Structural Investigation of a Dimeric Variant of Pyruvate Kinase Muscle

Nov 28, 2017 - Dhiraj Srivastava†, Mortezaali Razzaghi†, Michael T. Henzl‡, and Mishtu Dey†. † Department of Chemistry, University of Iowa, ...
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Structural Investigation of a Dimeric Variant of Pyruvate Kinase M2 Dhiraj Srivastava, Mortezaali Razzaghi, Michael T. Henzl, and Mishtu Dey Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b01013 • Publication Date (Web): 28 Nov 2017 Downloaded from http://pubs.acs.org on November 29, 2017

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Biochemistry

Structural Investigation of a Dimeric Variant of Pyruvate Kinase M2 Dhiraj Srivastava1,3, Mortezaali Razzaghi1,3, Michael T. Henzl2, Mishtu Dey1* 1 2

Department of Chemistry, University of Iowa, Iowa City, Iowa 52242

Biochemistry Department, University of Missouri-Columbia, Columbia, MO 65211 3

Authors contributed equally to the work

KEYWORDS: Pyruvate kinase, glycolysis, crystal structure, size-exclusion chromatography, small angle X-ray scattering, analytical ultracentrifuge Supporting Information Placeholder

ABSTRACT: Pyruvate kinase muscle isoform 2 (PKM2) catalyzes the terminal step in glycolysis, transferring a phosphoryl group from phosphoenolpyruvate to ADP, to produce pyruvate and ATP. PKM2 activity is allosterically regulated by fructose-1,6-bisphosphate (FBP), an upstream glycolytic intermediate. FBP stabilizes the tetrameric form of the enzyme. In its absence, the PKM2 tetramers dissociate, yielding a dimermonomer mixture having lower enzymatic activity. The S437Y variant of PKM2 is incapable of binding FBP. Consistent with that defect, we find that S437Y exists in a monomer-dimer equilibrium in solution, with a Kd of ~20 µM. Interestingly, however, the protein crystallizes as a tetramer, adopting a structure that resembles that of the FBP-bound wild-type PKM2.

Pyruvate kinase (PK) catalyzes the terminal step of glycolysis, transferring a phosphoryl group from phosphoenol pyruvate (PEP) to adenosine diphosphate (ADP), producing pyruvate and adenosine triphosphate (ATP). Four isoenzymes of PK, encoded by two genes,1 are expressed in mammalian cells. The L and R isoforms are splice variants derived from the PKLR gene. The PKL isoform is predominantly expressed in the liver, PKR is present in erythrocytes. The M1 and M2 isoforms are splice variants of the PKM gene.1 PKM1 is expressed in skeletal muscle, heart, and the brain. Although expression of PKM2 is normally restricted to embryonic cells, the protein commonly exists in rapidly proliferating cancer cells in adults.1 PKM1 and PKM2 differ only by 22 out of 531 amino acids within a 56 residue alternative splice exon, and the sequence differences are confined to the dimer-dimer interface.2 However, they have distinct catalytic and regulatory properties.3 While PKM1 exists in a constitutively active tetrameric state, PKM2 can adopt different oligomeric states: a highly active tetramer and a less active dimer/monomer.

