Human pyruvate kinase, muscle, in complex with activator NCGC00031955

Target ID:

PKM2

Aliases:

CTHBPMGC3932OIP3PK3PKMTCBTHBP1

Deposition Date:

Thursday 23rd April 2009

Families:

Non-protein Kinase

Related Diseases:

Cancer

Structure type:

protein-ligand

Download Coordinates:

3H6O

Authors:

Hong B, Dimov L, Tempel W, Auld D, Thomas C, Boxer M, Jianq JK, Skoumbourdis A, Min S, Southall N, Arrowsmith CH, Edwards AM, Bountra C, Weigelt J, Bochkarev A, Inglese J, Park H

Site:

Toronto

3H6O

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 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.

Materials and Methods

PKM2

PDB:3H6O

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:33286417
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal histag with thrombin cleavage site: mgsshhhhhhssglvprgs
Host:BL21 DE3

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsMSKPHSEAGTAFIQTQQLHAAMADTFLEHMCRLDIDSPPITARNTGIICTIGPASRSVETLKEMIKSGMNVARLNFSHGTHEYHAETIKNVRTATESFASDPILYRPVAVALDTKGPEIRTGLIKGSGTAEVELKKGATLKITLDNAYMEKCDENILWLDYKNICKVVEVGSKIYVDDGLISLQVKQKGADFLVTEVENGGSLGSKKGVNLPGAAVDLPAVSEKDIQDLKFGVEQDVDMVFASFIRKASDVHEVRKVLGEKGKNIKIISKIENHEGVRRFDEILEASDGIMVARGDLGIEIPAEKVFLAQKMMIGRCNRAGKPVICATQMLESMIKKPRPTRAEGSDVANAVLDGADCIMLSGETAKGDYPLEAVRMQHLIAREAEAAIYHLQLFEELRRLAPITSDPTEATAVGAVEASFKCCSGAIIVLTKSGRSAHQVARYRPRAPIIAVTRNPQTARQAHLYRGIFPVLCKDPVQEAWAEDVDLRVNFAMNVGKARGFFKKGDVVIVLTGWRPGSGFTNTMRVVPVP
Vector:p28a-LIC

Growth

Medium:
Antibiotics:
Procedure: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 37°C 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 20°C in a LEX bubbling system.

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.

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.
Concentration:30 mg/mL
Ligand
NCGC00031955MassSpec:
Crystallization:The protein was mixed with 5-10 fold molar excess of the activator and incubated overnight at room temperature. Crystals were obtained by the sitting drop vapor diffusion method, 1ul of the protein was mixed with 1 ul of unbuffered reservoir solution consisting of 25% PEG3350, 0.2M NH4 -Acetate, 0.1M Bis-Tris pH5.5. Crystal clusters appeared within two days. 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.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

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.
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
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.
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
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.