Mechanistic and Structural Insights into Cysteine-Mediated Inhibition

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Mechanistic and Structural Insights into CysteineMediated Inhibition of Pyruvate Kinase Muscle Isoform 2 Dhiraj Srivastava, Suparno Nandi, and Mishtu Dey Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.9b00349 • Publication Date (Web): 06 Aug 2019 Downloaded from pubs.acs.org on August 7, 2019

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Biochemistry

Mechanistic and Structural Insights into Cysteine-Mediated Inhibition of Pyruvate Kinase Muscle Isoform 2

Dhiraj Srivastava1, Suparno Nandi1, and Mishtu Dey1,2 *

1

Department of Chemistry, The University of Iowa, Iowa City, IA 52242, United States 2

Corresponding author

*Correspondence: [email protected]

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ABSTRACT Cancer cells regulate key enzymes in the glycolytic pathway to control the glycolytic flux, which is necessary for their growth and proliferation. One of the enzymes is pyruvate kinase muscle isoform 2 (PKM2), which is allosterically regulated by various small molecules. Using detailed biochemical and kinetic studies, we demonstrate that cysteine inhibits wild-type (wt) PKM2 by shifting from an active tetramer to a mixture of tetramer and less active dimer/monomer equilibrium and that the inhibition is dependent on cysteine concentration. The cysteine mediated PKM2 inhibition is reversed by fructose-1, 6-bisphosphate, an allosteric activator of PKM2. Furthermore, kinetic studies using two dimeric PKM2 variants, S437Y PKM2 and G415R PKM2, show that the reversal is caused by tetramerization of wtPKM2. The crystal structure of wtPKM2Cys complex was determined at 2.25 Å, which displayed that cysteine is held to the amino acid binding site via its main chain groups, similar to that observed for phenylalanine, alanine, serine, and tryptophan. Notably, ligand binding studies using fluorescence and isothermal titration calorimetry show that the presence of phosphoenolpyruvate alter the binding affinities of amino acids to wtPKM2 and vice versa, thereby unravelling the existence of a functionally bidirectional coupling between the amino acid binding site and active site of wtPKM2.

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Biochemistry

INTRODUCTION Pyruvate kinase (PK) catalyzes the terminal step of glycolysis and transfers a phosphoryl group from phosphoenol pyruvate (PEP) to adenosine diphosphate (ADP), generating adenosine triphosphate (ATP) and pyruvate. Pyruvate is further oxidized via the tricarboxylic acid (TCA) cycle resulting in the production of additional ATP. In mammals, there are 4 PK isoforms encoded by two genes.1 The PKLR gene encodes for PKL, which is mainly expressed in liver and PKR expressed in erythrocytes. The PKM gene encodes for the two isoforms PKM1 and PKM2 that are derived by alternative splicing and are identical in length, but differ only by 22 residues within a 56-amino acid (aa) region. PKM2 has four domains, namely- N (1-44 aa), A (44-166 aa and 218389 aa), B (166-218 aa), and C (318-589 aa).1-2 PKM1 is expressed in organs with high energy requirements such as, skeletal muscle, heart, and the brain. Whereas, PKM2 is primarily expressed in tissues with high anabolic functions such as normal proliferating cells, embryonic cells, and cancer cells. 1 Furthermore, while PKM1 always exists in a constitutively active tetrameric state, the other PKM isoforms, including PKM2, are allosterically regulated and enzymatically active as tetramers.3 PKM2 can adopt different oligomeric states: an active tetramer and a less active dimer/monomer. Active tetrameric wtPKM2 promotes glycolysis and ATP production. In contrast, a reduction in wtPKM2 activity, due to dimerization, results in reprogramming of the glycolytic flux where glucose-derived carbons are diverted towards biosynthesis of nucleic acids, lipids, and amino acids2 required for cancer cell growth and proliferation.4 The activity and oligomeric state of wtPKM2 are subject to complex allosteric regulation. Various metabolites (fructose-1,6bisphosphate

(FBP),5

succinyl-5-aminoimidazole-4-carboxamide-1-ribose

5’-phosphate

(SAICAR) 6, 7-8 amino acids (serine,9 phenylalanine,10 alanine, tryptophan,11 and cysteine12), and

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post-translational

modifications

(phosphorylation,13-15

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acetylation,16

oxidation,17

and

hydroxylation18 regulate the activity of wtPKM2. wtPKM2 is activated by serine, SAICAR, and the upstream glycolytic intermediate FBP. In contrast, a number of ligands and post-translational modifications, including alanine, ATP, and a thyroid hormone T3, inhibit wtPKM2 activity by causing subunit dissociation and thus converting tetrameric wtPKM2 into a dimeric/monomeric state.19 Dimeric wtPKM2 is translocated into the nucleus and exhibits various non-glycolytic functions.2, 20 Inside the nucleus, dimeric wtPKM2 interacts with multiple proteins, including transcriptional factors such as, HIF1a,18,

21

and b-catenin,14 and acts as a transcriptional

coactivator, thus regulating the transactivation of HIF1a and b-catenin target genes. In addition, dimeric wtPKM2 is known to use PEP to phosphorylate STAT3 and histone H3.22 With the exception of SAICAR and phenylalanine, most allosteric effectors modulate wtPKM2 activity through a mechanism that involves stabilization or destabilization of the tetrameric state. SAICAR has been shown to activate wtPKM2 in its dimeric form.6 Using the variant, G415R PKM2 that remains dimeric in solution, Yan et al 6 demonstrated that SAICAR binds to the less active dimeric variant and fully activates it without resulting in tetramer formation. In contrast, when phenylalanine binds to wtPKM2, a conformational change occurs shifting the equilibrium from an active R-state to an inactive T-state while maintaining the tetramer.

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The

presence of phenylalanine, however, lowers the substrate (PEP) binding affinity of wtPKM2. This allosteric inhibition effect of phenylalanine has also been observed on rabbit muscle PKM1.23 On the basis of ligand binding and kinetic studies of PKM1 with various amino acids, it has been proposed that main chain carboxylate and amine functionalities of the amino acids are primarily responsible for binding to PKM1 and groups beyond the β-carbon are responsible for eliciting the allosteric signal. 23 While PKM1 is poorly inhibited by phenylalanine with a Ki of 1

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Biochemistry

mM at pH 9.0, smaller amino acids such as alanine, cysteine, and serine did not have any influence on PKM1 activity.23 However, both phenylalanine and serine inhibit and activate wtPKM2 with an IC50 of 0.24 mM and AC50 of 1.3 mM respectively. 10 In contrast to the phenylalanine bound structure of wtPKM2 (PKM2-Phe), the binding of serine did not cause any noteworthy conformational change in the wtPKM2-Ser complex. Based on these structures, it has been proposed that the bulk of phenylalanine introduces steric clash that is responsible for the observed R-to-T conformational change.10 However, serine owing to its small size, fits well within the amino acid binding pocket without posing any steric constraints and maintains wtPKM2 in the active tetrameric state. Using cell-based assays and native SDS-PAGE and immunoblot analysis with mouse wtPKM2, it has been demonstrated that cysteine inhibited wtPKM2, with an IC50 of 0.059 mM, and this inhibition was attributed to subunit dissociation converting the active tetramer to a less active dimer/monomer form.12 Considering the similarity in the overall size of cysteine and serine, it is puzzling how cysteine causes a shift in the tetramer-to-dimer state and inhibits wtPKM2 activity, whereas serine does not appear to cause any conformational change,9 but serves as an activator. The mechanism underlying the allosteric regulation of wtPKM2 by cysteine and serine remains unclear and the findings described above indicates that additional factors, extending beyond bulk or size, might be responsible for modulating wtPKM2 activity. In the present study, we sought to gain insight into the mechanism of differential regulation of wtPKM2 activity by serine and cysteine. Kinetic studies of wtPKM2 and two variants, FBPfree S437Y PKM2 and the dimeric G415R PKM2, indicate that cysteine inhibits wtPKM2, but FBP is able to reverse the inhibitory effect of cysteine. Ligand binding studies by fluorescence and isothermal titration calorimetry (ITC) suggest that the presence of cysteine decreased the binding