The activity and oligomeric state of PKM2 are regulated by various metabolites (fructose-1,6-bisphosphate succinyl-5-aminoimidazole-4-carboxamide-1(FBP)4, ribose 5’-phosphate5), amino acids (serine,6 cysteine,7 and phenylalanine8), and posttranslational modifications (phosphorylation,9 acetylation,10 and oxidation11). There is increasing evidence that PKM2 can perform non-glycolytic functions with diverse implications. For example, dissociation of active tetrameric PKM2 into dimers promotes its translocation to the nucleus 9, where the dimeric form interacts with multiple proteins and transcription factors notably HIF1α,12, 13 PHD3,12 Oct414, β-catenin15 - thereby modulating gene expression. PKM2 expressed and purified from E. coli exists as a mixture of tetramer/dimer/monomer at low micromolar protein concentrations.8 Whereas, at higher concentration levels, isolated wild type (wt) PKM2 is predominantly tetrameric. The tetrameric fraction is due to FBP co-purifying with recombinant wtPKM2 and is bound with an occupancy of ~50%.16 Biochemical studies with PKM2 variants indicated that PKM2 dissociates into dimers in the absence of FBP.10 The dimeric form of PKM2 is predominant in tumor cells and is known to serve as a biomarker for gastrointestinal cancer.17-19 Because of the importance of dimeric PKM2 as a diagnostic tool and to investigate its interactions with various binding partner proteins, it would be useful to have access to a PKM2 variant that remains dimeric at the relatively high protein-concentrations typically employed for biophysical characterization. Based on in vitro activity studies, it has been demonstrated that a PKM2 variant, S437Y, incapable of binding FBP has compromised allosteric activation, which is believed to be due to the dissociation of the tetramer.20, 21 The wtPKM2-FBP structure (PDB 1T5A) shows that S437 is located in the FBP binding pocket and is hydrogen bonded with FBP (Figure S1A). We hypothesized that replacement of serine with tyrosine would introduce a steric clash with FBP significantly impairing the ability of S437Y PKM2 to bind FBP.

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In order to gain structural insight into the impact of the mutation on the quaternary structure of the enzyme, we examined the conformation and oligomeric state of S437Y PKM2. Although the S437Y variant crystallizes as a tetramer, analytical ultracentrifugation (AUC) and sizeexclusion chromatography (SEC)-small-angle X-ray scattering (SAXS) data indicate that in solution the protein exists as a monomer-dimer mixture. Preliminary SEC data (not shown) indicated that the average molecular weight (MW) of S437Y variant was smaller than that of wtPKM2. Accordingly, samples of S437Y PKM2 were examined by AUC to more rigorously evaluate the oligomeric state. When subjected to velocity analysis at 20 °C at a loading concentration of 1.5 mg/mL (25 µM monomer), the variant displayed a symmetric sedimentation-coefficient distribution centered at 4.79 S (Figure 1A). The corresponding MW distribution is presented

centrations of S437Y PKM2 (7, 14, and 28 µM) were sedimented to equilibrium at four rotor speeds. The data were treated globally with a reversible monomer-dimer model, to extract an estimate for the association constant for dimerization. The optimal fit yielded a value of 49,700 ± 3,300 M-1, corresponding to a dissociation constant (Kd) of 20.1 ± 1.3 µM. To obtain further insight into the shape and structure of S437Y PKM2 in solution, the variant was examined by SEC-coupled SAXS. The radius of gyration (Rg) obtained from Guinier analysis was constant across the elution volume, implying that the sample is monodisperse and that Rg

Figure 2. SEC-SAXS data for S437Y PKM2. (A) Plot of Rg calculated by AutoGnom and I0 vs. image number that corresponds to the elution volume. (B) Guinier plot showing linearity in the low q range. (C) Kratky plot suggesting that the protein is folded well. (D) Comparison of the experimental scattering profile with calculated scattering profiles for monomer, dimer and tetramer. I is scattering intensity, I0 is extrapolated scattering intensity at zero scattering angle, and q is momentum transfer.

Figure 1. (A) Sedimentation-coefficient distribution for S437Y PKM2. A 25 µM sample of the protein was sedimented at 30,000 rpm, collecting data continuously until 250 radial scans had been acquired. The resulting data set was analyzed with sedfit using the continuous c(s) model. (B) MW distribution for S437Y PKM2 obtained by analysis of the aforementioned velocity dataset, using the continuous c(M) model in sedfit. (C) Sedimentation equilibrium analysis. Samples of S437Y PKM2 (7, 14, and 28 µM) were sedimented to equilibrium at four rotor speeds. The resulting data set was subjected to least-squares analysis, employing a reversible monomer-dimer model. The red lines represent the optimal fit to the data.

