mutants of TIM [9] but this report defines more precisely the extent
Table 2 Summary of the effects seen in different mutants, compared to the wild-type enzyme. The table summarizes the results shown in Figs. 1-4.
Mutant
C41Y A62D G72A E104D G122R V154M I170V V231M F240L
Kinetic Km
kcat k kcat/Km X ND XX
ND XX
XX XX
XX
X XX X
Stability Tm
Dilution
Growth
XX ND XX XX
XX ND XX XX
X X
X
XX = strong, X = moderate, blank = no effect, ND = not determined.
1405
XX
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of the active site; the remaining seven mutations, which do have an effect, are located on the same side as the active site of the enzyme (Supplementary Fig. 4). In the following sections of the discussion, we will enlist, following the numbering in the sequence of HsTIM, the effects reported in the disease and the main findings observed in this study with corresponding recombinant mutant protein. In addition, we summarize the molecular contexts and the effects seen with each mutant (Supplementary Figs. 4 to 9) in order to help in the understanding of the molecular basis of TIM deficiency and to aid in the prediction of the severity of the mutations in a heterozygous situation. 4.1. C41Y 4.1.1. Occurrence Three cases have been reported in compound heterozygotes together with the E104D mutation [25,27]. 4.1.2. Severity in patients Chronic axonal neuropathy. The damage became evident at the age of 2 years [27]. 4.1.3. Previous experimental characterization In a yeast model with the deleted endogenous gene tim, this mutant showed the same activity as the WT enzyme [28]. In addition, the dimerization properties of this mutant were monitored using a yeast twohybrid system. It had an altered dimerization behavior, with increased interaction activity. 4.1.4. Findings in this work Thermal and dimer stability strongly affected (13.5 °C difference in the value of Tm and residual activity at 1 ng/mL, 32% lower than that of the WT enzyme). 4.1.5. Molecular context and predictions Residue 41 is in the vicinity of positions 62, 231 and 240 (Supplementary Fig. 7), which have critical, strong and moderate effects on the enzyme, respectively. A non-conservative change in this residue might have a strong effect on the stability of the enzyme, as was indeed seen for mutation C41Y. In combination with the E104D mutation, we predict that the heterodimer would have completely disruptive effects on the stability of the enzyme. The patient might have limited survival in the presence of the homodimers having each mutation.
Fig. 7. Structural basis of human TIM deficiency produced by mutation V231M. Mutation V231 M perturbs the active site region. A. Cα RMSD plot calculated using apo WT HsTIM (PDB ID: 2JK2) and the model of the crystal structure of mutant V231 M, solved at 2.3 Å resolution (PDB ID: 6C2G). The overall RMSD is 0.311 and 0.562 Å for the superpositions of the mutant V231 M with WT HsTIM apo and complexed with 2PG, respectively. The regions that are displaced are indicated by the residue numbers or structural feature that differ the most. Loop 6 (residues 169–173) is usually flexible in all TIMs. B. Superposition of the model of the crystal structure of mutant V231 M with WT HsTIM complexed with 2PGA, showing the regions of conformational movements depicted in panel A. The regions correspond to residues 134–141, loop 6, 212–219 and 231–234. The substrate 2PGA is shown as spheres. C. Perturbations at the active site. Regions 212–219 and 231–234 are located very close to the active site. The movement of these residues, with its peak at residue 233, which is displaced by 2.1 Å with respect to the WT structure. This correlates with the presence of the mutation and hinders the binding of the substrate.
4.2. A62D 4.2.1. Occurrence One case in a compound heterozygote, with E104D as the second mutation [22]. 4.2.2. Severity in patients Unknown. 4.2.3. Previous experimental characterization None.
and cause of the enzymatic deficiency at the biochemical level, in an all versus all comparative scenario. Although most of the mutants are present in humans as heterozygous proteins, as a first approach we studied single mutants in order to investigate their individual contribution to the kinetics and stability of human TIM. We were also able to classify the “unknown severity” mutants HsTIMA62D, HsTIMG72A, HsTIMG122R and HsTIMV154M [22–24]. Interestingly, HsTIMG122R and HsTIMV154M do not seem to have any major biochemical alteration, and only show a very modest effect on thermal stability. Residues 122 and 154 are located on the opposite side
4.2.4. Findings in this work The mutation impairs growth when expressed in bacterial strain E. coli Δtim. 4.2.5. Molecular context and predictions Residue 62 is located in a hydrophobic patch formed by residues V40, A42, A62 and V92 (Supplementary Fig. 5). It is possible that a conservative mutation in this position could be tolerated well, but a mutation that involves a charged residue, like A62D, completely 1406
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(Supplementary Fig. 7). A patient has limited survival having HsTIME104D homodimers.
disrupts this hydrophobic region, which in addition is close to residue 41, and compromises the survival of the organism. In combination with the E104D mutation, we predict that the heterodimer would have totally disruptive effects on the stability of the enzyme. The patient might have limited survival in the presence of only one mutant type of homodimer having mutation E104D.
4.5. G122R 4.5.1. Occurrence One individual [29].
4.3. G72A 4.5.2. Severity in patients No patients. The study was performed with a population of healthy heterozygotes.
4.3.1. Occurrence Found in a phenotypic heterozygote, for which TIM activity was reduced, together with the WT enzyme [24].
4.5.3. Previous experimental characterization Reported as a thermolabile enzyme [23]. In a yeast model, with a deleted endogenous gene for TIM, this mutant had the same activity compared to the WT enzyme [28]. In addition, the dimerization properties of this mutant were monitored with a yeast two-hybrid system, showing no effect.
4.3.2. Severity in patients No patients. Study in a population of healthy heterozygotes. 4.3.3. Previous experimental characterization None.
4.5.4. Findings in this work The enzyme is not affected. Interestingly, the residual activity at 1 ng/mL is 21.6% higher with respect to the WT enzyme.
4.3.4. Findings in this work Effects upon both kinetic (fourfold decrease in the catalytic efficiency) and stability parameters (11.7 °C difference in the Tm value and the residual activity at 1 ng/mL, 28% lower compared with the WT enzyme).
4.5.5. Molecular context and predictions Residue 122 is located on the opposite side of the active site, at the end of a loop and facing the solvent (Supplementary Fig. 9). Any change for a hydrophilic residue would have no effect on the enzyme, as is shown by non-conservative mutation G122R. Patients should show no symptoms or disease.
4.3.5. Molecular context and predictions Residue 72 is located in loop 3, which is very close to the active site and also forms part of the dimer interface. (Supplementary Fig. 6). Any change in this position would be severe for both the kinetic and stability parameters, as is shown by mutation G72A, which would now be too close to N15 of the dimeric partner (Supplementary Fig. 6). However, since both residues in positions 72 in both monomers are far apart from each other in the dimeric enzyme, if a dimer is formed in combination with one WT monomer, we predict that the heterodimer will have enough stability. The patient might survive in the presence of all types of homo and heterodimers involving this mutation.
4.6. V154M 4.6.1. Occurrence Found in a phenotypic heterozygote, for which TIM activity was reduced, together with the WT enzyme [24]. 4.6.2. Severity in patients No patients. The study was performed in a population of healthy heterozygotes.
4.4. E104D 4.4.1. Occurrence At least 15 cases from multiple unrelated families, although most of the patients are thought to have a common ancestor. HsTIME104D together with HsTIMV231M, are the only homozygote mutants reported to date.
4.6.3. Previous experimental characterization None. 4.6.4. Findings in this work The enzyme is not affected.
4.4.2. Severity in patients Hemolytic anemia, neuromuscular impairment and increased susceptibility to infection. No patient has survived beyond the age of six.