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affinity of wtPKM2 for PEP and the presence of PEP reduced the affinity of wtPKM2 for cysteine, which indicated the presence of a functionally bidirectional coupling mechanism. The finding that the bidirectional coupling occurs between the amino acid site and the active site was validated with serine. Not surprisingly, we observed that the presence of PEP indeed enhanced the affinity of wtPKM2 for serine, and vice versa, which is consistent with previous kinetic studies that demonstrated that serine activated wtPKM2 by lowering the Km for PEP.9 To gain structural insights into the mechanism of wtPKM2 inhibition by cysteine, the crystal structure of wtPKM2 in complex with cysteine (PKM2-Cys) was determined at 2.25 Å resolution. To rule out the possibility of crystal packing and FBP influencing the oligomeric state of wtPKM2, crystal structures of the FBP-free variant, S437Y PKM2, were determined in presence of either cysteine or serine. Crystals of the complexes of S437Y PKM2-Cys and S437Y PKM2Ser were obtained by soaking cysteine or serine to apo-S437Y PKM2 crystals, whose structure was previously determined.24 However, the structures of S437Y PKM2 with either cysteine or serine bound were quite similar with no noticeable conformational change. The solution structure of PKM2-Cys complex was analyzed by size-exclusion chromatography in line with small angle X-ray scattering (SEC-SAXS). However, due to higher concentration of wtPKM2 employed for the SAXS studies, PKM2-Cys complex remained in the tetrameric state. Further gel filtration studies using low concentrations of wtPKM2 in the presence of cysteine showed a shift in the oligomeric state from tetramer to a mixture of tetramer and dimer/monomer equilibrium, indicating cysteine inhibited wtPKM2 by causing a conformational change. The oligomeric state of S437Y PKM2 is an equilibrium of dimer and monomer with some tetramer as observed in gel filtration studies and as anticipated, it remains unchanged in the presence of cysteine or serine. These studies lead to the conclusion that cysteine inhibits wtPKM2 by causing a conformational change that

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Biochemistry

involves the conversion of the tetramer to a mixture of dimer/monomer equilibrium and tetramer.

RESULTS AND DISCUSSION Cysteine mediated wtPKM2 inhibition is reversible and dependent on FBP. To understand the mechanism of differential regulation of wtPKM2 by small amino acids, enzyme activity studies were performed with wtPKM2 and PKM2 variants in the presence of cysteine, serine, and FBP. The concentration of PEP was varied in the presence of different cysteine concentrations while keeping ADP concentration fixed at 0.8 mM. Cysteine inhibits wtPKM2 in a concentration dependent manner, with maximum inhibition observed at ~40 μM cysteine (Figure 1A). The data was replotted using the Lineweaver-Burk equation to show the change in enzyme activity in response to changing cysteine concentration (Figure S1). At 0.5 mM PEP, cysteine decreased the activity of wtPKM2 by ~3 fold (Vi of 0.25 mM/min, without cysteine vs. 0.093 mM/min, with 60 µM cysteine), whereas an increase in the activity of wtPKM2 was observed in the presence of serine (with Vi increasing from 0.02 mM/min to 0.034 mM/min) (Figure S2).

Figure 1. Effects of cysteine and FBP on the activities of wtPKM2 and cysteine inhibited wtPKM2 respectively. In all pyruvate kinase assays, the concentration of PEP was varied at different cysteine concentrations while ADP and wtPKM2 concentrations were kept constant at 0.8 mM and 50 nM respectively. (A) Kinetics of as-isolated wtPKM2 in the presence of varying 7


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concentration of cysteine, with Vi decreasing from 0.25 mM/min (without cysteine) to 0.093 mM/min (60 μM cysteine), at 0.5 mM PEP. (B) The effect of FBP on wtPKM2 inhibition by cysteine was evaluated using increasing concentration of FBP. Activity assays were performed in the presence of 1 mM cysteine and 0.8 mM ADP while varying PEP concentration at different concentrations of FBP. The kinetic parameters determined with 10 μM FBP: kcat/Km = 1.36*109 M-1 s-1; kcat = 5.1*103 min-1; Vmax = 0.17 mM/min; Khalf = 63 μM. Colors: 0 µM FBP (black), 1 µM FBP (magenta), 2 µM FBP (blue), 2.5 µM FBP (orange), 3 µM FBP (purple), 4 µM FBP (green), 6 µM FBP (cyan), 10 µM FBP (red). FBP is known to often co-purify with wtPKM2 with variable occupancy due to its high affinity for wtPKM2 (Kd ~ 0.21 μM). 6 To investigate if FBP concentration is important in restoring the activity of cysteine inhibited wtPKM2, kinetic studies were carried out in the presence of variable FBP concentration with excess cysteine (1 mM). Interestingly, FBP increased the activity of cysteine inhibited wtPKM2 in a concentration dependent manner (Figure 1B). These results indicate that cysteine mediated wtPKM2 inhibition is reversible and depends on the availability of FBP. To gain insight into the binding affinity of wtPKM2 for cysteine in the presence and absence of FBP, isothermal titration calorimetry (ITC) was used (Table 1). Cysteine binds wtPKM2 with a Kd of ~5 µM and the interaction is both entropically (TDS of -6.4 kcal/mol) and enthalpically (DH of -13.6 ± 0.6 kcal/mol) driven (Figure 2A). Binding studies in the presence of FBP shows that FBP reduced the affinity of wtPKM2 for cysteine (Kd of ~31 µM) by ~6 fold, which is consistent with the observed rescue effect of FBP on cysteine-inhibited wtPKM2 (Figure 1B). Table 1. Thermodynamic parameters obtained by isothermal titration calorimetry for wtPKM2, S437Y PKM2, and G415R PKM2 to cysteine, serine, and FBP binding in the presence and absence of various ligands. Ligand (Concn., mM)

Titrant

Kd (µM)

8

DH (kcal/mol)


TDS (kcal/mol)

n

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Biochemistry

wtPKM2

G415R PKM2

S437Y PKM2

⎯

Cysteine

5.5 ±0.5

-13.6 ± 0.6

-6.4

0.6±0.02

FBP (1)

Cysteine

31.2±2.78

-6.5±0.26

-0.39

1

⎯

FBP

0.0104 ±0.002

-14.27±0.11

-3.3674

0.950±0.00 3

PEP (1) + MgCl2 (5)

Cysteine

19.8 ±3.3

-5.17 ± 0.32

1.24564

1

Oxalate (2) +MgCl2 (10)

Cysteine

27.8±3.8

-4.11±0.234

2.1009

1

MgCl2 (5)

Cysteine

8.4±1.4

-8.2±0.74

-1.26

0.895±0.06

⎯

Serine

147 ± 24

--8.95±0.997

-3.725

1

PEP (1) + MgCl2 (5)

Serine

91.7±14.6

-4.93±0.35

0.57216

1

⎯

Cysteine

4.61±0.49

-11.3 ± 0.4

-3.9932

0.95±0.02

-

FBP

1.66±0.55

-5.89±0.35

-1.99

0.92±0.04

FBP (1)

Cysteine

7.14±0.95

-11.9±0.76

-4.9

0.9±0.04

⎯

Cysteine

7.04±1.83

-18.01±3.1

-10.9

0.67±0.09

Effect of cysteine on dimeric PKM2 variants, S437Y and G415R. As FBP is known to promote the active tetrameric state of wtPKM2, we hypothesized that FBP restores the activity of cysteine inhibited wtPKM2 by causing tetramerization. To understand the effect of cysteine in the absence of FBP, the dimeric variant of PKM2, S437Y, incapable of binding FBP, was used. In addition, a C-C interface variant, G415R PKM2, which binds FBP but remains in the dimeric state,6 was also

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examined for the inhibitory effect of cysteine.

