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Human pyruvate kinase, muscle, S437Y variant

PDB Code 3G2G Target Class Non-protein Kinase

Target PKM2
Alias CTHBP, MGC3932, OIP3, PK3, PKM, TCB, THBP1
Disease Area/Function Cancer
Date Deposited 2009-01-31
Authors Allali-Hassani A, Hong B, Dimov S, Tempel W, MacKenzie F, Arrowsmith CH, Edwards AM, Bountra C, Weigelt J, Bochkarev A, Park H, Vedadi M
Related Structure 1ZJH, 3GQY, 3GR4, 3H6O, 3ME3, 3U2Z

Struc Details Tabs

Structure Details

Pyruvate kinase (PK, EC 2.7.1.40) catalyzes the last step of glycolysis, where the phosphoryl group of phosphoenolpyruvate (PEP) is transferred to ADP to form pyruvate and ATP, and thus participates in the primary intersections of the energy methabolism. Lately, the enzyme was linked to other diseases related to both glucose and oxygen utilization, such as diabetes, blood and brain phenylketonuria, and angiogenesis[1]. Different isoenzymes of pyruvate kinase are expressed depending upon the metabolic responsibilities of the various cells and tissues. Pyruvate kinase type L (PK-L) is the characteristic pyruvate kinase isoenzyme of tissues with gluconeogenesis such as liver and kidney. Erythrocytes express the pyruvate kinase isoenzyme type R (PK-R). Pyruvate kinase type M1 (PKM1) is present in tissues in which large amount of energy have to be rapidly provided such as in muscle and brain. Pyruvate kinase type M2 (PKM2) is characteristic of lung tissues as well as all cells with high rates of nucleic acid synthesis, including all proliferating cells such as embryonic cells and especially tumor cells[2]. During tumor formation, a shift in the isoenzyme composition of pyruvate kinase always takes places in such a manner that the tissue specific isoenzyme, such as PKM1 in brain or PK-L in the liver, disappears and PKM2 is expressed[3].

Of these isoforms, M2, L and R isozymes are regarded as allosterically regulated via feed-forward activation by fructose-1,6-bisphosphate (FBP), the product of the phosphofructokinase reaction. The architecture of PK is evolutionally highly conserved and is organized as a homotetramer with four distinct domains in each subunit. The activity of the enzyme is a combination of domain and subunits rotations coupled to active site geometry. Residues, located in the domain interfaces, play a crucial role in function and communication between the subunits of the PK[4].

There are two major forms of PKM2 in cells, a highly active tetramer and a less active dimer. The balance between these two forms can be shifted and FBP induces tetramer formation[5]. One of reported PKM2 SNPs, S437Y, is located in the FBP-binding pocket, thereby resulting in poor interaction with the allosteric activator. We have determined the crystal structure of PKM2-S437Y variant. This structure will provide the basis for understanding the mechanism of PKM2 activity by FBP-induced allosteric regulation and addressing the underlying principles of PK-related human diseases by mutations.