4.6.5. Molecular context and predictions Residue 154 is located on the opposite side of the active site, in a hydrophobic patch formed by residues L177, V154, W157 and V160 (Supplementary Fig. 9). Most changes for a hydrophobic residue would have no effect on the enzyme, as is shown by conservative mutation V231M. Patients should not show any symptom or disease.
4.4.3. Previous experimental characterization The molecular basis of the deficiency has been reported elsewhere [5,28] and is caused by alterations in dimer stability, rather than on the effects in catalytic activity.
4.7. I170V 4.4.4. Findings in this work Thermal and dimeric stability are strongly affected (13.5 °C difference in Tm and the residual activity at 1 ng/mL is 32% lower compared to the WT enzyme).
4.7.1. Occurrence Found in one case in a compound heterozygote together with the E104D mutation [25] [26].
4.4.5. Molecular context and predictions Residue 104 is located very close to the interface and forms a network of interactions with other highly conserved residues (Supplementary Fig. 7). Any greater change in this position would compromise the stability of the enzyme, as is implied by the highly conservative mutation E104D. This is worsened by the fact that the counterpart of this region in the other monomer is in close proximity.
4.7.2. Severity in patients One patient with hemolytic anemia, but no muscular impairment [26]. A model in Drosophila has shown behavioral abnormalities [30]. 4.7.3. Previous experimental characterization This mutant had very decreased activity in the patient (10% or less) when compared to the wild type enzyme, and decreased activity 1407
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4.9.4. Findings in this work The mutation has moderate effects upon the kinetics (less than a twofold decrease in the catalytic efficiency) and stability parameters (10 °C difference in the Tm value, but a relatively high residual activity value of 22.6% compared to 35.6% of the WT enzyme).
(around 30%) in a yeast model with a deleted endogenous TIM gene. In addition, the dimerization properties of this mutant were examined using a two-hybrid system in yeast, with a slight enhancement of the stability [28]. The mutation lowered Km and kcat values 20 and 30fold, respectively, and increased enzyme stability (Tm = 59.4 °C, compared to 46.5 °C of the WT enzyme) [30].
4.9.5. Molecular context and predictions Residue 240 is close to positions 41 and 231 (Supplementary Fig. 8). A conservative change in this residue might have an effect on the stability and perhaps the catalytic performance of the enzyme because of its vicinity with V231. The effect is indeed moderate for mutation F240L and perhaps it would be more severe for mutation F240S in combination with mutation E104D. We predict that the heterodimer would be functional, but severely affected in its stability. The patient might have limited survival in the presence of the heterodimers, with little contribution to the deficiency because of the F240S homodimers. The combination of F240L with E145Stop is probably not functional as a heterodimer, but the F240L homodimer should be functional. Overall, it is clear that the perturbation of the stability of the quaternary structure may define some TIM deficiencies (mutations C41Y, E104D) as well as disturbances mainly in kinetic parameters (mutations I170V, V231M), effects in kinetics and stability (mutation G72A) or completely aberrant enzymes (mutation A62D) for others. Notably, this study is in general agreement with experimental studies dealing with the in vitro characterization of one or several mutants [28]; [5]; [30]. Further studies will be needed to define the molecular effects of some mutations, notably C41Y, as well as the effects seen in heterodimeric constructions, which can be expected from the compound heterozygous mutations found in most cases of this disease.
4.7.4. Findings in this work Enzyme kinetics are moderately affected. The Km and the kcat values decrease 15 and 25 times, respectively. However, the overall efficiency of the enzyme is not strongly affected (less than twofold). The stability of the enzyme is unchanged. 4.7.5. Molecular context and predictions Residue 170 is very close to the active site, in loop 6, which is important for catalysis (Supplementary Fig. 8). A non-conservative change in this residue might have a strong effect on the catalysis of the enzyme. The effect is indeed moderate for mutation I170V, because the enzyme also shows an increased substrate affinity. The structural basis of the deficiency caused by this mutant has been analyzed in [30]. In combination with the E104D mutation, we predict that the heterodimer should be functional, but with severe effects on its kinetic parameters and stability. Yet the patient with the hetero dimers does survive at least until late childhood and, patients having homo dimers would probably not be severely affected. 4.8. V231M 4.8.1. Occurrence Homozygous mutation linked to clinical deficiency in two cases [31] [32]
Data accessibility The atomic coordinates and structure factors of mutant V231M of human triosephosphate isomerase have been deposited in the Protein Data Bank (PDB) with the accession code 6C2G.
4.8.2. Severity in patients Hemolytic anemia and neurological dysfunction.
Conflict of interests
4.8.3. Previous experimental characterization None.
The authors declare they have no conflict of interest. 4.8.4. Findings in this work Enzyme kinetics are strongly affected. The Km increases nine-fold and the catalytic efficiency decreases accordingly. The crystal structure of the mutant shows that the active site is perturbed and hinders the binding of the substrate (Fig. 7).
Authors' contributions NC, ATL and RPM conceived and designed the experiments; NC, ATL, IGT and SEF performed the experiments; NC, ATL, IGT, SEF and RPM analyzed the data; NC, ATL and RPM wrote the manuscript. All authors gave final approval for publication.
4.8.5. Molecular context and predictions Patients might have limited survival (and see above).
Transparency document
4.9. F240L
The Transparency document associated this article can be found, in the online version.
4.9.1. Occurrence Found in two brothers who are compound heterozygotes (E145Stop) [33,34]; reviewed in [8]. A mutation in the same position (F240S), is found in a compound heterozygote together with mutation E104D [35].
Acknowledgements We thank Gloria Saab-Rincón, PhD and Leticia Olvera-Rodríguez, BSc (Instituto de Biotecnología-UNAM, México) for providing the E. coli Δtim-BL21-Gold (DE3) strain. We also thank Gabriel Del Río, PhD, who kindly allowed the use of the Synergy MX equipment, María-Teresa Lara-Ortiz, Jessica Díaz-Salazar, Beatriz Aguirre, Manuel Ortinez Benavides, Aurey Galván Lobato and Unidad de Biología Molecular at Instituto de Fisiología Celular-UNAM (Dr. Laura Ongay and Guadalupe Codiz) for technical support. Use of the Advanced Photon Source, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02-06CH11357. Use of the LS-CAT
4.9.2. Severity in patients Neurological symptoms for only one of the brothers with the F240 L mutation and also for the patient with the F240S mutation. 4.9.3. Previous experimental characterization Decreased catalytic activity [36]. In a yeast model with a deleted TIM endogenous gene, this mutant showed the same activity as compared to the wild type enzyme [28]. In addition, the dimerization properties of this mutant were measured using a yeast two-hybrid system, and no effect was detected. 1408
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Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor, Grant 085P1000817. This work was supported by grant 254694 from Consejo Nacional de Ciencia y Tecnología, México, to RPM.