Figure 2. Cysteine binds wtPKM2 and influences the activity of FBP-free S437Y PKM2 and the dimeric variant, G415R PKM2. (A) ITC data for a representative titration of cysteine into wtPKM2. 20 µM wtPKM2 was titrated with first injection of 0.4 µl cysteine (0.4 mM) and 19 consecutive injections of 2 µl each. The top panel shows the raw measured heat changes versus time. The area under each spike is proportional to the heat produced with each injection. The lower panel shows the integrated areas normalized to the number of moles of wtPKM2 injected at each injection step. The data were fitted to a single-binding site model, and the resulting thermodynamic parameters for the complex formation are summarized in Table 1. (B) Kinetics of FBP-free S437Y

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Biochemistry

PKM2 with varying concentrations of cysteine. Cysteine inhibits S437Y PKM2 (50 nM) significantly, with 20 µM cysteine decreasing the Vi from 0.46±0.02 to 0.28±0.01 mM/min, at 3 mM PEP. (C) Fold inhibition of wtPKM2, G415R PKM2, and S437Y PKM2 with increasing concentration of cysteine. The concentration for all the enzymes were kept constant at 50 nM. (D) Increase in the activity of cysteine inhibited wtPKM2 (50 nM) in the presence of FBP. FBP increases the activity of wtPKM2 by about 6-fold. Inset: Cysteine inhibited G415R PKM2 (50 nM) and S437Y PKM2 (50 nM) were titrated with increasing concentration of FBP and the data shows that FBP does not rescue the variants from cysteine inhibition. The kinetics data show that cysteine inhibits S437Y PKM2 significantly, with 20 µM cysteine decreasing the Vi from 0.46±0.02 to 0.28±0.01 mM/min, at a PEP concentration of 3 mM (Figure 2B). Whereas, both wtPKM2 and G415R PKM2 are inhibited to a similar extent (Figure 2C), with 80 µM cysteine inhibiting S437Y PKM2 and wtPKM2/G415R PKM2 by about 4 fold and 3 fold respectively. These studies indicate that cysteine inhibits S437Y PKM2 much more effectively than wtPKM2 or G415R PKM2, which can be attributed to the fact that FBP is unable to bind S437Y PKM2, thus leading to a more pronounced inhibition effect unlike wt and G415R PKM2 that can bind FBP. Further, the effect of FBP on the activity of cysteine inhibited variant enzymes were examined. While FBP was able to increase the activity of cysteine inhibited wtPKM2 by ~6-fold, it is incapable of restoring the activities of G415R and S437Y variants from cysteine inhibition (Figure 2D). These results support the hypothesis that FBP restores the activity of cysteine inhibited wtPKM2 by promoting the tetrameric state of wtPKM2. The binding affinities of the dimeric PKM2 variants, G415R and S437Y, for cysteine were similar to that of wtPKM2, as determined by ITC (Table 1). The presence of FBP has little to no effect on cysteine binding affinity of G415R PKM2 (Kd values of ~4.6 and ~7 µM without and with FBP respectively), unlike wtPKM2, where the affinity to cysteine decreases in the presence of FBP. While both wtPKM2 and G415R PKM2 can bind FBP, the variant is not influenced by

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FBP to undergo tetramerization unlike wtPKM2. This confirms that the mechanism of FBPmediated activation of cysteine-inhibited wtPKM2 is by stabilization of the tetrameric state.

Cysteine inhibits wtPKM2 by altering PEP binding. PKM2 is known to display positive homotropic cooperativity for substrate PEP and most allosteric modulators affect PEP binding. 5 In order to gain a mechanistic understanding of how cysteine influences substrate binding affinity of wtPKM2, ligand binding studies were conducted by monitoring changes in the intrinsic tryptophan fluorescence. Using wtPKM2, the binding affinities for ADP and PEP were determined in the presence and absence of cysteine. As expected, cysteine does not influence ADP binding affinity of wtPKM2 (Kd of ~0.9 mM, with and without cysteine) (Figure 3A).

Figure 3. Cysteine affects the binding of PEP to wtPKM2. (A) Binding of ADP in the absence (black) and presence (magenta) of 1 mM cysteine, showing cysteine does not influence ADP binding to wtPKM2.(B) Binding of PEP in the absence (black) and presence (magenta) of 1 mM cysteine. Cysteine binding alters the Khalf of PEP from 46 μM to 329 μM, with an increase in the Hill coefficient (h) from 1.1 to 2, thereby suggesting that binding of PEP to wtPKM2 is cooperative in nature. The concentration of wtPKM2 was fixed at 1.6 μM in all experiments. The results further indicate that wtPKM2 binds ADP non-cooperatively and ADP binding is insensitive to allosteric regulation by cysteine (Figure 3A). Whereas, the presence of 1 mM 12


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Biochemistry

cysteine reduces the affinity of wtPKM2 for PEP, with Khalf increasing from 46±11 μM to 329±59 μM. In addition, the Hill coefficient (h) is increased from 1.1 to 2, indicating PEP binds with positive cooperativity (Figure 3B). These results indicate that cysteine inhibits wtPKM2 activity by affecting wtPKM2-PEP binary complexation.

Functionally bidirectional allosteric coupling between the amino acid binding site and the active site of wtPKM2. Many allosteric processes are known to be intrinsically bidirectional in nature.

25-26

To

further examine potential communication between amino acid and substrate binding sites, additional binding studies were conducted. The presence of cysteine lowered the PEP binding affinity of wtPKM2 as described in the previous section (Figure 3B). To probe the affinity of wtPKM2 for cysteine in presence of PEP ligand binding studies were conducted using ITC (Table 1). Interestingly, PEP reduced the affinity of wtPKM2 for cysteine by about ~4 fold (Kd values of ~6 µM and ~20 µM in absence and presence of PEP respectively). In addition, the PEP mimic, oxalate alters the cysteine binding affinity of wtPKM2 by ~6 fold, which might be due to the high binding affinity of oxalate for wtPKM2. These results indicate that the carboxylate moiety of PEP is possibly involved in allosteric coupling. Furthermore, the affinity of wtPKM2 for cysteine was only slightly perturbed in the presence of magnesium (Kd ~8 µM), suggesting that magnesium ion and corresponding active site residues (Glu272 and Asp296) associated in magnesium binding are probably not involved in allosteric communication. 23 To understand the mechanism of differential regulation effects of cysteine and serine and to further investigate the functionally bidirectional coupling effect, serine binding affinity of wtPKM2 was explored in the presence and absence of PEP using ITC. While the presence of PEP

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or oxalate lowered the affinity of wtPKM2 for cysteine, PEP promoted the affinity of wtPKM2 for serine, with the Kd shifting from ~147 µM (without PEP) to ~92 µM (with PEP) (Table 1). These results further confirm that cysteine inhibits wtPKM2 by reducing PEP binding affinity, but serine activates wtPKM2 by enhancing the affinity for PEP. It has been reported that, for yeast pyruvate kinase, a PEP analogue and competitive inhibitor of pyruvate kinase, phosphoglycolate, acts as an activator at sub-saturating PEP concentrations, likely by promoting the active conformation of pyruvate kinase.

27

Therefore, based on these findings, it is reasonable to conclude that when

oxalate (or PEP) binds to wtPKM2, it promotes the active tetrameric conformation that is more amenable to binding serine rather than cysteine.

Crystal structure of PKM2-Cysteine Complex. To understand the molecular mechanism of inhibition of wtPKM2 by cysteine, crystals of wtPKM2 with bound cysteine (PKM2-Cys) were obtained by co-crystallization of wtPKM2 and cysteine (PDB:6NU1). The structure of PKM2-Cys complex was determined in the P1211 space group at 2.25 Å resolution with Rwork and Rfree of 19.1 % and 23.5 % respectively (Figures 4 and S3, Table 2). There are four molecules in the asymmetric unit (asu) and each monomer contains one molecule of bound cysteine, oxalate, Mg2+, and FBP (known to co-purify with recombinant wtPKM2) 8 (Figure 4A). The overall structure of a PKM2Cys tetramer is similar to the structures of wtPKM2 in complex with FBP (PKM2-FBP, PDB: 1T5A and 3SRD) (Figure S3A and B), or, serine (PKM2-Ser, PDB:4B2D) (Figure S3C), or phenylalanine (PKM2-Phe, PDB:4FXJ) (Figure S3D). Structural alignment of a PKM2-Cys monomer over 430 Cα atoms, excluding the B-domain, with monomers of PKM2-FBP, PKM2Ser, PKM2-Phe resulted in rmsd values of 0.323 Å (PDB:1T5A), 0.215 Å (PDB:3SRD), 0.284 Å (PDB:4B2D), and 0.521 Å (PDB:4FXJ). Alignment of residues around 5 Å from the cysteine

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Biochemistry

ligand of PKM2-Cys structure with the corresponding residues of the amino acid free PKM2-FBP (PDB:1T5A) shows little to no structural differences (Figure S4A).

Figure 4. Crystal structure of PKM2-Cys complex. (A) Overall structure of a PKM2-Cys monomer (chain D). Cysteine binds at the bottom of α8-β8 barrel of domain A and in the interface of A and C domains. Oxalate bound at the active site is on the opposite end of the α8-β8 barrel between the clefts of A and B domains. Colors: domain B, green; domain C, wheat; domain A, cyan with β-strands in magenta; with ligands being O, red; S, yellow, C, green. Cysteine, FBP, and oxalate are shown as spheres. (B) Close-up view of cysteine bound wtPKM2 (cyan), where cysteine is bound in the amino acid binding pocket of wtPKM2. PKM2-Cys complex superimposed on apo-PKM2 (tan) (PDB:1T5A), with S in yellow, O in red, N in blue, C in cartoon color. Cysteine and residues involved in hydrogen bond interactions are shown as sticks. The FoFc omit map is contoured at 3σ and the omit map (green mesh) of the cysteine indicating the position of the sulfur is contoured at 8σ. Table 2: Data collection and refinement statistics.