in Figure 1B. The maximum MW appears at 96,000 Da, intermediate between the monomer (60.2 kDa) and dimer (120.4 kDa) masses. This result suggests that the protein sediments as a rapidly equilibrating monomer-dimer mixture. This conclusion was supported by sedimentationequilibrium data (Figure 1C). Three distinct loading con-

is concentration-independent over the range of concentrations examined (Figure 2A). The resulting Guinier curve for the peak in the elution profile displayed linear behavior in the low q range, indicating the absence of aggregation or radiation damage (Figure 2B). The Rg value of 32.84±0.15 Å is consistent with the theoretical Rg value of 30.09 Å calculated for a dimeric PKM2 structure extracted from wtPKM2-FBP structure (PDB 1T5A). The Kratky plot suggests that S437Y PKM2 is well-folded (Figure 2C). The pair-distribution function, calculated from the experimental scattering profile with AUTOGNOM22, yielded a maximum dimension of 105 Å. The Rg value (32.76 ±0.07 Å) calculated with AUTOGNOM agrees well with that estimated by Guinier analysis (Table S1). When the experimental scattering profile is compared with theoretical scattering profiles for the PKM2 monomer, dimer, and tetramer generated by the FOXS server23, 24, using the wtPKM2-FBP crystal structure (PDB 1T5A), the dimeric structure yields the best agreement (Figure 2D). The χ2-value for the dimer (1.16) is substantially lower than the values obtained for the monomer (7.78) and tetramer (4.21).

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Biochemistry Ab-initio shape reconstruction was performed using DAMMIF, and a volumetric bead map was generated with SITUS25 (Figure 3). The scattering volume calculated by DAMMIF (220 x 103 Å3) yielded an estimated MW of

loop that covers the FBP-binding site (residues 515-521) in wtPKM2 (Figure 4B). The configuration of this loop is chain-dependent in S437Y PKM2. Although the loop is disordered in chain A, with unmodeled residues due to poor electron density, it is structured in chain B. In chain B of S437Y PKM2, the carboxylate group of Glu223 from the neighboring asu occupies the site occupied by P1 phosphoryl group of FBP in wtPKM2-FBP structure (not

Figure 3. SAXS derived ab-initio shape reconstructions depicting solution conformation of S437Y PKM2, with two different views being displayed. The envelope generated from the shape reconstruction of SAXS data fits well with a dimeric species (blue and green cartoon) extracted from the crystal structure of the wtPKM2-FBP tetramer (PDB 1T5A). Shape reconstructions statistics are provided in Table S1.

~110 kDa, which agrees well with the sequence-derived value for dimeric PKM2 (120 kDa). Moreover, a wtPKM2FBP dimer extracted from the tetrameric structure (PDB 1T5A) fits well into the SAXS-envelope of S437Y PKM2. In summary, the SAXS data indicate that S437Y PKM2 exists as a dimer in solution under the conditions employed for the analysis. The elevated dimer population may reflect the lower temperature and higher protein concentration employed in the SAXS analysis. If the dimerization reaction were exothermic, lowering the temperature from 20 °C (AUC) to 12 °C (SEC-SAXS) would promote dimer formation. In addition, the loading concentration (12.5 mg/ml) employed in the SEC-SAXS measurement greatly exceeded the concentrations present in the AUC experiments, even though the sample underwent substantial dilution during the chromatographic step. However, if the kinetics of dimer dissociation are slow, the size distribution observed in SAXS might be more reflective of the dominant oligomeric state at the 12.5 mg/ml loading concentration. The crystal structure of S437Y variant of PKM2 was solved in C2 space group at 1.35 Å resolution (Figures 4, S1B, Table S2). There are two monomers in the asymmetric unit (asu). Each monomer contains one molecule of oxalate, Mg2+, K+, Cl−, and bis-tris propane. The overall structure of the variant is very similar to wtPKM2-FBP structure (PDB 1T5A), which was solved in a different space group (P212121) at resolution of 2.8 Å (Figure 4A). Structural alignment of the A-domain from chain A of S437Y PKM2 with the A-domain of wtPKM2-FBP (PDB 1T5A) has an rmsd of 0.24 Å. Likewise, alignment of the entire monomeric chain resulted in an rmsd of 0.38 Å (Figure 4B), and the alignment of dimer gives an rmsd of 0.5 Å (Figure S1C). The minor structural differences between wtPKM2FBP and S437Y PKM2 are confined to the B- and Cdomains (Figure 4B). The alteration observed in the Bdomain is reasonable, considering that it is known to be highly mobile.2, 21 The B-domain undergoes hinge-bending motion causing rotation and translation relative to the Adomain.2 The conformation of the B-domain with respect to the A-domain may be influenced by crystal packing. Not surprisingly, the deviation in the C-domain occurs in the

