Structures of pyruvate kinases display evolutionarily divergent allosteric strategies

The transition between the inactive T-state (apoenzyme) and active R-state (effector bound enzyme) of Trypanosoma cruzi pyruvate kinase (PYK) is accompanied by a symmetrical 8° rigid body rocking motion of the A- and C-domain cores in each of the four subunits, coupled with the formation of additional salt bridges across two of the four subunit interfaces. These salt bridges provide increased tetramer stability correlated with an enhanced specificity constant (k_cat/S_0.5). A detailed kinetic and structural comparison between the potential drug target PYKs from the pathogenic protists T. cruzi, T. brucei and Leishmania mexicana shows that their allosteric mechanism is conserved. By contrast, a structural comparison of trypanosomatid PYKs with the evolutionarily divergent PYKs of humans and of bacteria shows that they have adopted different allosteric strategies. The underlying principle in each case is to maximize (k_cat/S_0.5) by stabilizing and rigidifying the tetramer in an active R-state conformation. However, bacterial and mammalian PYKs have evolved alternative ways of locking the tetramers together. In contrast to the divergent allosteric mechanisms, the PYK active sites are highly conserved across species. Selective disruption of the varied allosteric mechanisms may therefore provide a useful approach for the design of species-specific inhibitors.

2. Background

Pyruvate kinase (PYK, EC 2.7.1.40) catalyses the final step in glycolysis, namely the transfer of a phospho group from phosphoenolpyruvate (PEP) to ADP to form pyruvate and ATP. PYK exists primarily as a homotetramer with subunits of 50–60 kDa (depending on species), each of which is composed of four domains: the N-terminal (not present in bacteria), A-, B- and C-domains (figure 1a). An additional C'-domain is also found in PYKs from Geobacillus stearothermophilis[2] (figure 2) and Staphylococcus aureus [6]. The active site is nestled between the A- and B-domains and is located approximately 39 Å from the effector site which is located in the C-domain. In the tetramer, adjacent C-domains form the C–C or ‘small’ interface, and neighbouring A-domains form the A–A or ‘large’ interface. The B-domain contributes a mobile lid at one end of the (α/β)₈-barrelled A-domain and modulates access to the active site.

The overall tetrameric structure of PYK is highly conserved across distant phylogenetic groups; however, strategies for the regulation of PYK activity vary substantially between species [2,7–11]. Three distinct ligand-binding sites with affinities for a range of small molecules have been identified in PYK tetramers and are particularly well characterized in mammals, trypanosomatids, yeast and bacteria (figure 1b). These sites are designated here as (i) the active site, (ii) the effector site, and (iii) the amino acid binding site. Most PYKs display cooperative binding of the substrate PEP at the active site, with characteristic sigmoid binding curves and Hill coefficients>1 (e.g. table 1). In this way, glycolytic flux can be augmented in response to the presence of elevated levels of PEP.

Modulation of PYK activity by activators and inhibitors which bind to the effector site is well characterized (table 1). Fructose 1,6-bisphosphate (F16BP) is the allosteric activator of three of the four mammalian isoenzymes: RPYK (erythrocyte), LPYK (liver) and M2PYK (embryonic or tumour). The fourth isoenzyme is the constitutively active M1PYK found in skeletal muscle. The activity of M1PYK can also be attenuated by the binding of amino acids such as proline or phenylalanine at the amino acid binding site [9,23]. PYK from Escherichia coli is also activated by F16BP, but PYK from Geobacillus stearothermophilus is allosterically activated by AMP or ribose 5-phosphate (R5P) [8] which probably bind at a position corresponding to the effector site [2]. PYK from baker's yeast Saccharomyces cerevisiae is also activated by F16BP [17] (table 1). Interestingly, PYKs from plants and archaea appear to be unresponsive to effectors such as F16BP or R5P [24,25]. There are currently no structural data for plant or archaeal PYKs.

The ‘TriTryp’ group of trypanosomatid parasites are responsible for diseases, including sleeping sickness (caused by Trypanosoma brucei), leishmaniasis (caused by various species of Leishmania) and Chagas' disease (caused by Trypanosoma cruzi), and T. brucei PYK is a validated drug target [26]. Trypanosomatid PYKs are allosterically activated by micromolar concentrations of fructose 2,6-bisphosphate (F26BP), instead of F16BP which is effective only at millimolar concentrations [27] (table 1). The X-ray structures of PYK from L. mexicana [10] and T. brucei [12] have been determined. Here, the first apoenzyme and R-state structures for T. cruzi PYK (TcPYK) are presented, thereby providing a detailed structural and enzymatic comparison for each of the ‘TriTryp’ family members. Analysis of the R- and T-state PYK structures from trypanosomatids, mammals and bacteria also provides an explanation of the evolutionary divergence of allosteric regulation.