PDB code Space group Unit cell dimensions a b c (Å) α β γ (deg) Beamline Wavelength (Å) Oscillation range (deg) Resolution range (Å)a

PKM2-Cys 6NU1 P1211

S437Y-Cys 6NU5 C121

S437Y-Ser 6NUB C121

81.83 155.92 94.14 90 103.51 90 APS 19-ID 0.9786032 180

110.0 94.13 109.26 90 95.61 90 ALS 4.2.2 1.000030 180

29.70-2.25 (2.372.25)

41.98-1.60 (1.69-1.60)

110.11 94.23 109.13 90 95.63 90 ALS 4.2.2 1.000030 180 41.96-1.70 (1.79-1.70)

15


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Page 16 of 43

Observations reflections a Unique reflections a Redundancy a CC1/2 a Rpim a Completeness (%)a I/σ (I)a Rmerge (%)a Rmeas (%)a Wilson B factor (Å2) Refinement Rwork (%)a, c Rfree (%)a, d No. of Reflectionsa No. of Molecules per asu Number of Atoms: Protein Cysteine Serine FBP Oxalate Magnesium Potassium Water Thiocyanate 1, 2-ethanediol Tetraethylene glycol Polyethylene glycol Bis-Tris propane Average B-Factor (Å2) Protein

388129 (58736) 106138 (15673) 3.7 (3.7) 0.993 (0.768) 0.084 (0.448) 97.8 (99.2) 10.2 (3.5) 8.7 (46.6) 12.7 (61.9) 33.57

1016922 (131889) 145145 (20906) 7.0 (6.3) 1.000 (0.897) 0.029 (0.267) 99.6 (98.4) 25.2 (3.2) 4.7 (41.2) 5.7 (50.1) 14.7

848148 (101409) 121214 (17221) 7.0 (5.9) 0.999 (0.947) 0.022 (0.140) 99.6 (97.3) 25.9 (4.9) 5.4 (31.2) 5.9 (34.3) 13.3

19.1 (23.6), 19.1e 23.5 (30.3), 23.5e 106046 (10721) 4

16.6 (21.7), 16.6e 18.2 (24.5), 18.2e 145131 (14182) 2

15.4 (18.7), 15.3e 17.4 (20.9), 17.3e 121211 (11670) 2

14366 28 80 24 4 2 616 -

8018 14 -

8093 14 38 12 2 2 1017 15 4 13 7 38

39.9, 39.9e

Cysteine

33.8

18.2, 18.1e 12.1

Serine

-

FBP

48.3

Oxalate

40.2

Magnesium

31.5

Potassium

68.9

12 2 2 971 6 8 38

12.9 11.5 12.7 16


16.6, 16.6e 10.4 11.4 10.3 11.1

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Biochemistry

a

Water

39.7

27.5 28.4

26.0 27.5

Thiocyanate

-

1, 2-ethanediol

-

20.8

19.7

Tetraethylene glycol

-

-

Polyethylene glycol

-

-

Bis-Tris propane

-

25.3

28.1

RMSD Bond Lengths (Å) RMSD Bond Angles (deg) Ramachandran favored (%) Ramachandran allowed (%) Ramachandran outliers (%)

0.003, 0.003e

0.006, 0.006e

0.007, 0.007e

0.602, 0.606e

0.797, 0.798e

0.858, 0.862e

97.30, 97.30e

98.16, 98.16e

98.4, 98.4e

2.39

1.55

1.45

0.3, 0.3e

0.29, 0.29e

0.19, 0.19e

32 29.6

Values in parentheses are the highest-resolution shell. bRmerge = ΣΣj|Ihj - |/ΣΣjIhj Rmeas = Σ√(n/(n-1)) Σj|Ihj -

|/ΣΣjIhj multiplicity-weighted Rmerge. cRwork= Σh{|Fo|-|Fc|}/Σh|Fo| Where Fo and Fc are the observed and calculated structure factor amplitudes respectively. dRfree = as R but for an independent 5% test set of reflections excluded from the modelling and refinement process. edenotes values obtained from molprobity. asu, asymmetric unit.

The PKM2-Cys structure shows that cysteine is located at the amino acid binding pocket previously reported for serine,

9

phenylalanine,

10

and other amino acids (Figure 4).

9-10, 23

The

carboxylate group of cysteine makes direct contact with the sidechains of Asn70 and Arg106, but interacts with the backbone carbonyl group of Arg43 through water-mediated hydrogen bonding. The amine group of cysteine interacts directly with the sidechain of His464 and backbone carbonyl of Ile469, in addition to forming water mediated hydrogen bonds with backbone carbonyl groups of His464 and Tyr466. The binding of cysteine resulted in a subtle change in the amino acid binding pocket, with Arg106 flipping inwards to interact with the carboxylate group of cysteine 17


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(Figure 4B). Because main chain carboxylate and amine moieties of amino acid ligands are required for binding, as demonstrated for PKM1,23 it is not surprising that cysteine binds wtPKM2 similar to other amino acids, such as serine and phenylalanine. Both cysteine and serine are anchored to wtPKM2 through multiple hydrogen bond interactions. While the thiol group of cysteine is not involved in any hydrogen bond interaction, the sidechain hydroxyl of serine forms direct and water mediated hydrogen bonds with backbone carbonyl of Ile469 and Asn44 respectively (Figures 4 and S4B).

9

Regardless of their size similarity, cysteine and serine exhibit differential effect on wtPKM2 activity. An overlay of PKM2-Cys and PKM2-Ser structures show that there is no conformational change associated with the binding of the two amino acids and residues around the active site and amino acid binding site superimpose well (Figure S3C). The distance between the active site and the amino acid binding site is ~24 Å. One explanation for the differential regulation of wtPKM2 activity can be attributed to the additional hydrogen bond interactions with serine, which might be responsible for altering the dynamics of residues in the amino acid binding pocket (Figures 4B and S4B). Given that the less polar cysteine binds wtPKM2 in a largely hydrophilic and polar pocket comprising of flexible amino acids, such as, arginine and glutamine, it is conceivable that cysteine binding to wtPKM2 could perturb the dynamics of polar amino acids located in the amino acid binding pocket. Nonetheless, both cysteine and serine results in a longrange allosteric effect upon binding to wtPKM2, where the signal is being transmitted to the active site, causing a decrease and increase in the affinity of PEP respectively. Although, the specific residues involved in the long-range allosteric communication are unknown, one can speculate that the two β-strands that connect the active site and the amino acid binding site might be involved in the allosteric regulation of wtPKM2 by amino acids (Figure 5A).

18


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Biochemistry

Figure 5. (A) A proposal for long-range allosteric communication between the active site (left) and the allosteric site (right), based on the crystal structure of PKM2-Cys complex. Oxalate present in the active site can be involved in long-range communication with Cys located in the allosteric site via K270, D113, β-strand, V108, and R106. Another potential communication route between oxalate and Cys might involve T328, N75, β-strand, and N70. (B, C) Structures of the complexes of S437Y PKM2 with: (B) cysteine (S437Y-Cys) and (C) serine (S437Y-Ser). 2Fo-Fc electron density maps, contoured at 1s are shown around cysteine and serine. Colors: O in red, N in blue, S in yellow, C in backbone color. Mg2+, K+, and water molecules are shown as green, purple, and red spheres respectively. The dashed lines represent hydrogen bonds.

19


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

It has been suggested by Morgan et al 10 that Arg342 plays an important role in allosteric communication by stabilizing α6’ helix in the active site. The backbone atoms of two residues (Gly295 and Asp296) located in the α6’ helix make hydrogen bonding interactions with the carboxylate group of oxalate (PDB: 1T5A) or, PEP (PDB: 4HYV). In addition, Thr328 also interacts with the carboxylate group of PEP/oxalate while Lys270 interacts with the enol oxygen of PEP (Figure 5A).23 All these residues in the active site are conserved among PKM1, PKM2, PKL/R, yeast pyruvate kinase, E. coli pyruvate kinase, and Trypanosoma pyruvate kinase. In the active site of wtPKM2, the carboxylate of oxalate also interacts with Lys270, which in turn interacts with Asp113.23 Asp113 is the first amino acid in a string of residues, which connects the active site to Arg106 located in the amino acid binding site. Arg106 interacts with cysteine via a H-bond (Figure 5A). Likewise, Asn75 located near the active site interacts with oxalate via Thr328 and is connected to Asn70 in the amino acid binding site by a β-strand. Asn70 also forms H-bond interaction with cysteine. Thus, the ITC data along with PKM2-Cys crystal structure (Table 1 and Figure 5A) suggest that residues interacting with the carboxylate and enol oxygen of PEP might be involved in allosteric communication between active site and amino acid binding site.