Figure 4. Crystal structure of S437Y PKM2. (A)-(C) Superimposition of S437Y and wtPKM2-FBP complex (PDB 1T5A, colored in wheat). (A) Overall structures. (B) Chain B of S437Y monomer shows minor differences in the FBP binding site and in the B-domain. (C) A close-up view of S437Y site in chain B, with 2fo-fc map contoured at 1.0 σ, shows open conformation of the FBP-free variant and potential steric clash between Y437 and FBP. In (B), FBP and oxalate are shown as spheres and in (C) FBP and Y437 are shown as sticks, with carbon in cartoon color, oxygen in red, and phosphorus in orange.

shown). As a consequence of crystal packing constraints, the FBP-binding loop in chain B of S437Y PKM2 adopts a more open conformation compared to that of wtPKM2-FBP (Figures 4C, S1B). In chain A, while the FBP binding loop is disordered, due to crystal packing effects, this site is occupied by Glu7 from the neighboring asu, which evidently does not exert the same stabilizing influence on the loop (not shown). With the exception of PKM1, all PK isoforms, PKL, PKR and PKM2 are allosterically regulated by FBP. PKM1 and PKM2 differ only by 23 amino acids between residues 388-434, which are located at the dimer-dimer interface. All residues lining the FBP binding pocket are identical between PKM2 and PKM1, except K433 in PKM2, which is glutamate in PKM1 (Figure S1D). However, the mechanism by which FBP affects the oligomeric state of PKM2 is not known. Ishwar et al26 reported that the binding of FBP to PKL primarily impacts the orientation of residues 489, 482, and 515-521. As observed in S437Y PKM2, the FBP binding loop encompassing residues 515521 adopts a more open conformation in both human and rabbit PKM1.27 In contrast, there is little movement of Trp482 towards the FBP binding site in S437Y PKM2 structure. The tetrameric structure of S437Y PKM2 generated by crystallographic symmetry resembles an unpublished structure of this variant (PDB 3G2G) solved at