[18] R.P. Joosten, F. Long, G.N. Murshudov, A. Perrakis, The PDB_REDO server for macromolecular structure model optimization, IUCrJ. 1 (2014) 213–220. [19] T.V. Borchert, R. Abagyan, K.V. Kishan, J.P. Zeelen, R.K. Wierenga, The crystal structure of an engineered monomeric triosephosphate isomerase, monoTIM: the correct modelling of an eight-residue loop, Structure 1 (1993) 205–213. [20] T.V. Borchert, J.P. Zeelen, W. Schliebs, M. Callens, W. Minke, R. Jaenicke, R.K. Wierenga, An interface point-mutation variant of triosephosphate isomerase is compactly folded and monomeric at low protein concentrations, FEBS Lett. 367 (1995) 315–318. [21] V. Mainfroid, S.C. Mande, W.G. Hol, J.A. Martial, K. Goraj, Stabilization of human triosephosphate isomerase by improvement of the stability of individual alpha-helices in dimeric as well as monomeric forms of the protein, Biochemistry 35 (1996) 4110–4117. [22] L. Manco, M.L. Ribeiro, Novel human pathological mutations. Gene symbol: TPI1. Disease: triosephosphate isomerase deficiency, Hum. Genet. 121 (2007) 650. [23] B.A. Perry, H.W. Mohrenweiser, Human triosephosphate isomerase: substitution of Arg for Gly at position 122 in a thermolabile electromorph variant, TPI-Manchester, Hum. Genet. 88 (1992) 634–638. [24] M. Watanabe, B.C. Zingg, H.W. Mohrenweiser, Molecular analysis of a series of alleles in humans with reduced activity at the triosephosphate isomerase locus, Am. J. Hum. Genet. 58 (1996) 308–316. [25] R. Arya, M.R. Lalloz, A.J. Bellingham, D.M. Layton, Evidence for founder effect of the Glu104Asp substitution and identification of new mutations in triosephosphate isomerase deficiency, Hum. Mutat. 10 (1997) 290–294. [26] A. Ationu, A. Humphries, M.R.A. Lalloz, R. Arya, B. Wild, J. Warrilow, J. Morgan, A.J. Bellingham, D.M. Layton, Reversal of metabolic block in glycolysis by enzyme replacement in triosephosphate isomerase-deficient cells, Blood 94 (1999) 3193–3198. [27] J.M. Wilmshurst, G.A. Wise, J.D. Pollard, R.A. Ouvrier, Chronic axonal neuropathy with triosephosphate isomerase deficiency, Pediatr. Neurol. 30 (2004) 146–148. [28] M. Ralser, G. Heeren, M. Breitenbach, H. Lehrach, S. Krobitsch, Triose phosphate isomerase deficiency is caused by altered dimerization–not catalytic inactivity–of the mutant enzymes, PLoS One 1 (2006) e30. [29] J. Asakawa, H.W. Mohrenweiser, Characterization of two new electrophoretic variants of human triosephosphate isomerase: stability, kinetic, and immunological properties, Biochem. Genet. 20 (1982) 59–76. [30] B.P. Roland, C.G. Amrich, C.J. Kammerer, K.A. Stuchul, S.B. Larsen, S. Rode, A.A. Aslam, A. Heroux, R. Wetzel, A.P. VanDemark, M.J. Palladino, Triosephosphate isomerase I170V alters catalytic site, enhances stability and induces pathology in a Drosophila model of TPI deficiency, Biochim. Biophys. Acta 1852 (2015) 61–69. [31] B.A. Neubauer, A. Pekrun, S.W. Eber, M. Lakomek, W. Schröter, Relation between genetic defect, altered protein structure, and enzyme function in triose-phosphate isomerase (TPI) deficiency, Eur. J. Pediatr. 151 (1992) 232. [32] G. Serdaroglu, Y. Aydinok, S. Yilmaz, L. Manco, E. Ozer, Triosephosphate isomerase deficiency: a patient with Val231Met mutation, Pediatr. Neurol. 44 (2011) 139–142. [33] M.L. Chang, P.J. Artymiuk, X. Wu, S. Hollan, A. Lammi, L.E. Maquat, Human triosephosphate isomerase deficiency resulting from mutation of Phe-240, Am. J. Hum. Genet. 52 (1993) 1260–1269. [34] S. Hollan, H. Fujii, A. Hirono, K. Hirono, H. Karro, S. Miwa, V. Harsanyi, E. Gyodi, M. Inselt-Kovacs, Hereditary triosephosphate isomerase (TPI) deficiency: two severely affected brothers one with and one without neurological symptoms, Hum. Genet. 92 (1993) 486–490. [35] E. Fermo, P. Bianchi, C. Vercellati, D.C. Rees, A.P. Marcello, W. Barcellini, A. Zanella, Triose phosphate isomerase deficiency associated with two novel mutations in TPI gene, Eur. J. Haematol. 85 (2010) 170–173. [36] F. Orosz, J. Olah, M. Alvarez, G.M. Keseru, B. Szabo, G. Wagner, Z. Kovari, M. Horanyi, K. Baroti, J.A. Martial, S. Hollan, J. Ovadi, Distinct behavior of mutant triosephosphate isomerase in hemolysate and in isolated form: molecular basis of enzyme deficiency, Blood 98 (2001) 3106–3112.
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bbagen.2018.03.019. References [1] S.V. Rieder, I.A. Rose, The mechanism of the triosephosphate isomerase reaction, J. Biol. Chem. 234 (1959) 1007–1010. [2] J.R. Knowles, Enzyme catalysis: not different, just better, Nature 350 (1991) 121–124. [3] A.R. Katebi, R.L. Jernigan, The critical role of the loops of triosephosphate isomerase for its oligomerization, dynamics, and functionality, Protein Sci. 23 (2014) 213–228. [4] S.C. Mande, V. Mainfroid, K.H. Kalk, K. Goraj, J.A. Martial, W.G. Hol, Crystal structure of recombinant human triosephosphate isomerase at 2.8 A resolution. Triosephosphate isomerase-related human genetic disorders and comparison with the trypanosomal enzyme, Protein Sci. 3 (1994) 810–821. [5] C. Rodriguez-Almazan, R. Arreola, D. Rodriguez-Larrea, B. Aguirre-Lopez, M.T. de Gomez-Puyou, R. Perez-Montfort, M. Costas, A. Gomez-Puyou, A. Torres-Larios, Structural basis of human triosephosphate isomerase deficiency: mutation E104D is related to alterations of a conserved water network at the dimer interface, J. Biol. Chem. 283 (2008) 23254–23263. [6] A.S. Schneider, Triosephosphate isomerase deficiency: historical perspectives and molecular aspects, Baillieres Best Pract. Res. Clin. Haematol. 13 (2000) 119–140. [7] F. Orosz, J. Olah, J. Ovadi, Triosephosphate isomerase deficiency: facts and doubts, IUBMB Life 58 (2006) 703–715. [8] F. Orosz, J. Olah, J. Ovadi, Triosephosphate isomerase deficiency: new insights into an enigmatic disease, Biochim. Biophys. Acta 1792 (2009) 1168–1174. [9] C. Oliver, D.J. Timson, In silico prediction of the effects of mutations in the human triose phosphate isomerase gene: towards a predictive framework for TPI deficiency, Eur. J. Med. Genet. 60 (2017) 289–298. [10] R.B. Kapust, D.S. Waugh, Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused, Protein Sci. 8 (1999) 1668–1674. [11] B. Plaut, J.R. Knowles, pH-dependence of the triose phosphate isomerase reaction, Biochem. J. 129 (1972) 311–320. [12] F.H. Niesen, H. Berglund, M. Vedadi, The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability, Nat. Protoc. 2 (2007) 2212–2221. [13] T.G. Battye, L. Kontogiannis, O. Johnson, H.R. Powell, A.G. Leslie, iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM, Acta Crystallogr. D Biol. Crystallogr. 67 (2011) 271–281. [14] P.R. Evans, An introduction to data reduction: space-group determination, scaling and intensity statistics, Acta Crystallogr. D Biol. Crystallogr. 67 (2011) 282–292. [15] A.J. McCoy, Solving structures of protein complexes by molecular replacement with Phaser, Acta Crystallogr. D Biol. Crystallogr. 63 (2007) 32–41. [16] P. Emsley, K. Cowtan, Coot: model-building tools for molecular graphics, Acta Crystallogr. D Biol. Crystallogr. 60 (2004) 2126–2132. [17] P.D. Adams, P.V. Afonine, G. Bunkoczi, V.B. Chen, I.W. Davis, N. Echols, J.J. Headd, L.W. Hung, G.J. Kapral, R.W. Grosse-Kunstleve, A.J. McCoy, N.W. Moriarty, R. Oeffner, R.J. Read, D.C. Richardson, J.S. Richardson, T.C. Terwilliger, P.H. Zwart, PHENIX: a comprehensive python-based system for macromolecular structure solution, Acta Crystallogr. D Biol. Crystallogr. 66 (2010) 213–221.