Furthermore, it has been shown that when phenylalanine binds wtPKM2, it causes a rigid body rotation of a monomer, consequently forming the inactive, less compact tensed state (T-state) of wtPKM2. 10 The PKM2-Phe structure (PDB:4FXJ) displayed significant conformational change in the monomer, which is associated with the rigid body rotation of A and C domains.10 Whereas, in cysteine and serine bound structures of wtPKM2 no such conformational change was observed. A close inspection of the amino acid binding site of wtPKM2 indicates that the bulk of phenylalanine sidechain causes significant steric crowding, but smaller amino acids such as

20


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Biochemistry

alanine, serine,9 and cysteine can bind wtPKM2 without introducing any significant steric clash. On the basis of kinetic studies of PKM1 with amino acids, it has been suggested that moieties beyond the β-carbon of amino acids are primarily responsible for allostery because smaller sidechain containing amino acids such as alanine, cysteine, or serine did not elicit any effect on the activity of PKM1, unlike phenylalanine, which inhibits the enzyme.23 Because PKM1 and PKM2 differ only by 22 out of 531 amino acids within a 56-aa region covering residues 378-434, and the sequence differences are only confined to the dimer-dimer interface, therefore, it is conceivable that cysteine inhibits wtPKM2 by a mechanism, different from that adopted by phenylalanine, potentially involving the dimer-dimer interface. Indeed, our gel filtration data shows that wtPKM2 is converted from its native tetrameric form to a mixture of tetramer and dimer/monomer equilibrium in the presence of cysteine (Figure 6A). The monomer and dimer merge into one peak because of a rapid equilibrium between the two species. The tetramer and monomer equilibrium occurs in 15 min.11 However, the rate of the equilibria between dimer and monomer remains to be determined.

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Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 6. Gel filtration and SEC-SAXS data demonstrating the effect of cysteine binding to wtPKM2. (A) Gel filtration chromatograms depicting increased dimer/monomer formation upon increasing Cys concentration. An overlay of 0.5 mg/ml wtPKM2 in the absence of Cys (a, black), presence of: 50 µM Cys (b, red), 2 mM Cys (c, blue), and 5 mM Cys (d, green). The standards (158 kDa and 44 kDa) along with the theoretical mobility of wtPKM2 dimer (120 kDa) and monomer (60 kDa) are shown as ticks on the upper axis. (B) Guinier plot of wPKM2 with different ligands, showing slight differences in the slope corresponding to difference in radius of gyration (Rg). (C) Comparison of x-ray scattering profile of wtPKM2 (black), and complexes of wtPKM2Cys (blue), wtPKM2-FBP (green), and wtPKM2-FBP-Cys (magenta). Each curve represents an average of 10 different curves collected around the peak from gel filtration chromatogram. (D) Pair distance distribution (Pr) of wtPKM2 and the complexes, showing only slight difference in maximum dimensions (Dmax) beyond 80 Å. For SAXS experiments, the concentrations of wtPKM2, cysteine, and FBP were kept at 12.5 mg/ml, 2 mM, and 0.5 mM respectively.

22


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Biochemistry

It has been proposed that wtPKM2 effectors modulate its activity by switching it from an active tetramer to a less active dimer/monomer, and various activators and inhibitors have been shown to bind wtPKM2 at different sites.

5, 10, 23, 28-32

Structural comparison of the PKM2-Cys

complex with previously reported amino acid-free PKM2-FBP structures (PDB:1T5A, 3SRD) display quaternary structure similarities, with little to no difference in the relative orientation of various tetrameric subunits (Figure S3A, S3B). These cysteine-free PKM2-FBP structures contain various bound ligands, such as oxalate, Mg2+, and FBP, similar to the PKM2-Cys structure. The overall structural similarity, including subtle differences observed between PKM2-Cys and PKM2-FBP structures might be due to the difference in the space groups (P1211 for PKM2-Cys and 3SRD versus P212121 for 1T5A). While it is proposed that cysteine binding causes wtPKM2 subunit dissociation in cells, 12 the lack of any quaternary structural changes in PKM2-Cys might be a consequence of crystal packing. Because FBP is able to reverse the inhibitory effect of cysteine and given recombinant wtPKM2 co-purifies8 and crystallizes with bound FBP, to rule out the possibility of FBP influencing the tetrameric structure of PKM2-Cys, the effect of cysteine and serine were investigated using the FBP-free variant, S437Y PKM2 in the same crystal packing environment. The crystal structures of S437Y PKM2 in complex with cysteine (S437Y-Cys, PDB:6NU5) or serine (S437Y-Ser, PDB:6NUB) were determined at 1.6 and 1.7 Å resolution (Figure 5B and C, Table 2) by soaking respective ligand into the amino acid-free S437Y PKM2 (apo-S437Y) crystallized in the C2 space group (PDB:6B6U).

24

The overall structures of S437Y-Cys and

S437Y-Ser are tetrameric as generated by crystallographic symmetry and closely resemble that of apo-S437Y. 24 Structural alignment of monomers of S437Y-Cys and S437Y-Ser with apo-S437Y resulted in rmsd values of 0.108 Å and 0.133 Å respectively. Little to no difference was observed

23


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

in the structures of monomers and dimers of the amino acid bound complexes of S437Y PKM2 compared to that of apo-S437Y PKM2 structure, with highly similar B-factor values for all Cα and side chain atoms, thus confirming that FBP does not influence the PKM2-Cys structure. PKM2 exhibits positive cooperativity for one of its natural substrates, PEP (Figure 3B). It has been demonstrated that in the absence of FBP and at subsaturating PEP concentrations, a PEP structural analogue phosphoglycolate (PG), acts as an allosteric activator (by causing active conformation) of yeast pyruvate kinase.

33

Ligand binding studies presented here indicate that

oxalate/PEP, similar to PG, may promote the active tetrameric conformation of wtPKM2, which leads to decrease/increase in the affinity of wtPKM2 to cysteine/serine (Table 1). Given that the FBP-free structures of S437Y PKM2 have oxalate bound to the active site, it is possible that oxalate might be preventing the effect of cysteine and serine in causing any conformational changes of wtPKM2. Attempts to crystallize S437Y PKM2 without bound oxalate were unsuccessful. On the basis of our detailed biophysical analysis of apo-S437Y PKM2, 24 we believe that the observed tetrameric crystal structure of apo-S437Y is likely driven by a combination of high protein concentration employed for crystallization, crystallization solution used, and crystalpacking forces. Thus, the tetrameric form of S437Y PKM2 in the crystalline lattice could potentially exclude the possibility of cysteine and serine causing conformational changes.

Solution studies of PKM2-Cys complex. To probe if cysteine binding alters the conformational state of wtPKM2 in solution, wtPKM2 was examined by SEC-SAXS in the presence of cysteine, FBP, and FBP-cysteine (Figures 6B-D and S5). In addition, the cysteine bound form of the FBP-free variant, S437Y-Cys, was analyzed by SAXS (Figure S6). Preliminary analysis of scattering profiles indicated the absence of any aggregation and radiation damage.