3. Material and methods

3.1 Materials

ADP, PEP, oxalate (OX), F16BP, F26BP, lactate dehydrogenase (LDH; rabbit muscle), polyethylene glycol (PEG) 8000, antibiotics and buffers were obtained from Sigma-Aldrich. NADH and EDTA-free protease inhibitor mixture tablets were from Roche, glycerol was from BDH Prolabo, IPTG was from Melford and salts were from Fisher Scientific. Restriction enzymes, vector and E. colicompetent cells were from Novagen.

N-terminal His₆-tagged TcPYK was prepared as described previously [28]. Briefly, the expression of His₆-tagged TcPYK was achieved by the T7lac promoter-driven system in E. coli BL21 (DE3) cells after adding IPTG to a final concentration of 1 mM. Pure protein was obtained by immobilized metal ion affinity chromatography (IMAC) followed by gel-filtration chromatography. N-terminally His₆-tagged human pyruvate kinases (M1PYK and M2PYK) were expressed in E. coli BL21(DE3) cells and purified using IMAC and gel-filtration chromatography as described previously [16].

3.2 Enzyme activity assay and kinetics study

The activity and kinetics of TcPYK were determined as described for T. bruceiPYK (TbPYK) previously [12]. Briefly, the activity of TcPYK was measured by following the decrease in NADH absorbance at 339 nm, where one activity unit is defined as the conversion of 1 μmol substrate per minute under standard conditions. The assay was performed at 298 K in 100 μl reaction mixtures containing 50 mM triethanolamine (TEA) buffer, pH 7.2, 50 mM KCl, 10 mM MgCl₂, 10% glycerol, 0.5 mM NADH and 5 μg (3.2 U) of LDH. The turnover number (k_cat) of TcPYK was calculated by the enzyme specific activity divided by the subunit molar mass of 56769.1 (g mol⁻¹). Enzyme kinetics with regard to ADP was studied at saturating concentrations of PEP (10 mM) and variable concentrations of ADP (from 0 to 2.5 mM). Enzyme kinetics with regard to PEP was studied at saturating concentrations of ADP (2.5 mM) and variable concentrations of PEP (from 0 to 10 mM) in the presence or the absence of 1 μM F26BP, or in the presence or the absence of 4.5 mM F16BP. Enzyme kinetics with regard to the effector F26BP was studied at 2.5 mM ADP, 0.4 mM PEP and variable concentrations of F26BP (from 0 to 1 μM), while enzyme kinetics with regard to the effector F16BP was studied at 2.5 mM ADP, 0.7 mM PEP and variable concentrations of F16BP (from 0 to 4.5 mM). The Michaelis–Menten equation or its extended equations were fitted to the experimental data to calculate the values of the kinetic parameters. The parameter S_0.5 is used instead of K_m when the substrate binding shows cooperativity. The parameter Ka_0.5 refers to the concentration of activator at which half the maximal activation is observed.

3.3 Thermal shift assay (differential scanning fluorimetry)

Melting temperature (T_m) was determined by a thermal shift assay using a fluorescence method as described for TbPYK previously [12]. For TcPYK and TbPYK, the assay buffer contained 50 mM TEA, pH 7.2, 100 mM KCl, 10 mM MgCl₂, 10× SYPRO Orange dye (the dye was supplied by Invitrogen (catalogue number S6650) at 5000× concentration in DMSO, and diluted in assay buffer for use), 4 μM TcPYK or TbPYK, in the presence or the absence of 1 μM F26BP.

3.4 Dynamic light scattering

The T_m values for human M2PYK and human M1PYK were also determined by monitoring changes in light scattering with increasing temperature. Five microlitres of human M2PYK or human M1PYK (10 mg ml⁻¹), 5 μl of 10× metals buffer (500 mM TEA, pH 7.2, 500 mM MgCl₂, 1 M KCl) and 1 μl of each ligand (50 mM stocks prepared in 100 mM TEA buffer, pH 7.2), the final volume was then adjusted to 50 μl using dilution buffer (10 mM TEA (pH 7.2) and 2% glycerol).

A Zetasizer Auto Plate Sampler was used to determine the Z-average molecular ‘size’ in terms of the hydrodynamic diameter in solution. Changes in the Z-average or particle diameter with increased temperature (293–353 K in increments of 1 K) were monitored in automated mode (typically requiring a measurement duration of 150 s; 13 acquisitions were determined for each run and repeated in triplicate). The resulting data were then analysed using the manufacturer's software provided (Malvern Instruments Ltd, Malvern, UK).

3.5 Crystallization, data collection and structure determination

Purified TcPYK aliquots (30 mg ml⁻¹) were diluted to 15 mg ml⁻¹ using a buffer containing 50 mM TEA (pH 7.2) or supplemented with 3.5 mM ATP, 3.5 μM F26BP and 3.5 mM oxalate. Single crystals of TcPYK or TcPYK complexed with F26BP, Mg²⁺ and oxalate (there was no evidence of ATP from the crystal structure) were obtained at 277 K by vapour diffusion using the hanging drop technique. The drops were formed by mixing 1.5 μl of protein solution with 1.5 μl of a well solution composed of 8–18% PEG 8000, 20 mM TEA buffer (pH 7.2), 50 mM MgCl₂, 100 mM KCl and 10–20% glycerol. The drops were equilibrated against a reservoir filled with 0.5 ml of well solution.