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Differential effects on enzyme stability and kinetic parameters of mutants related to human triosephosphate isomerase deficiency
T
Nallely Cabreraa, Alfredo Torres-Lariosa, Itzhel García-Torresb, Sergio Enríquez-Floresb, ⁎ Ruy Perez-Montforta, a
Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Av. Universidad 3000, Coyoacán, 04510, Ciudad de México, Mexico b Laboratorio de Bioquímica-Genética, Instituto Nacional de Pediatría, Insurgentes Sur 3700-C, Col. Insurgentes Cuicuilco, Coyoacán, 04530, Ciudad de México, Mexico
A R T I C LE I N FO
A B S T R A C T
Keywords: Triosephosphate isomerase deficiency Site directed mutagenesis Recombinant enzyme Activity Stability Crystal structure
Human triosephosphate isomerase (TIM) deficiency is a very rare disease, but there are several mutations reported to be causing the illness. In this work, we produced nine recombinant human triosephosphate isomerases which have the mutations reported to produce TIM deficiency. These enzymes were characterized biophysically and biochemically to determine their kinetic and stability parameters, and also to substitute TIM activity in supporting the growth of an Escherichia coli strain lacking the tim gene. Our results allowed us to rate the deleteriousness of the human TIM mutants based on the type and severity of the alterations observed, to classify four “unknown severity mutants” with altered residues in positions 62, 72, 122 and 154 and to explain in structural terms the mutation V231M, the most affected mutant from the kinetic point of view and the only homozygous mutation reported besides E104D.
1. Introduction The fifth enzyme of the glycolytic pathway, triosephosphate isomerase (TIM, E.C. 5.3.1.1), catalyzes the reversible conversion between dihydroxyactetone 3 phosphate (DHAP) and glyceraldehyde 3 phosphate (GAP). After this enzymatic reaction, GAP can be further metabolized by subsequent enzymes in the pathway, continuing the breakdown of glucose, which produces two ATPs per molecule of the carbohydrate [1,2]. Almost all organisms have this enzyme, which, in most cases, is a homodimer with two identical subunits with a molecular mass of 27 kDa composed of approximately 250 amino acids. Both monomers have all catalytic residues but TIM is only active as a dimer. In some archaea TIM has an active and stable homotetrameric quaternary structure, but, in all cases, monomers of this enzyme are inactive and unstable, emphasizing that the enzyme acts as either a dimer or a tetramer [3]. Human triosephosphate isomerase (HsTIM) is formed by two subunits, which have 248 amino acids, that associate to form a 54 kDa homodimer. The enzyme with the wild type (WT) sequence of HsTIM has kinetic parameters that make it a nearly perfect enzyme, because the reaction is a diffusion-limited process. It also has a high thermal
resistance and its monomers interact with high affinity making it a very stable dimer [4,5]. The sequence of TIM has high homology for many species. A few key residues are completely conserved among all known sequences, and in some other positions have relatively little variation. In the sequence of HsTIM, very few mutations occur naturally that allow an individual to survive. The extremely small number of persons who have their HsTIM with an altered sequence suffer from HsTIM deficiency. This condition represents the most severe glycolytic enzyme defect in humans which is almost always lethal in early childhood. Although this disease, and the mutations in HsTIM that produce it, have been extensively reviewed [6–8], biochemical characterization using purified recombinant mutant enzymes has not been investigated. There have been several attempts to predict in silico the effect of mutations in HsTIM as they might reflect in the patients with TIM deficiency. Schneider [6] and Oliver and Timson [9] proposed that mutant proteins associated with pathological phenotypes are less stable than those associated with a less severe disease. Schneider [6], based on structural information, also predicted that catalytic abnormalities might be associated with the severity of the disease. Oliver and Timson [9] could not include kinetic parameters of enzyme function into their bioinformatics investigations. So, both these analyses lack the direct study of the
⁎ Corresponding author at: Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Circuito Exterior S/N, Delegación Coyoacán, 04510, Ciudad de México, Mexico. E-mail address: [email protected] (R. Perez-Montfort).
https://doi.org/10.1016/j.bbagen.2018.03.019 Received 8 February 2018; Received in revised form 14 March 2018; Accepted 19 March 2018 Available online 20 March 2018 0304-4165/ © 2018 Elsevier B.V. All rights reserved.
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function of the mutant HsTIMs having the mutated amino acids described in patients with HsTIM deficiency and the characterization of these enzymes in vitro. In this study, we offer such a comparison of several mutants related to HsTIM deficiency. We rate these mutants in terms of their deleterious effects according to kinetic, thermal and dimerization stability assays, and also bacterial growth inhibition. In addition, using X-ray crystallography, we analyze the molecular basis of the deficiency produced by mutant HsTIMV231M, the only other known homozygous mutant reported besides HsTIME104D.
Table 1 Enzymatic kinetic parameters for WT HsTIM and the mutant enzymes. The mean of three independent experiments is shown. For simplicity, a common color code is followed in all tables and figures to describe the behavior of the enzymes.
2. Materials and methods 2.1. Cloning and purification The DNA sequence X69723.1 (NCBI database) for HsTIM was used to make the single mutants. All mutants were constructed on a modified plasmid pET-3a [5] using the QuikChange protocol (Agilent Technologies, CA). The plasmids containing the different mutants were transformed into the Escherichia coli BL21 Codon Plus (DE3)-RIL strain (Agilent Technologies, CA). Cells were grown at 37 °C in Luria-Bertani medium containing ampicillin and chloramphenicol until an A600 of 0.8 was obtained. At that time, they were induced with 1 mM isopropyl-D-1thiogalactopyranoside following the conditions described in Supplementary Table 1, which were obtained after testing two periods of time for induction (3 and 18 h), and three different temperatures (18, 30 and 37 °C). The cell pellet from 1 L of culture was suspended in 20 mL of buffer A containing 50 mM sodium phosphate pH 8.0, 10 mM imidazole and different concentrations of NaCl (Supplementary Table 1). Cells were lysed by sonication and centrifuged at 20,000 ×g for 20 min. The supernatant was loaded on a 5 mL HisTrap (GE Healthcare) column. A 1 mL column was used, for the HsTIMA62D mutant. The proteins were eluted with a linear gradient of buffer A containing 500 mM imidazole and dialyzed for 2 h against a buffer containing 50 mM Tris pH 8.0, 0.5 mM EDTA and 1 mM dithiothreitol (DTT). The proteins were then cleaved using purified recombinant His tagged tobacco etch virus (TEV) protease expressed using vector pRK508 [10]. The protease was added in a proportion of 1:20 (w/w) and incubated at 30 °C for 18 h. Subsequently, the His-tagged TEV protease was removed using a HisTrap column equilibrated with buffer A. The enzymes were precipitated with ammonium sulfate at 75% saturation and maintained at 4 °C until their use.