24


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Biochemistry

Experimental X-ray scattering profiles of wtPKM2, wtPKM2-Cys, wtPKM2-FBP and wtPKM2FBP-Cys (Figure 6C) match well with the calculated scattering profiles of wtPKM2-Cys and cysteine-free FBP-wtPKM2 (PDB:1T5A) crystal structures respectively, indicating that under the given experimental conditions and concentrations of protein used, the overall solution and crystal structures are similar. A comparison of the experimental scattering profiles of wtPKM2 and the complexes of wtPKM2-Cys, wtPKM2-FBP, and wtPKM2-FBP-Cys show minor differences in the low scattering angle range, which is suggestive of little to no conformational changes occurring in solution (Figure S5A). The radius of gyration (Rg) calculated from Guinier analysis for all samples was in the range of 41.9 - 43.3 Å, which is consistent with that obtained from pair distribution function, confirming that wtPKM2-Cys is tetrameric in solution (Figure 6B, Table S1). The Guinier plot shows linearity in the low q range, suggesting the absence of aggregation and any interparticle interaction (Figure 6B). The Rg and the maximum dimension (Dmax) of wtPKM2-FBP complex are smaller than as-isolated wtPKM2 by about 2 Å and 10 Å respectively, suggesting that wtPKM2-FBP complex is more compact (Figure 6D, Table S1). For wtPKM2-FBP-Cys and wtPKM2-FBP complexes, both Rg and Dmax are very similar, which indicates that FBP maintains wtPKM2 in a tetrameric state, regardless of whether cysteine is present or not. However, there is a slight increase in Rg and Dmax of wtPKM2-Cys complex relative to as-isolated wtPKM2, indicating a less compact structure in solution when cysteine is present. Ab-initio shape reconstruction of wtPKM2 and complexes of wtPKM2-Cys, wtPKM2-FBP, and wtPKM2-FBPCys using DAMMIF resulted in low-resolution shapes. Docking of tetrameric PKM2-Cys crystal structure into the SAXS envelope generated for wtPKM2-Cys complex aligns well, thus confirming that wtPKM2 exists in tetrameric form in solution upon binding cysteine (Figure S5B and S5C), under the conditions employed for SAXS studies. It has been reported using small angle

25


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Page 26 of 43

neutron scattering that phenylalanine increases the Rg and Dmax of PKM1, and this increase is attributed to the open state of the B domain.

34

As described in the previous section, the crystal

structure of PKM2-Phe (PDB:4FXJ) shows that the presence of phenylalanine alters the conformation and orientation of A and C domains in the tetramer, causing wtPKM2 to adopt a less compact T-state. 10 Using the reported crystal structures of phenylalanine-bound (PDB:4FXJ) and phenylalanine-free PKM2 (PDB:4FXF), our calculations of Rg and Dmax results in higher values than when phenylalanine is bound to wtPKM2. Thus, the observed conformational changes in PKM2-Phe crystal structure leading to a less compact T-state appears to have a net effect on the increased Rg and Dmax compared to the active relaxed state (R-state) of wtPKM2. Our SAXS results indicate that the presence of cysteine slightly increased the Rg and Dmax of wtPKM2 and FBP has the reverse effect. We can only speculate that the increase in Rg and Dmax might be either due to an open state of B domain, or that wtPKM2 adopts a T-state similar to that of PKM2-Phe. It is likely that the mechanism of inhibition of wtPKM2 by cysteine and phenylalanine are different. Within the resolution limits of SAXS experiments, the results indicate that there might be minor conformational changes associated with cysteine binding. However, cysteine binding does not cause subunit disruption of wtPKM2 tetramer. It is reported that several ligands, mutations, and post-translational modifications modulate the activity of wtPKM2 by switching it from an active tetramer to a less active dimer/monomer.

10, 35

Our SAXS results combined with the our

reported biophysical studies 24 indicate that the tetramer-to-dimer/monomer shift is dependent on the concentration of wtPKM2. At low concentrations (~0.1 mg/ml), wtPKM2 is known to exist in the dimer/monomer form, 11 whereas, our SAXS studies have been done at relatively higher protein concentrations (~12.5 mg/ml). This concentration effect was validated with SEC studies using 0.5 mg/ml wtPKM2, revealing that the elution profile of wtPKM2 changes upon addition of cysteine

26


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Biochemistry

(Figure 6A). While wtPKM2 has an elution peak corresponding to an average molecular weight (MW) of 206 kDa, in the presence of 50 µM cysteine, a shoulder corresponding to a MW of 100 kDa appears in the chromatogram. The shoulder becomes more prominent when cysteine concentration is increased to 2 mM and subsequently to 5 mM (Figure 6A, Table S2). This result confirms that cysteine inhibits wtPKM2 by shifting the equilibrium from tetramer to a mixture of tetramer and dimer/monomer equilibrium. Previous studies suggest that wtPKM2 attains quaternary structure equilibrium in 15 minutes. 11 Therefore, to avoid dissociation of wtPKM2 in the gel filtration column, the enzyme was pre-incubated with and without cysteine for 15 minutes prior to the experiment. Moreover, as the expected elution time of wtPKM2 is ~30 minutes, it provides sufficient time for quaternary structure stabilization (Table S2). In order to avoid any effect of FBP, the FBP-free variant, S437Y PKM2, was used to understand how cysteine influences its oligomeric state in solution. The solution structure of S437Y PKM2-Cys was determined by SEC-SAXS and the results were compared with that of the dimeric apo-S437Y PKM2 published by us.

24

SAXS data analysis showed no significant

difference in the solution structure of S437Y PKM2 in the absence and presence of cysteine, indicating S437Y PKM2-Cys predominantly exists as a dimer under the experimental conditions of SAXS (Figure S6A & B). Size exclusion analysis of S437Y PKM2 at 0.5 mg/ml showed that the enzyme exists predominantly in an equilibrium between dimer and monomer with a very small population in the tetrameric state (Figure S6C). The MW of the tetramer and dimer/monomer peak was calculated to be 218 kDa and 89 kDa, respectively. The oligomeric state of the enzyme was unchanged in the presence of cysteine, which is consistent with the dimeric form observed in SAXS (Figure S6C, Table S2). The gel filtration results thus confirm that while cysteine shifts tetrameric wtPKM2 to a mixture of tetramer and dimer/monomer equilibrium (Figure 6A),

27


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

addition of cysteine to S437Y PKM2 does not have the same effect, as this variant pre-exists in a dimer:monomer equilibria. Incubation of S437Y PKM2 with serine did not alter the oligomeric state of the enzyme (Figure S6C, Table S2), even though serine has been shown to activate S437Y PKM2. 9 The size exclusion experiments of S437Y PKM2 with cysteine and serine lead us to the conclusion that while cysteine inhibits wtPKM2 by converting it to a mixture of tetramer and dimer/monomer equilibrium from tetramer, the mechanism of serine mediated wtPKM2 activation does not involve a change in the oligomeric state and perhaps follows a yet to be determined mechanism. A control gel filtration experiment with S437Y PKM2 and FBP shows the enzyme to be predominantly a dimer/monomer equilibrium (100 kDa ) with some tetramer fraction (243 kDa), which is expected as it is known that FBP is incapable of binding and activating S437Y PKM2 (Figure S6). 9, 36-37

Conclusions In summary, the biochemical and kinetic studies described here show that cysteinemediated inhibition of wtPKM2 occurs by shift in the active tetrameric conformation to a mixture of tetramer and dimer/monomer equilibrium. The inhibitory effect of cysteine was shown to be reversed by FBP. The mechanism of FBP dependent activation is demonstrated using the variants, FBP-free S437Y PKM2 and dimeric G415R PKM2, which shows that FBP activates cysteine inhibited wtPKM2 by promoting tetramerization. Ligand binding studies show that the cysteine mediated inhibition of wtPKM2 is due to reduced affinity for PEP. Furthermore, the presence of PEP lowered the affinity of wtPKM2 for cysteine, which illustrates the existence of a functionally bidirectional coupling mechanism between the active site and amino acid binding site. While crystal structures of wtPKM2 and S437Y PKM2 with cysteine/serine, and SAXS studies with

28


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Biochemistry

wtPKM2-Cys complex displayed a tetrameric conformation, gel filtration data with relatively lower concentration of the enzyme showed a clear shift from tetramer to a mixture of tetramer and dimer/monomer equilibrium in presence of cysteine, thus confirming that the high concentrations employed in crystallization and SAXS studies is responsible for shifting the equilibrium to the tetrameric state. In conclusion, the present study allowed us to decipher the mechanism by which cysteine inhibits wtPKM2. The study also raises the question as to why cysteine acts as an inhibitor while serine is an activator despite being similar in size and binding in the same allosteric pocket and in a similar orientation within the enzyme. Thus, further studies with other amino acids involving different functional groups might provide insight into the cause of differential regulation of wtPKM2.