References

  1. Jill D. Dombrauckas, Bernard D. Dantarsiero, and Andrew D. Mesecar. 2005. Structural basis for tumor pyruvate kinase M2 allosteric regulation and catalysis. Biochemistry. 44, 9417-9429.
  2. Heather R. Christofk, Matthew G. Vander Heiden, Ning Wu, John M. Asara, and Lewis C. Cantley. 2008. Pyruvate kinase M2 is a phosphotyrosine-binding protein. Nature. 452, 181-186
  3. Sybille Mazurek, C. Bruce Boschek, Ferdinand Hugo, and Erich Eigenbrodt. 2005. Pyruvate kinase type M2 and its role in tumor growth and spreading. Seminars in Cancer Biology. 15, 300-308.
  4. Sybile Mazurek. 2008. Pyruvate kinase type M2: A key regulator within the tumor metabolome and a tool for metabolic profiling of tumours. Ernst Schering Foundation Symposium Proceedings. 4, 99-124
  5. Ashizawa K, Willingham MC, Liang CM, Cheng SY. 1991. In vivo regulation of monomer-tetramer conversion of pyruvate kinase subtype M2 by glucose is mediate via fructose 1,6-bisphosphate. Journal of Biological Chemistry. 266, 16842-16846.
Materials & Methods
StructurePKM2
PDB Code3G2G
Entry clone accession GI:33286417
Entry clone source MGC
Tag N-terminal histag with thrombin cleavage site: mgsshhhhhhssglvprgs
Construct sequencemgsshhhhhhssglvprgsMSKPHSEAGTAFIQTQQLHAAMADTFLEHMCRLDIDSPPITARNTGIICTIGPASRSVETL
KEMIKSGMNVARLNFSHGTHEYHAETIKNVRTATESFASDPILYRPVAVALDTKGPEIRTGLIKGSGTAEVELKKGATLK
ITLDNAYMEKCDENILWLDYKNICKVVEVGSKIYVDDGLISLQVKQKGADFLVTEVENGGSLGSKKGVNLPGAAVDLPAV
SEKDIQDLKFGVEQDVDMVFASFIRKASDVHEVRKVLGEKGKNIKIISKIENHEGVRRFDEILEASDGIMVARGDLGIEI
PAEKVFLAQKMMIGRCNRAGKPVICATQMLESMIKKPRPTRAEGSDVANAVLDGADCIMLSGETAKGDYPLEAVRMQHLI
AREAEAAIYHLQLFEELRRLAPITSDPTEATAVGAVEASFKCCSGAIIVLTKSGRYAHQVARYRPRAPIIAVTRNPQTAR
QAHLYRGIFPVLCKDPVQEAWAEDVDLRVNFAMNVGKARGFFKKGDVVIVLTGWRPGSGFTNTMRVVPVP
Vector p28a-LIC
Expression host BL21 DE3
Growth method We prepared the seeds by inoculating freshly transforming E. coli cells (BL21 DE3) into 80 mL of Luria-Bertani medium. After overnight, all of the seeds were inoculated into 1.8 L of Terrific Broth medium in the presence of 50 µg/mL of kanamycin at 37ºC and grown to an OD600 of 4.0. Cells were then induced by isopropyl-1-thio-D-galactopyranoside at the final concentration of 1.5 mM and grown overnight at 20ºC in a LEX bubbling system.
Extraction buffersBinding buffer: 10 mM Tris pH 7.5, 0.5 M NaCl, 5% glycerol, 5 mM imidazole
Extraction procedure Cultures were centrifuged and the cell pellets were suspended in 100 mL of the binding buffer with a protease inhibitor cocktail (0.1 mM M benzamidine-HCl and 0.1 mM phenylmethyl sulfonyl fluoride) and flash frozen. The thawed cell pellet was lysed by a combination of 0.5% CHAPS (Sigma) and sonication. The lysate was centrifuged at 15000 rpm for 30 min and the supernatant was used for subsequent steps of purification.
Purification buffersWash buffer: 10mM Tris pH 7.5, 0.5 M NaCl, 5% glycerol, 30 mM imidazole
Elution buffer: 10mM Tris pH 7.5, 0.5 M NaCl, 5% glycerol, 250 mM imidazole
Purification procedure The supernatant was passed through DE52 (Whatman) column equilibrated with the binding buffer and then loaded onto 3 mL Ni-NTA column (Qiagen) equilibrated with the same binding buffer at 4 ºC. The Ni-NTA column was washed with 150 mL of the wash buffer and the protein was eluted with 15 mL of the elution buffer. The eluate was dialyzed overnight against a buffer containing 10 mM HEPES pH 7.5, 150mM KCl, 2mM TCEP, 5% glycerol, and 5mM MgCl2. The protein concentration was estimated based on the extinction coefficient of the protein, 29190 at 280 nm. Five molar equivalents of ADP, 5 mM TCEP and 5 mM MgCl2 were added to the purified protein before concentration. The protein was concentrated using an Amicon Ultra centrifugal filter to the final volume of 1 mL and the concentration of 30 mg/mL. About 55 mg of protein was obtained from 1.8 L of cell culture.
Protein stock concentration 30 mg/mL
Crystallization Crystals were obtained by the sitting drop vapor diffusion method, 1 µL of the protein was mixed with 1 µL of unbuffered reservoir solution consisting of 25% PEG1500, 0.2M NH4SO4, 0.1M Na-cacodylate (pH5.5). Diamond-like crystals were grown within a week. For data collection a single crystal was separated from the cluster and cryoprotected in a 50:50 mixture of Paratone-N and mineral oil before flash cooling in liquid nitrogen.