Enzyme
Km (mM)
kcat (105 min-1)
kcat/ Km (106 M- 1 s-1)
kcat/ Km relative
WT C41Y A62D G72A E104D G122R V154M I170V V231M F240L
0.46 + 0.07 0.62 + 0.03 nd 3.0 + 0.36 0.57 + 0.08 0.57 + 0.08 0.49 + 0.04 0.03 + 0.004 4.02 + 0.99 0.83 + 0.06
2.73 2.16 nd 4.11 3.31 3.57 2.7 0.11 2.67 3.32
9.8 5.8 nd 2.3 9.6 10.4 9.0 5.8 1.1 6.6
1 0.59 nd 0.2 0.97 1.06 0.91 0.59 0.1 0.6
corresponding mutant enzymes (Table 1). To calculate the kinetic parameters, GAP concentration was varied between 0.05 and 3 mM. The data were adjusted to the Michaelis-Menten model and the values of Km and Vmax were calculated using non-linear regression. Activity was measured in a Cary 60 spectrophotometer (Agilent Technology, CA) with a multi-cell attachment. All assays were performed in triplicate.
2.4. Thermal shift assay (differential scanning fluorimetry) We followed the protocol described by Niesen and collaborators [12]. Briefly, the assay system had 0.2 μg of each mutant protein, 8 μl of 100 mM TEA pH 7.4,10 mM EDTA and a 1:100 dilution of SYPRO Orange dye (Invitrogen, CA), in a final volume of 10 μl. The dye was excited at 490 nm and the emitted light intensity was recorded at 575 nm. Data were collected at 1 °C intervals from 25 to 99 °C on a StepOnePlus real time PCR system using a 96-well reaction plate (Applied Biosystems 4346907, MA) and analyzed with the Protein Thermal Shift Software v1.3 from Applied Biosystems to define the thermal melting temperature (Tm). All assays were performed in triplicate.
2.5. Dimer stability All enzymes we analyzed were incubated at the following concentrations: 0.01, 0.05, 0.1, 0.5, 1, 2 and 5 μg of protein/mL for 2 h at 36 °C. At that time, the specific activities of the samples were determined. The amount of enzyme used to measure the specific activity was different for each mutant (Supplementary Table 1). All assays were performed in triplicate. The percentage of residual catalytic activity was plotted against the logarithm of protein concentration and the data were adjusted to a non-linear-fit-three-parameter equation, included in the GraphPad Prism 7.0 software.
2.2. Growth curves of E. coli Δtim- BL21-gold (DE3) cells complemented with WT HsTIM and different mutants We used a genetically manipulated strain BL21-Gold (DE3) without the tim gene (E. coli Δtim-BL21-Gold (DE3)) [Saab et al., unpublished data]. Cells were grown at 37 °C in M9 minimal medium supplemented with glucose (0.2%), casamino acids (0.006%) and ampicillin (100 μg/ mL). The assay was performed in triplicate in 96-well, clear, flatbottom, plates (Corning Costar 3697), in a total volume of 80 μl inoculated either with a single colony of WT bacteria, mutants or cells containing the plasmid without the gene, using a Synergy MX equipment (BioTek, VT). The growth was followed for 16 h and the results were monitored with the software of the equipment (Gen 1.11).
2.6. Crystallization and data collection of HsTIMV231M HsTIMV231M was crystallized via vapor diffusion using the sitting drop method. One microliter of a solution at 35 mg/ of protein mL was mixed with 1 μL of reservoir solution. Crystals were obtained after three weeks of incubation at 20 °C in condition A6 of the Crystal Screen HT kit from Hampton Research (200 mM MgCl2, 100 mM Tris pH 8.5, 30% polyethylene glycol 4000). The crystals were cryoprotected in a solution prepared with the mother liquor supplemented with 20% glycerol; they were immediately frozen in liquid nitrogen. Diffraction data were collected at 100 K using a wavelength of 0.9785 Å at the Life Sciences Collaborative Access Team (LS-CAT) 21-ID-G beamline at the Advanced Photon Source (Argonne National Laboratory, IL). The data were processed with iMOSFLM [13] and reduced with Aimless [14].
2.3. Activity assay Enzyme activity was measured at 25 °C following the conversion of glyceraldehyde 3 phosphate (GAP) to dihydroxyacetone phosphate using α-glycerolphosphate dehydrogenase (α-GDH) as a coupling enzyme, as described by [11]. NADH oxidation was monitored at 340 nm. The assay system (1 mL) had 100 mM triethanolamine (TEA) pH 7.4, 10 mM EDTA,1 mM GAP, 0.2 mM NADH and 20 μg/mL of α-GDH. The reaction was initiated by the addition of different quantities of the 1402
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2.7. Structure determination and refinement The structure was solved by the molecular replacement method with the program PHASER [15] using the coordinates of WT HsTIM, at a resolution of 1.7 Å (Protein Data Bank code 2JK2), as the search model. The existence of the V231 M mutation was initially confirmed by difference Fourier maps calculated using the structure of the wild type enzyme. Refinement was alternated with manual building/refinement in COOT [16], PHENIX [17] and the PDB_REDO server [18]. At the start, non-crystallographic symmetry restraints were used for the refinement, using one group that included the four monomers of the asymmetric unit. The restraints were released during the last cycles of refinement, before the addition of water molecules. The electron density of monomer A is very well defined. For monomers B and C, residues 170–177 (in loop 6 or the flexible loop) are less clear in the electron density map. These regions are normally poorly defined in all apo-TIMs. In addition, one of the monomers (D) is somewhat more disordered than the other three (residues 130–240). The regions that comprise the active sites were well defined for all monomers. Five percent of the data were used to validate the refinement. Data collection and refinement statistics are given in Supplementary Table 2. Figures were generated with PyMOL (the Pymol Molecular Graphics System, Version 1.7.4, Schrödinger, LCC). 3. Results 3.1. Cloning and purification All the mutant enzymes and WT HsTIM were cloned and expressed in Escherichia coli strain BL21-CodonPlus (DE3)-RIL. To optimize protein expression, the time and temperature of induction was fine tuned for each mutant, as well as the amount of NaCl used in the lysis buffer (Supplementary Table 1). The enzymes were expressed with a His-tag at the N-terminal position, which was cleaved with TEV protease, as part of the purification process. In general, the yield of protein was between 20 and 200 mg/L of culture. The purity and homogeneity of all the proteins (which was more than 95%) was analyzed by SDS-PAGE and size exclusion chromatography coupled to a multi-angle light scattering instrument, which showed a dimeric behavior for all the enzymes at a concentration of 2 mg/mL (data not shown). HsTIMA62D had a very low yield after purification (5 mg/L of culture) and had a very strong tendency to precipitate. This behavior was due to an inherent low stability of the enzyme (Fig. 1, and growth curves described below). Thus, most of the described assays could not be performed with this mutant, and will be shown as not determined (nd).
Fig. 2. The enzymatic kinetic constants are affected differently for each mutant. A. Km is increased for mutants G72A (6.5 fold) and V231 M (8.7 fold). It decreases 15-fold for mutant I170V. B. kcat is affected for mutant I170V (25 fold). C. The catalytic efficiency (kcat/Km) is only strongly affected for mutants G72A (4.2 fold) and V231 M (9 fold). The assays were performed in triplicate.