Materials and Methods Human wild-type PKM2 and S437Y PKM2 mutant. Human PKM2 gene in pET28a vector was a generous gift from Matthew G. Vander Heiden. The plasmid was used as the template for site directed mutagenesis using mutagenic oligonucleotides yielding the S437Y PKM2 or G415R PKM2 mutant plasmid. Wild-type PKM2 (wt PKM2), S437Y PKM2, and G415R PKM2 mutants plasmids were transformed into E. coli BL21 (DE3) (Life Technologies, Grand Island, NY) for protein expression. Starter cultures were grown in Luria-Bertani (LB) media from single colonies and were used to inoculate 0.5 liter terrific broth (TB) media. The cells were grown at 37 °C/200 rpm to an optical density at 600 nm (OD600) of 1.2 and induced with 0.2 mM isopropyl-Dthiogalactopyranoside (IPTG). After induction, the cells were further grown at 22 °C/150 rpm overnight. Cells were harvested by centrifugation at 4,000 rpm for 20 min at 4 °C and the cell

29


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

pellet was re-suspended in 20 mM HEPES, pH 7.5, 300 mM NaCl, 5 % glycerol (buffer A). The cell suspension was lysed by sonication and the cell debris were cleared by centrifugation at 18,000 rpm for 30 minutes. His6-PKM2 (both wt and mutant forms) were purified using an immobilized Ni-affinity resin pre-equilibrated with buffer A. The column loaded with protein was first washed with buffer A followed by buffer A containing 30 mM imidazole. The protein was eluted with buffer A containing 300 mM imidazole, analyzed by SDS-PAGE for purity, and pure fractions were dialyzed overnight in buffer containing 20 mM HEPES, pH 7.5, 100 mM KCl, 5 % glycerol, 0.5 mM tris(3-hydroxypropyl)phosphine (THP) (storage buffer). Protein samples were concentrated and loaded onto a HiLoad Superdex 200 16/600 gel filtration column (GE Healthcare) pre-equilibrated with storage buffer.

Pyruvate Kinase Activity Assays. wtPKM2 activity was measured with wtPKM2-lactate dehydrogenase coupled assay using an Epoch microplate spectrophotometer (BioTeK, Winooski, VT). The assays were performed in the presence of 20 mM HEPES, pH 7.5, 100 mM KCl, 5 mM MgCl2, 4 units/ml lactate dehydrogenase, 0.4 mM NADH, and either 50 nM wtPKM2, S437Y PKM2, and G415R PKM2. Varying concentrations of PEP and cysteine were used at a fixed concentration of ADP (0.8 mM). For the inhibition and activation studies at fixed concentration of PEP, 166 nM of wtPKM2 was used. Reactions (100 μL) were initiated by PEP and monitored for 3 mins by following the decrease in absorbance at 340 nm corresponding to oxidation of NADH to NAD+. For the rescue assay with FBP, 33.2 nM wtPKM2 was incubated on ice for 30 min with fixed concentrations of ADP and cysteine, but varying FBP concentrations and reactions were monitored similar to that described above. The initial velocities were calculated by GraphPad

30


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Biochemistry

Prism and were plotted against PEP concentrations. The resulting curves were fit to an allosteric sigmoidal equation (1), Y=

&'() *+,

(1)

, -, ,(./ 0+

where X and Y are PEP concentration and initial velocity respectively, Vmax is the maximum velocity of product formation, Khalf is the concentration of PEP at half-maximal velocity, and h is the Hill coefficient.

Ligand Binding Assays. Fluorescence. All binding studies were performed in 20 mM HEPES, pH 7.5, 100 mM KCl, 5 mM MgCl2. The binding of ADP and PEP in the presence and absence of 1 mM cysteine was determined by quenching of intrinsic tryptophan fluorescence of wtPKM2. Fluorescence data were collected on a Cary eclipse fluorescence spectrophotometer (Agilent Technologies, Santa Clara, CA). The wavelengths used for excitation and emission of wtPKM2 were set at 295 nm and 340 nm with slit widths of 5 nm and 10 nm respectively. Titration experiments were performed using 1.6 μM wtPKM2 and varying concentrations of appropriate ligand. The data were converted into fractional saturation and fitted to the one-site specific binding (equation 2, for ADP) or the Hill equation (equation 3, for PEP binding) using GraphPad Prism, Y=

1'() *+

(2)

-2 0+ 1'() *+,

Y=-

,(./ *30+

,

(3)

where Bmax is maximum specific binding, Kd is the equilibrium dissociation constant of the binding ligand, Khalf is the equilibrium constant at which ligand concentration occupying half of the binding

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sites, h is the Hill coefficient, and X and Y are the total ligand concentration and fractional saturation respectively. Isothermal titration calorimetry (ITC). The binding of wtPKM2, S437Y PKM2, and G415R PKM2 with various ligands was measured using an iTC200 microcalorimeter (Microcal, Inc., Northampton, MA), equipped with a temperature-controlled cell (200 µl cell volume), and a 40 µl syringe. Prior to performing the titrations, protein sample was dialyzed in 50 mM Tris, 100 mM KCl, 5 % glycerol, 0.2 mM THP, pH 7.0 with or without respective ligand. Furthermore, in order to prevent the effect of exogenous magnesium on cysteine binding affinity of wtPKM2, EDTA (10 mM) was added to the enzyme and dialyzed overnight in 50 mM Tris pH 7.0, 100 mM KCl, 5 % glycerol, 0.2 mM THP containing 1 mM EDTA. The dialyzed enzyme was used to determine the cysteine binding affinity of PKM2. Protein concentrations were measured using extinction coefficient of 29910 M-1 cm-1 at 280 nm. Titration experiments were performed at 25 °C, with a stirring speed of 700 rpm. The initial injection of 0.2 µl was followed by several 2 µl injections. 15-30 µM of wtPKM2 and PKM2 variants in 50 mM Tris, 100 mM KCl, 5 % glycerol, 0.2 mM THP, pH 7.0 were used in the cells. Titrant concentrations ranged from 250 µM (for strongly binding ligands) to 5 mM (for weakly binding ligands). The heat of dilution obtained by injecting wtPKM2 into the buffer was subtracted from the heats of binding. The titration experiments were performed in triplicates and the data were analyzed with Microcal ORIGIN 7.0 software using a single-site-binding model to determine the equilibrium association constant, KA (=1/Kd), and the binding enthalpy, ΔH°. For all calculations and data fitting, the concentration of the monomer was used, which resulted in a stoichiometry of 1. Furthermore, instances where the binding affinity was relatively weak and protein concentration used in the experiment was either close to or less than Kd value, the stoichiometry was fixed to 1 during data analysis.

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The Gibbs free energy of binding, ΔG°, and the entropic contribution to the binding free energy, -TΔS°, were calculated using equation 4. ΔG° = ΔH° − TΔS°

(4)

Crystallization. To crystallize the cysteine bound complex of wtPKM2 (referred as, PKM2-Cys), wtPKM2 at 10 mg/mL was mixed with cysteine, the inhibitor oxalate, and MgCl2 at 2 mM, 2 mM, and 5 mM final concentrations respectively. This solution was incubated at 4 °C for 30 min and crystal drops were set at 20 °C using the sitting-drop vapor diffusion method with 1 μL of the above protein solution and 1 μL of a precipitant solution containing 0.2 M sodium thiocyanate, 100 mM Bis-Tris propane pH 8.0, and 16-20% PEG 3350. Crystals with dimensions of 50 × 100 × 100 μm appeared in a day and were further optimized with an additive screen (Hampton Research). The crystals obtained from 0.2 mM non-detergent sulfobetaine 221 (additive screen 66) were used for data collection. For cryocooling, crystals were looped, and washed through a drop of precipitant solution containing mother liquor and 25% (vol/vol) glycerol, and then submerged in liquid nitrogen. S437Y PKM2 crystals were obtained as described earlier by Srivastava et al. 24 To obtain cysteine and serine bound crystal structures, S437Y PKM2 crystals were soaked with 2 mM serine or cysteine for 2 hours. Crystals were cryo-protected with mother liquor containing 25 % sucrose and then submerged in liquid nitrogen.

