Human pyruvate kinaseM2 in complex with soluble activator

PDB Code 3ME3 Target Class Non-protein Kinase

Target PKM2
Alias CTHBP, MGC3932, OIP3, PK3, PKM, TCB, THBP1
Disease Area/Function cancer
Date Deposited 2010-03-31
Authors Hong B, Dimov S, Tempel W, Auld D, Thomas C, Boxer M, Jianq J-K, Skoumbourdis A, Min S, Southall N, Arrowsmith CH, Edwards AM, Bountra C, Weigelt J, Bochkarev A, Inglese J, Park H
Related Structure 1ZJH, 3G2G, 3GQY, 3GR4, 3H6O, 3U2Z

Struc Details Tabs

Structure Details
Pyruvate kinase (PK, EC 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 subunit 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]. The structures of PKM2 in the presence of various activators and inhibitors are needed to understand the nature of allosteric modulation of the subunits. These structures will provide the basis for understanding the mechanism of PKM2 activity and addressing the underlying principles of PK-related human diseases.


  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
PDB Code3ME3
Ligands3-{[4-(2,3-dihydro-1,4-benzodioxin-6-ylsulfonyl)- 1,4-diazepan-1-yl]sulfonyl}aniline
Entry clone accession BC007952
Entry clone source MGC: AU36-H1
SGC clone accession HPC002-A01
Tag N-terminal His6-tag, not removed
Construct comments PANK3: M1-P531
Vector pET28a-LIC
Expression host BL21-V2R
Growth method The seeds were grown in 80 mL Luria-Bertani broth media supplemented with 50 g/mL kanamycin at 37 C overnight. The following morning, all of the seeds were inoculated 1800 mL of Terrific Broth media supplemented with 50 g/mL kanamycin, 8 g/l glycerol and approximately 500 l antifoam in glass flasks in the Large Scale Expression System (LEX). Cells were grown at 37 C until OD600nm of 4.0 and were then induced by addition of IPTG to a concentration of 0.5 mM. Protein expression was allowed to continue over night at 18 C.
Extraction buffers10 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 buffersWashing Buffer: 10 mM Tris pH 7.0, 0.5 M NaCl, 5% glycerol, 30 mM imidazole
Elution Buffer: 10 mM Tris pH 7.0, 0.5 M NaCl, 5% glycerol, 250 mM imidazole
Size exclusion Buffer: : 10mM HEPES (pH7.5), 100mM KCl, 2mM TCEP, 5mM MgCl2, 5% Glycerol
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 further purified by size-exclusion chromatography (Superdex 200) equilibrated with 10 mM HEPES pH 7.5, 150mM KCl, 2mM TCEP, 5% glycerol, and 5mM MgCl2. The protein was concentrated using an Amicon Ultra centrifugal filter to the concentration of 50 mg/mL.
Protein stock concentration 50 mg/mL
Crystallization Crystallization trials were set up using the vapor diffusion method and the protein drop was equilibrated against a reservoir solution with 1:1 volume ratio. Prior to crystallization, the purified protein was incubated overnight at 4 °C in the presence of the activator (to 5 ~10 mM final concentration). Crystals of activator bound PKM2 were grown at 25% PEG-3350, 0.1M ammonium sulfate, 0.1M Bis-Tris, pH6.5.
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