3.2. Growth of E. coli Δtim-BL21 gold (DE3) cells complemented with the different enzymes In order to test for major survival impairments due to the mutations of HsTIM, we monitored the growth of bacterial cells lacking their endogenous tim gene and transformed them with the plasmids overexpressing WT HsTIM or the mutant enzymes (Fig. 1). Notably, there is a clear difference in the growth rate of mutant A62D, when compared to all the other mutants and the WT enzyme. No growth at all is seen for HsTIMA62D, when the assay is performed at 42 °C (Supplementary Fig. 1). Fig. 1. Mutant A62D impairs the growth of a Δtim bacterial strain. Growth curves of Escherichia coli strain Δtim BL21-Gold (DE3) transformed with the overexpression plasmid pET-3a (Novagen) encoding WT HsTIM or the mutant enzymes or the empty plasmid. The logarithm of OD600 is plotted against time of incubation in M9 minimal medium supplemented with 0.006% casamino acids and 0.2% glucose at 37 °C. The assay was performed in triplicate on a Synergy MX microplate reader on 96-well plates. No growth is seen for the A62D mutants when the assay is performed at 42 °C (Supplementary Fig. 1).
3.3. Enzyme activity Steady state kinetics assays for all enzymes were determined in the direction of glyceraldehyde 3-phosphate to dyhydroxyacetone-phosphate (Table 1 and Fig. 2). All enzymes exhibited a Michaelis-Menten behavior. No activity was detected with HsTIMA62D. 1403
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3.3.1. Km Regarding this parameter, we found mainly two groups of mutant enzymes (depicted in Fig. 2A): 1) those that had a “WT-like” behavior, including HsTIMC41Y, HsTIME104D, HsTIMG122R, HsTIMV154M and HsTIMF240L and 2) severely affected enzymes, including HsTIMG72A and HsTIMV231M, which have a Km 6 and 9 times higher than the WT enzyme, respectively. Interestingly, the Km value of HsTIMI170V is 15 times lower than that of WT HsTIM.
assays (Fig. 5). This is the case for the most affected enzymes in these parameters: HsTIMG72A, HsTIME104D and HsTIMC41Y. There is another group of enzymes that are only slightly affected: HsTIMG122R, HsTIMV154M and HsTIMI170V. HsTIMV231M has a residual activity similar to WT HsTIM, but its Tm value is affected. Together with HsTIMF240L, both mutants represent the borderline between the “WTlike” group and the most affected mutants. 3.6. Overall comparison and rating of the deleteriousness of the mutant enzymes
3.3.2. kcat As shown in Fig. 2B, the only mutant that is strongly affected in this parameter is HsTIMI170V. Its kcat value is 25 times lower as compared to that of the WT enzyme. The rest of the mutants have a similar behavior to the WT enzyme, with a kcat that is 1.5 times higher than the WT enzyme for HsTIMG72A and 1.2 times lower for HsTIMC41Y.
In order to provide an overall and comparative view of this study, the strength of the effects seen in the different measured parameters described in Figs. 1-4, compared with the WT enzyme, is summarized in Table 2. According to these results, we propose a rating of the deleteriousness of the mutants, based on their effects on growth (g), kinetics (k, represented by Km, kcat or kcat/Km) or stability (s, represented by the thermal or dilution stability) (Fig. 6). The critical mutations are those whose three parameters (g,k,s) are affected. This is the case for HsTIMA62D, which was extremely difficult to purify and whose precipitation behavior precluded the measurement of any parameter. This mutant cannot support the growth of the Δtim strain of E. coli. HsTIMG72A is a severe mutant for which two parameters (k and s) are strongly affected. Other important mutations are those that show a strong effect in one parameter. This is the case for HsTIMC41Y, HsTIME104D (with an effect on s) and HsTIMV231M (with an effect on k). HsTIMI170V and HsTIMF240L have moderate effects, since their kinetic or stability parameters are only somewhat affected. Finally, HsTIMG122R and HsTIMV154M do not have any effect on these parameters of enzyme function.
3.3.3. Catalytic efficiency (kcat/Km) Fig. 2C shows that HsTIMG72A and HsTIMV231M are strongly affected in this global parameter, with a respective four- and nine-fold decrease when compared to the WT enzyme. The catalytic efficiency values of HsTIMC41Y, HsTIMI170V and HsTIMF240L decreases by half with respect to the WT enzyme. 3.4. Thermostability We used thermal shift assays to measure the thermal stability of the mutants (Fig. 3 and Supplementary Fig. 2). HsTIMG122R, HsTIMV154M and HsTIMI170V display a melting temperature (Tm) similar to the WT enzyme (60 to 63 °C). HsTIMV231M and HsTIMF240L have a Tm of around 53 °C. HsTIMC41Y, HsTIMG72A and HsTIME104D show a difference of more than 10 °C compared to the WT enzyme, with Tm values ranging between 49.7 and 51.5 °C.
3.7. Structural basis for the kinetic effects of mutation HsTIMV231M
3.5. Dimer stability
HsTIMV231M is the most affected mutant from the kinetic point of view, considering the catalytic efficiency parameter. In addition, this enzyme is, together with E104D, the only homozygous mutation reported to date. We therefore solved the crystal structure of this mutant in the apo conformation at 2.3 Å resolution (Fig. 7, Supplementary Table 2 and Supplementary Fig. 8). The inspection of the four molecules that constitute the asymmetric unit revealed the structural basis of TIM deficiency for this mutant (Fig. 7). Comparing it with the available crystal structures of the WT enzyme, both in the apo conformation (PDB ID: 2JK2) and in complex with the substrate 2-phosphoglycerate (2PG or PGA), allowed us to define the conformational changes that occur as a consequence of the V231 M mutation (Figs. 7A, B). The most important changes are found just around residue 231 (Fig. 7C), which is located very close to the active site (Figs. 7A, B). These changes are
Stability to dilution experiments measure the reduced activity of the TIM enzymes related to monomerization events [19–21]. We incubated the HsTIM mutants at different decreasing concentrations for 2 h and measured the remaining activity (Fig. 4 and Supplementary Fig. 3). At the lowest enzyme concentration assayed (1 ng/mL), we noticed that the residual activity showed an important gap for the enzymes that are still active (between 31 and 57%) and another group of enzymes: HsTIMC41Y, HsTIMG72A and HsTIME104D, which are strongly affected by the dilution effect and had between 2.6 and 7.7% activity of their original activity. Remarkably, we found a strong correlation for some of the mutants between thermostability data and the results of the dimer stability
Fig. 3. Mutants C41Y, G72A and E104D are strongly affected in thermal stability. The Thermal Shift Assay (TSA) was used to measure the thermal stability of the WT and the mutant enzymes. Each assay had 0.2 μg of protein in 100 mM TEA pH 7.4 and SYPRO Orange dye in a final volume of 10 μl. The experiment was performed using a 96-well plate in a real time PCR system. The assay was performed in triplicate. The thermal melting temperature was calculated using the peak of the first derivative plot shown in Supplementary Fig. 2.
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Fig. 4. Mutants C41Y, G72A and E104D are strongly affected in dimeric stability. The enzymes were incubated at different protein concentrations (see Supplementary Fig. 3) in 100 mM TEA pH 7.4 and 10 mM EDTA for 2 h at 36 °C. After that time, their residual catalytic activity was determined. This residual catalytic activity was plotted against the logarithm of protein concentration. The apparent dissociation constants (Kd app) were determined for the WT enzyme and each mutant. The values of each enzyme were adjusted using the Three-parameter Logistic equation of Prism 7.
Fig. 6. Deleteriousness rating of the HsTIM mutants. The rating is based upon the severity of the effects on kinetic parameters (k), stability (s) and growth (g), also shown in Table 2. Critical: implicates that all these parameters are affected. Severe: both k and s are affected. Important: either k or s are affected. Moderate: a mild effect on k or s.