Prior to data collection, crystals were equilibrated for 14 h over a well solution composed of 8–18% PEG 8000 (2% above the crystallization conditions), 20 mM TEA buffer (pH 7.2), 50 mM MgCl₂, 100 mM KCl and 25% glycerol, which eliminated the appearance of ice rings. Intensity data were collected at the Diamond synchrotron radiation facility in Oxfordshire, UK on beamline I04 (TcPYK) or I03 (TcPYK-F26BP-OX-Mg) to a resolution of 2.50 Å (TcPYK), or 2.80 Å(TcPYK-F26BP-OX-Mg). All datasets were obtained from a single crystal flash frozen in liquid nitrogen at 100 K. The TcPYK structures were solved as described previously [28].

Superpositions of PYK structures were performed using both PyMOL [29] and CCP4 superpose [30,31]. CCP4 superpose was also used to calculate the allosteric rigid body rotations of TcPYK from the superposition of T- and R-state tetramers (AC cores) as described previously [10]. Both RMS difference numbers and rotation matrices were calculated in the superposition process.

4. Results and discussion

4.1 TcPYK and TbPYK have similar kinetic parameters

Values of TcPYK kinetic parameters were determined for PEP in the presence and absence of both the trypanosomatid PYK allosteric activator F26BP and the more general activator F16BP at 298 K and are summarized in table 1, together with the parameters previously reported for TbPYK [12]. In the absence of F26BP, the S_0.5 values for PEP were 1.23 mM and 1.03 mM for TcPYK and TbPYK, respectively, both exhibiting positive cooperativity (h>1) and similar specificity constants (k_cat/S_0.5 values of 137 and 141 s⁻¹ mM⁻¹, respectively). In the presence of F26BP, the S_0.5 value for PEP decreased ninefold and the specificity constant increased 11-fold (average k_cat/S_0.5 value of 1514 mM⁻¹ s⁻¹) for both TcPYK and TbPYK, with positive cooperativity being completely abolished (n=∼1). By contrast, addition of the general effector F16BP decreased the S_0.5value for PEP fourfold and increased the specificity constant fivefold, although these effects were observed only at concentrations over 1000 times higher than that of F26BP. There are no data available for the intracellular concentrations of F16BP and F26BP in T. cruzi; however, in vivo concentrations for F16BP and F26BP have been determined for the bloodstream form of T. brucei as approximately 600 μM for F16BP and approximately 10 μM for F26BP (calculated from data in [32] and assuming 175 mg total cellular protein per millilitre total cellular volume [33]). In T. cruzi, there is an interestingly large difference in the Ka_0.5 values for F26BP (0.03 μM) and F16BP (1239 μM) (table 1). The corresponding Ka_0.5 values for T. brucei are 0.01 and 50 μM. Based on the suggested relative cellular concentrations of the FBPs and their PYK affinities, it is not out of the question that F16BP may also contribute to PYK activation in trypanosomatids.

4.2 Sequence identity explains different effector recognition

The TcPYK amino acid sequence is 81% identical to TbPYK and 76% to LmPYK (electronic supplementary material, table S1), with residues involved in substrate and effector binding conserved (figure 2). This high degree of sequence conservation between trypanosomatids is reflected in their similar kinetic parameter values (table 1). TcPYK and the F16BP-regulated human M2PYK are only 47% identical (electronic supplementary material, table S1), although the residues involved in substrate binding exhibit 100% sequence conservation (figure 2). The side chains involved in effector binding are much more variable and show only 57% sequence identity.

4.3 T- to R-state transition: a common pivot region

Both T- and R-state TcPYK X-ray crystal structures were obtained under near identical (pH 7.2) conditions (see table 2 for data collection and refinement statistics). In the R-state structure (TcPYK-F26BP-OX-Mg), the active and effector sites are occupied by Mg²⁺-oxalate and F26BP, respectively (figures 3and 4). In the T-state structure of TcPYK (apo-TcPYK), the effector loop (Ala482-Gly488) is disordered in the absence of F26BP binding. A superposition of the T- and R-state tetramers shows that the T- to R-state transition involves a rigid body rotation of subunits (excluding B-domains; AC cores; figure 5a–c), whereby the subunits pivot 8° (electronic supplementary material, table S2) around a region (residues 430–433) located at the base of the αβ barrel of domain A (figure 1b). In a similar way, LmPYK experiences a rigid body rotation of its AC cores during the transition between T- and R-states [10]. A superposition of the TcPYK and LmPYK AC cores gave a C^α RMS fit of 0.7 and 0.6 Å for the T- and R-states, respectively.