Data Collection and Structure Determination. X-ray diffraction data of PKM2-Cys complex (Table 2) were collected at 100 K at beamline 19-ID at the Advanced Photon Source (Argonne, IL) with ADSC QUANTUM 315r detector using 0.25° oscillations, 1 s exposure time, and 250

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mm detector distance. Diffraction data on S437Y PKM2 -Cys and S437Y PKM2 -Ser complex were collected at beamline 4.2.2 at Advanced Light Source (Berkley, CA) with RDI CMOS_8M detector using 0.2° oscillation and 0.3 second exposure. For S437Y PKM2-Cys complex, detector distance was 200 mm while detector distance for S437Y PKM2 -Ser complex was 220 mm. The data were processed using XDS package 38 and SCALA (CCP4). 39 For the PKM2-Cys complex, molecular replacement was performed with PHASER 40 using two different monomers of wtPKM2 structures (one from PDB:4B2D and the other from PDB:3SRH) as the search models while looking for 2 copies of each. For the S437Y-complexes, rigid body refinement was done using crystal structure of apo form of S437Y (PDB:6B6U) to preserve the origin of the unit cell. The Rfree flags from apo S437Y (PDB:6B6U) were used to determine the structures of cysteine- and serine-bound complexes. Refinement and manual model building were carried out in PHENIX 41 and COOT

42

respectively. In PKM2-Cys structure, the N-terminal His tag and first 23 residues

are disordered and were not modelled in all chains. The B domain comprising of residues 117-218 was modelled completely in two (chains C and D) out of four chains. In chain A, a majority of backbone atoms of the B domain could be traced, however 42 residues were not modelled. Whereas, for chain B due to poor density only part of the B domain was modelled and about 66 residues are missing. For remaining residues in the B domain of chain B, only backbone atoms were modelled. All four cysteine molecules were refined as independent ligands. In S437Y-Cys and S437Y-Ser structure, first 13/14 residues in the N-terminus along with the His tag is missing in both chain A and B owing to poor electron density. Initial rounds of refinement were performed using rigid body, TLS, individual isotropic ADP, and restrained coordinate refinement coupled with simulated annealing. At later stages, rigid body, simulated annealing, and TLS refinement were turned off and only individual isotropic ADP and restrained coordinate refinement was used.

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Addition of cysteine/serine into distinct positive electron density was based on a simulated annealing omit map generated by omitting cysteine and sidechain of Arg160. Water molecules were added into clear densities in later rounds of refinement and composite omit maps were generated to verify the final models. The structures were validated with MolProbity

43

and the

wwPDB validation server. 44 All structural figures were made using PyMOL. Protein coordinates have been deposited in the Protein Data Bank (PDB:6NU1, 6NU5, 6NUB). Details of data processing, refinement, and validation are included in Table 2. Size-Exclusion Chromatography – Small Angle X-ray scattering (SEC-SAXS). Experiments were performed at BioCAT (beamline 18ID, at the Advanced Photon Source, Chicago). The setup for SEC-SAXS included a camera with a focused 12 KeV (1.03 Å) X-ray beam, a 1.5 mm quartz capillary sample cell, a sample to detector distance of ~3.5 m, and a Pilatus 3 1M detector (Dectris). 45

The q-range sampled was ~0.004 – 0.4 Å-1. In order to ensure sample monodispersity, an in-line

SEC setup was used, which included an AKTA-pure FPLC unit and a Superdex-200 Increase 10/300 GL column (GE Healthcare Life Sciences). The gel filtration column was equilibrated with 20 mM HEPES, pH 7.5, 100 mM KCl, 5 % glycerol, 0.5 mM THP. Cysteine and FBP were added in the equilibration buffer to final concentrations of 2 mM and 0.5 mM for the respective wtPKM2ligand complex. A 200 μl solution of wtPKM2 at 12.5 mg/mL was loaded on to the column. The elution trajectory after the UV monitor was redirected to the SAXS sample flow-cell. 1 second exposures were collected every 2 seconds during the gel-filtration chromatography run. Appropriate exposures were averaged and used as the buffer curve for each run, and the exposures during elution (co-incident with the UV peak on the chromatogram) were treated as protein+buffer curves. Data were corrected for background scattering by subtracting the buffer curve from protein+buffer curves.

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Preliminary data analyses were performed using Primus 46 and ATSAS packages, 47 which produced the protein scattering profile, I(q), as a function of the momentum transfer q, where q = 4π/λ sin θ (2θ is the scattering angle and λ is the X-ray wavelength). Guinier plots were made in GraphPad Prism6 (GraphPad Software Inc., La Jolla, CA) using the equation described by Putnam et al. 48 and radius of gyration (Rg) was calculated by fitting Guinier plot to the Guinier equation. 48

The pair distribution function, P(r) was calculated using AUTOGNOM

reconstruction was performed using DAMMIF. SITUS

50

49

47

and ab-initio shape

A volumetric bead map was created using

followed by rigid body docking of a wtPKM2 tetramer crystal structure (PKM2-Cys)

into the volumetric bead map.

Gel filtration Analysis. Solutions of 0.5 mg/ml wtPKM2 in the presence and absence of cysteine (50 µM, 2 mM and 5 mM) and 0.5 mg/ml S437Y PKM2 in the presence and absence of 5 mM Cys, 10 mM Ser, and 0.5 mM FBP were injected onto a Superdex 200 10/300 GL gel-filtration column (24 ml, GE Healthcare) with a 0.2 ml loop at 0.5 mL/min using an AKTA Pure FPLC system (GE Healthcare). The enzyme solutions were incubated with the corresponding amino acid and FBP for 15 minutes prior to injection. Furthermore, to control the redox state of the enzyme, a buffer containing 50 mM Tris-Cl, pH 7.5, 150 mM KCl, 5% glycerol, 0.5 mM EDTA, and 10 mM THP was used for experiments with wtPKM2. For S437Y PKM2 the same buffer containing 0.5 mM THP was used. The column was calibrated with gel filtration molecular weight (MW) standards (Bio-Rad) containing thyroglobulin (670 kDa, bovine), g-globulin (158 kDa, bovine), ovalbumin (44 kDa, chicken), myoglobin (17 kDa, horse), and vitamin B12 (1.35 kDa). The eluting protein and the complexes were detected by monitoring the UV absorbance at

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280 nm. The intensity data was normalized for comparison and plotted using GraphPad Prism 8.0.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: XXXX/acs. biochem.xxxxxx. Supporting Figures S1-S6 and Table S1-S2: Lineweaver-Burk plot PKM2 inhibition by cysteine (Figure S1). Activation of PKM2 by serine (Figure S2), Structural alignment (Figure S3), Comparison of PKM2-Cys structure with PKM2-FBP and PKM2-Ser (Figure S4), Comparison of scattering profiles of ligand bound PKM2 and shape reconstruction (Figure S5), SAXS and gelfiltration profiles of S437Y PKM2 (Figure S6). SAXS data statistics (Table S1) and Gel filtration data (Table S2).

ACCESSION CODE. Atomic coordinates and structure factors have been deposited in the Protein Data Bank as entries 6NU1, 6NU5, and 6NUB. UniProt entry for PKM2 P14618.

AUTHOR INFORMATION Corresponding Author. Department of Chemistry, University of Iowa, Iowa City, IA 522421727. Telephone: 734-747-2311. Fax: 319-335-1270. E-mail: [email protected]. ORCID. Mishtu Dey: 0000-0003-4763-2921 AUTHOR CONTRIBUTIONS D.S. and M.D. designed the experiments. D.S. and S.N. performed the experiments; D.S., S.N., and M.D. analyzed the data and wrote the manuscript.

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Funding. M.D. was supported by the University of Iowa College of Liberal Arts and Sciences and funding from the National Science Foundation (CLP 1506181).

Notes. The authors declare no competing financial interests.

ACKNOWLEDGEMENTS M.D. was supported by funding from the National Science Foundation (CLP 1506181) and from the University of Iowa College of Liberal Arts and Sciences. X-ray were collected at the Advanced Light Source (4.2.2) and Advanced Photon Source (APS) (beamline 19-ID), and SEC-SAXS data was collected at APS (beamline 18-ID). This research used resources of the APS, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. This project was supported by grant 9 P41 GM103622 from the National Institute of General Medical Sciences of the National Institutes of Health. Use of the Pilatus 3 1M detector was provided by grant 1S10OD018090-01 from NIGMS. We would like to acknowledge use of resources at the Carver College of Medicine’s Protein Crystallography Facility at the University of Iowa. We would like to thank Dr. Lokesh Gakhar for helping with PDB deposition. ALS is a DOE Office of Science User Facility under contract no. DE-AC02-05CH11231. The content is solely the responsibility of the authors and does not necessarily reflect the official views of the National Institute of General Medical Sciences or the National Institutes of Health. ABBREVIATIONS. PKM2, Pyruvate Kinase muscle isoform 2; FBP, Fructose-1, 6bisphosphate; wt, wild-type; ITC, isothermal titration calorimetry; SEC, size-exclusion

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Biochemistry

For Table of Contents Use Only. Manuscript Title: Mechanistic and Structural Insights into Cysteine-Mediated Inhibition of Pyruvate Kinase Muscle Isoform 2 Authors: Dhiraj Srivastava, Suparno Nandi, and Mishtu Dey TOC Graphic.

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Mechanistic and Structural Insights into Cysteine-Mediated Inhibition

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