Fig. 5. Correlation between thermal and dimeric stability of mutants C41Y, G72A, E104D and F240L. Double plot of the residual activity at 1 ng/ml (depicted in Fig. 4) and the Tm temperatures (depicted in Fig. 3). The order of the proteins in the x-axis follows the residual activity values, from highest to lowest. In general, a mild effect is seen for both parameters in mutants G122R, V154 M and I170V, and a strong effect in mutants G72A, E104D and C41Y. Mutants V231 M and F240 L represent a borderline between these two extremes.
substrate. This effect is correlated with the change in the kinetic parameter Km, described in this work.
represented by movements in the main chain around residues 212–219 and 231–234, which show a maximal displacement of 1.1 and 2.1 Å, respectively, when compared to both conformations of the WT enzyme (apo and 2PG, Fig. 7). In particular, the movement of residues 231–234 would approach the main chain of the protein to 2PGA by 0.5 Å (Fig. 7C), which is now too close, and hinders the binding of the
4. Discussion In this work, we performed an experimental analysis of most of the reported mutants related to human triosephosphate isomerase deficiency. Other attempts have been made to evaluate the range of effects on mutants of TIM [9] but this report defines more precisely the extent
Table 2 Summary of the effects seen in different mutants, compared to the wild-type enzyme. The table summarizes the results shown in Figs. 1-4.
Mutant
C41Y A62D G72A E104D G122R V154M I170V V231M F240L
Kinetic Km
kcat k kcat/Km X ND XX
ND XX
XX XX
XX
X XX X
Stability Tm
Dilution
Growth
XX ND XX XX
XX ND XX XX
X X
X
XX = strong, X = moderate, blank = no effect, ND = not determined.
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of the active site; the remaining seven mutations, which do have an effect, are located on the same side as the active site of the enzyme (Supplementary Fig. 4). In the following sections of the discussion, we will enlist, following the numbering in the sequence of HsTIM, the effects reported in the disease and the main findings observed in this study with corresponding recombinant mutant protein. In addition, we summarize the molecular contexts and the effects seen with each mutant (Supplementary Figs. 4 to 9) in order to help in the understanding of the molecular basis of TIM deficiency and to aid in the prediction of the severity of the mutations in a heterozygous situation. 4.1. C41Y 4.1.1. Occurrence Three cases have been reported in compound heterozygotes together with the E104D mutation [25,27]. 4.1.2. Severity in patients Chronic axonal neuropathy. The damage became evident at the age of 2 years [27]. 4.1.3. Previous experimental characterization In a yeast model with the deleted endogenous gene tim, this mutant showed the same activity as the WT enzyme [28]. In addition, the dimerization properties of this mutant were monitored using a yeast twohybrid system. It had an altered dimerization behavior, with increased interaction activity. 4.1.4. Findings in this work Thermal and dimer stability strongly affected (13.5 °C difference in the value of Tm and residual activity at 1 ng/mL, 32% lower than that of the WT enzyme). 4.1.5. Molecular context and predictions Residue 41 is in the vicinity of positions 62, 231 and 240 (Supplementary Fig. 7), which have critical, strong and moderate effects on the enzyme, respectively. A non-conservative change in this residue might have a strong effect on the stability of the enzyme, as was indeed seen for mutation C41Y. In combination with the E104D mutation, we predict that the heterodimer would have completely disruptive effects on the stability of the enzyme. The patient might have limited survival in the presence of the homodimers having each mutation.
Fig. 7. Structural basis of human TIM deficiency produced by mutation V231M. Mutation V231 M perturbs the active site region. A. Cα RMSD plot calculated using apo WT HsTIM (PDB ID: 2JK2) and the model of the crystal structure of mutant V231 M, solved at 2.3 Å resolution (PDB ID: 6C2G). The overall RMSD is 0.311 and 0.562 Å for the superpositions of the mutant V231 M with WT HsTIM apo and complexed with 2PG, respectively. The regions that are displaced are indicated by the residue numbers or structural feature that differ the most. Loop 6 (residues 169–173) is usually flexible in all TIMs. B. Superposition of the model of the crystal structure of mutant V231 M with WT HsTIM complexed with 2PGA, showing the regions of conformational movements depicted in panel A. The regions correspond to residues 134–141, loop 6, 212–219 and 231–234. The substrate 2PGA is shown as spheres. C. Perturbations at the active site. Regions 212–219 and 231–234 are located very close to the active site. The movement of these residues, with its peak at residue 233, which is displaced by 2.1 Å with respect to the WT structure. This correlates with the presence of the mutation and hinders the binding of the substrate.
4.2. A62D 4.2.1. Occurrence One case in a compound heterozygote, with E104D as the second mutation [22]. 4.2.2. Severity in patients Unknown. 4.2.3. Previous experimental characterization None.
and cause of the enzymatic deficiency at the biochemical level, in an all versus all comparative scenario. Although most of the mutants are present in humans as heterozygous proteins, as a first approach we studied single mutants in order to investigate their individual contribution to the kinetics and stability of human TIM. We were also able to classify the “unknown severity” mutants HsTIMA62D, HsTIMG72A, HsTIMG122R and HsTIMV154M [22–24]. Interestingly, HsTIMG122R and HsTIMV154M do not seem to have any major biochemical alteration, and only show a very modest effect on thermal stability. Residues 122 and 154 are located on the opposite side
4.2.4. Findings in this work The mutation impairs growth when expressed in bacterial strain E. coli Δtim. 4.2.5. Molecular context and predictions Residue 62 is located in a hydrophobic patch formed by residues V40, A42, A62 and V92 (Supplementary Fig. 5). It is possible that a conservative mutation in this position could be tolerated well, but a mutation that involves a charged residue, like A62D, completely 1406
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(Supplementary Fig. 7). A patient has limited survival having HsTIME104D homodimers.
disrupts this hydrophobic region, which in addition is close to residue 41, and compromises the survival of the organism. In combination with the E104D mutation, we predict that the heterodimer would have totally disruptive effects on the stability of the enzyme. The patient might have limited survival in the presence of only one mutant type of homodimer having mutation E104D.
4.5. G122R 4.5.1. Occurrence One individual [29].
4.3. G72A 4.5.2. Severity in patients No patients. The study was performed with a population of healthy heterozygotes.
4.3.1. Occurrence Found in a phenotypic heterozygote, for which TIM activity was reduced, together with the WT enzyme [24].
4.5.3. Previous experimental characterization Reported as a thermolabile enzyme [23]. In a yeast model, with a deleted endogenous gene for TIM, this mutant had the same activity compared to the WT enzyme [28]. In addition, the dimerization properties of this mutant were monitored with a yeast two-hybrid system, showing no effect.
4.3.2. Severity in patients No patients. Study in a population of healthy heterozygotes. 4.3.3. Previous experimental characterization None.
4.5.4. Findings in this work The enzyme is not affected. Interestingly, the residual activity at 1 ng/mL is 21.6% higher with respect to the WT enzyme.
4.3.4. Findings in this work Effects upon both kinetic (fourfold decrease in the catalytic efficiency) and stability parameters (11.7 °C difference in the Tm value and the residual activity at 1 ng/mL, 28% lower compared with the WT enzyme).
4.5.5. Molecular context and predictions Residue 122 is located on the opposite side of the active site, at the end of a loop and facing the solvent (Supplementary Fig. 9). Any change for a hydrophilic residue would have no effect on the enzyme, as is shown by non-conservative mutation G122R. Patients should show no symptoms or disease.
4.3.5. Molecular context and predictions Residue 72 is located in loop 3, which is very close to the active site and also forms part of the dimer interface. (Supplementary Fig. 6). Any change in this position would be severe for both the kinetic and stability parameters, as is shown by mutation G72A, which would now be too close to N15 of the dimeric partner (Supplementary Fig. 6). However, since both residues in positions 72 in both monomers are far apart from each other in the dimeric enzyme, if a dimer is formed in combination with one WT monomer, we predict that the heterodimer will have enough stability. The patient might survive in the presence of all types of homo and heterodimers involving this mutation.