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2.0 Å resolution in P212121 space group. The FBP binding loops in three out of four chains in the asu of the 3G2G structure are disordered, and in the fourth chain it exists in a slightly different conformation than the structure solved by us. Regardless, our S437Y PKM2 structure presented here is similar to 3G2G, with an rmsd of 1.04 Å. To summarize, sedimentation data unequivocally demonstrate that S437Y PKM2 exists in a monomer-dimer equilibrium in solution. At the higher protein concentration and relatively lower temperature employed in the SAXS analysis, the protein is predominantly dimeric. Nevertheless, the S437Y variant crystallizes as a tetramer, adopting a structure that closely resembles that of FBP-bound wtPKM2 (Figure 4) and constitutively active PKM18. The C-C’ interfaces of S437Y and wtPKM2 are highly similar, with seemingly identical surface areas (Figure 4A). Furthermore, the few significant structural differences are localized to the FBP-binding site (Figures 4C, S1B). Presumably, the high protein concentration employed for crystallization, in concert with crystal-packing forces, induced the tetrameric form of S437Y. It should be noted that the effective protein concentration would have been further elevated, through the excluded volume effect, by the 16-20 % PEG 3350 present in the crystallization solution and could potentially influence the observed tetrameric structure in the crystalline lattice of S437Y PKM2. Although, the crystal structure of our FBP-free S437Y offers no direct insight into the FBP-driven tetramerization of PKM2, it provides a structural basis of impaired FBP binding of S437Y. The mechanism of allosteric regulation of PKM2 by FBP remains a topic for further inquiry. In conclusion, PKM2 associates with, and modulates the activities of various proteins. Because these interactions are often specific to a particular oligomeric state, the availability of PKM2 variants with restricted oligomeric flexibility could facilitate investigation of the dimeric enzyme properties, which is noted as a diagnostic marker for gastrointestinal cancer. Yan et al5 previously reported that the G415R PKM2 variant is dimeric in solution. We have herein demonstrated that S437Y variant is likewise dimeric at concentrations routinely employed to study protein-protein interactions. Although both mutations prevent adoption of the canonical tetrameric structure, they evidently disrupt quaternary structure via distinct mechanisms. Because the G415 residue is located at the C-C’ interface, replacement with arginine could perturb the PKM2-target interactions. By contrast, the crystal structure of S437Y PKM2 suggests that the mutation should have a negligible impact on the CC’ interface structure and, in turn, PKM2-target protein interactions. Moreover, while the G415R mutation has a minimal impact on FBP binding, S437Y is incapable of binding FBP. Thus, the consequences of the G415R and S437Y mutations on PKM2 oligomeric structure are, at some level, complementary to each other. In our opinion, detailed examination of the two variants could provide substantial insight into the mechanism of allosteric regulation of PKM2. ASSOCIATED CONTENT

Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: XXXX. Experimental details and Supporting Tables 1 and 2, and Figure 1 (PDF). ACCESSION CODE. Atomic coordinates and structure factors have been deposited in the Protein Data Bank as entry 6B6U. AUTHOR INFORMATION Corresponding Author *Department of Chemistry, University of Iowa, W285 Chem. Bldg., Iowa City, IA 52242-1727. Telephone: 319384-1319. Fax: 319-335-1270. E-mail: [email protected]. ORCID Mishtu Dey: 0000-0003-4763-2921 Author Contributions D.S. and M. R. contributed equally to this work. D.S. and M.D. designed the experiments. D.S., M.R., and M.H. performed the experiments and D.S., M.H., and M.D. analyzed the data. D.S., M.H., and M.D. wrote the manuscript. 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). M. H. was supported by the University of Missouri Department of Biochemistry. Notes. The authors declare no competing financial interests. ACKNOWLEDGMENTS X-ray and SEC-SAXS data were collected at the Advanced Light Source (4.2.2) and Advanced Photon Source (18-ID) respectively. Support for the synchrotron sources is provided by the U.S. Department of Energy. ALS is a DOE Office of Science User Facility under contract no. DE-AC0205CH11231. ABBREVIATIONS PKM2, Pyruvate Kinase muscle isoform 2; FBP, Fructose1, 6-bisphosphate; wt, wild-type; AUC, analytical ultracentrifugation; SEC, size-exclusion chromatography; SAXS, small-angle X-ray scattering, rmsd, root-mean-squaredeviation; PDB, Protein Data Bank. REFERENCES [1] Filipp, F. V. (2013) J. Carcinog. 12(1), 1-14. [2] Dombrauckas, J. D., Santarsiero, B. D., and Mesecar, A. D. (2005) Biochemistry 44 (27), 9417-9429. [3] Noguchi, T., Inoue, H., and Tanaka, T. (1986) J. Biol. Chem. 261 (29), 13807-13812. [4] Taylor, C. B. a. B., E. (1967) Biochem. J. 102, 32C. [5] Yan, M., Chakravarthy, S., Tokuda, J. M., Pollack, L., Bowman, G. D., and Lee, Y. S. (2016) Biochemistry 55, 4731-4736. [6] Chaneton, B., Hillmann, P., Zheng, L., Martin, A. C., Maddocks, O. D., Chokkathukalam, A., Coyle, J. E.,

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