4.6. V154M 4.6.1. Occurrence Found in a phenotypic heterozygote, for which TIM activity was reduced, together with the WT enzyme [24]. 4.6.2. Severity in patients No patients. The study was performed in a population of healthy heterozygotes.
4.4. E104D 4.4.1. Occurrence At least 15 cases from multiple unrelated families, although most of the patients are thought to have a common ancestor. HsTIME104D together with HsTIMV231M, are the only homozygote mutants reported to date.
4.6.3. Previous experimental characterization None. 4.6.4. Findings in this work The enzyme is not affected.
4.4.2. Severity in patients Hemolytic anemia, neuromuscular impairment and increased susceptibility to infection. No patient has survived beyond the age of six.
4.6.5. Molecular context and predictions Residue 154 is located on the opposite side of the active site, in a hydrophobic patch formed by residues L177, V154, W157 and V160 (Supplementary Fig. 9). Most changes for a hydrophobic residue would have no effect on the enzyme, as is shown by conservative mutation V231M. Patients should not show any symptom or disease.
4.4.3. Previous experimental characterization The molecular basis of the deficiency has been reported elsewhere [5,28] and is caused by alterations in dimer stability, rather than on the effects in catalytic activity.
4.7. I170V 4.4.4. Findings in this work Thermal and dimeric stability are strongly affected (13.5 °C difference in Tm and the residual activity at 1 ng/mL is 32% lower compared to the WT enzyme).
4.7.1. Occurrence Found in one case in a compound heterozygote together with the E104D mutation [25] [26].
4.4.5. Molecular context and predictions Residue 104 is located very close to the interface and forms a network of interactions with other highly conserved residues (Supplementary Fig. 7). Any greater change in this position would compromise the stability of the enzyme, as is implied by the highly conservative mutation E104D. This is worsened by the fact that the counterpart of this region in the other monomer is in close proximity.
4.7.2. Severity in patients One patient with hemolytic anemia, but no muscular impairment [26]. A model in Drosophila has shown behavioral abnormalities [30]. 4.7.3. Previous experimental characterization This mutant had very decreased activity in the patient (10% or less) when compared to the wild type enzyme, and decreased activity 1407
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4.9.4. Findings in this work The mutation has moderate effects upon the kinetics (less than a twofold decrease in the catalytic efficiency) and stability parameters (10 °C difference in the Tm value, but a relatively high residual activity value of 22.6% compared to 35.6% of the WT enzyme).
(around 30%) in a yeast model with a deleted endogenous TIM gene. In addition, the dimerization properties of this mutant were examined using a two-hybrid system in yeast, with a slight enhancement of the stability [28]. The mutation lowered Km and kcat values 20 and 30fold, respectively, and increased enzyme stability (Tm = 59.4 °C, compared to 46.5 °C of the WT enzyme) [30].
4.9.5. Molecular context and predictions Residue 240 is close to positions 41 and 231 (Supplementary Fig. 8). A conservative change in this residue might have an effect on the stability and perhaps the catalytic performance of the enzyme because of its vicinity with V231. The effect is indeed moderate for mutation F240L and perhaps it would be more severe for mutation F240S in combination with mutation E104D. We predict that the heterodimer would be functional, but severely affected in its stability. The patient might have limited survival in the presence of the heterodimers, with little contribution to the deficiency because of the F240S homodimers. The combination of F240L with E145Stop is probably not functional as a heterodimer, but the F240L homodimer should be functional. Overall, it is clear that the perturbation of the stability of the quaternary structure may define some TIM deficiencies (mutations C41Y, E104D) as well as disturbances mainly in kinetic parameters (mutations I170V, V231M), effects in kinetics and stability (mutation G72A) or completely aberrant enzymes (mutation A62D) for others. Notably, this study is in general agreement with experimental studies dealing with the in vitro characterization of one or several mutants [28]; [5]; [30]. Further studies will be needed to define the molecular effects of some mutations, notably C41Y, as well as the effects seen in heterodimeric constructions, which can be expected from the compound heterozygous mutations found in most cases of this disease.
4.7.4. Findings in this work Enzyme kinetics are moderately affected. The Km and the kcat values decrease 15 and 25 times, respectively. However, the overall efficiency of the enzyme is not strongly affected (less than twofold). The stability of the enzyme is unchanged. 4.7.5. Molecular context and predictions Residue 170 is very close to the active site, in loop 6, which is important for catalysis (Supplementary Fig. 8). A non-conservative change in this residue might have a strong effect on the catalysis of the enzyme. The effect is indeed moderate for mutation I170V, because the enzyme also shows an increased substrate affinity. The structural basis of the deficiency caused by this mutant has been analyzed in [30]. In combination with the E104D mutation, we predict that the heterodimer should be functional, but with severe effects on its kinetic parameters and stability. Yet the patient with the hetero dimers does survive at least until late childhood and, patients having homo dimers would probably not be severely affected. 4.8. V231M 4.8.1. Occurrence Homozygous mutation linked to clinical deficiency in two cases [31] [32]
Data accessibility The atomic coordinates and structure factors of mutant V231M of human triosephosphate isomerase have been deposited in the Protein Data Bank (PDB) with the accession code 6C2G.
4.8.2. Severity in patients Hemolytic anemia and neurological dysfunction.
Conflict of interests
4.8.3. Previous experimental characterization None.
The authors declare they have no conflict of interest. 4.8.4. Findings in this work Enzyme kinetics are strongly affected. The Km increases nine-fold and the catalytic efficiency decreases accordingly. The crystal structure of the mutant shows that the active site is perturbed and hinders the binding of the substrate (Fig. 7).
Authors' contributions NC, ATL and RPM conceived and designed the experiments; NC, ATL, IGT and SEF performed the experiments; NC, ATL, IGT, SEF and RPM analyzed the data; NC, ATL and RPM wrote the manuscript. All authors gave final approval for publication.
4.8.5. Molecular context and predictions Patients might have limited survival (and see above).
Transparency document
4.9. F240L
The Transparency document associated this article can be found, in the online version.
4.9.1. Occurrence Found in two brothers who are compound heterozygotes (E145Stop) [33,34]; reviewed in [8]. A mutation in the same position (F240S), is found in a compound heterozygote together with mutation E104D [35].
Acknowledgements We thank Gloria Saab-Rincón, PhD and Leticia Olvera-Rodríguez, BSc (Instituto de Biotecnología-UNAM, México) for providing the E. coli Δtim-BL21-Gold (DE3) strain. We also thank Gabriel Del Río, PhD, who kindly allowed the use of the Synergy MX equipment, María-Teresa Lara-Ortiz, Jessica Díaz-Salazar, Beatriz Aguirre, Manuel Ortinez Benavides, Aurey Galván Lobato and Unidad de Biología Molecular at Instituto de Fisiología Celular-UNAM (Dr. Laura Ongay and Guadalupe Codiz) for technical support. Use of the Advanced Photon Source, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02-06CH11357. Use of the LS-CAT
4.9.2. Severity in patients Neurological symptoms for only one of the brothers with the F240 L mutation and also for the patient with the F240S mutation. 4.9.3. Previous experimental characterization Decreased catalytic activity [36]. In a yeast model with a deleted TIM endogenous gene, this mutant showed the same activity as compared to the wild type enzyme [28]. In addition, the dimerization properties of this mutant were measured using a yeast two-hybrid system, and no effect was detected. 1408
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Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor, Grant 085P1000817. This work was supported by grant 254694 from Consejo Nacional de Ciencia y Tecnología, México, to RPM.
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