PKM2 - Human Pyruvate Kinase, Muscle

PDB Code 1ZJH Target Class Non-protein Kinase

Target PKM2
Alias CTHBP, MGC3932, OIP3, PK3, PKM, TCB, THBP1
Disease Area/Function metabolism
Date Deposited 2005-04-28
Authors J.CHOE, A.ATANASSOVA, C.ARROWSMITH, A.EDWARDS, M.SUNDSTROM, A.BOCHKAREV, H.PARK, STRUCTURAL GENOMICS CONSORTIUM (SGC)
Related Structure 3G2G, 3GQY, 3GR4, 3H6O, 3ME3, 3U2Z

Struc Details Tabs

Structure Details
Pyruvate kinase (PK, EC 2.7.1.40) has been extensively studied since the mid 1960s, as the enzyme's deficiency in human erythrocytes is the most common cause of hemolytic anemia. More than 150 mutations in the gene coding PK in erythrocytes, designated as RPK, have been identified so far. The enzyme 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.

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. Much progress has been made in the understanding of the structure and property for RPK, which is expressed in erythrocytes and liver. Less is known about the two other iso-forms, PKM1 and PKM2, which are expressed in muscle, kidney and lung, and are the products of alternative splicing of the same mRNA.

Cations, such as H+, K+, Mg2+ and/or Mn2+, modulate the activity of PKs. Mono- or biphosphorylated sugars as well as PEP activate the enzyme and alter its properties. Phenylalanine is known as a physiological inhibitor of the enzyme. Indeed, we obtained well diffracting crystals when PKM2 was crystallized in presence of phenylalanine, Mg2+ and ADP. However, these ligands and Mg2+ were not located in the structure of PKM2, suggesting that they acted as additives for the crystallization of PMK2. 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 & Methods
StructurePKM2
PDB Code1ZJH
Entry clone accession GI:33286417
Entry clone source MGC
Tag N-terminal histag with thrombin cleavage site: mgsshhhhhhssglvprgs
Construct sequencemgsshhhhhhssglvprgsKPHSEAGTAFIQTQQLHAAMADTFLEHMCRLDIDSPPITARNTGIICTIGPASRSVETLKE
MIKSGMNVARLNFSHGTHEYHAETIKNVRTATESFASDPILYRPVAVALDTKGPEIRTGLIKGSGTAEVELKKGATLKIT
LDNAYMEKCDENILWLDYKNICKVVEVGSKIYVDDGLISLQVKQKGADFLVTEVENGGSLGSKKGVNLPGAAVDLPAVSE
KDIQDLKFGVEQDVDMVFASFIRKASDVHEVRKVLGEKGKNIKIISKIENHEGVRRFDEILEASDGIMVARGDLGIEIPA
EKVFLAQKMMIGRCNRAGKPVICATQMLESMIKKPRPTRAEGSDVANAVLDGADCIMLSGETAKGDYPLEAVRMQHLIAR
EAEAAIYHLQLFEELRRLAPITSDPTEATAVGAVEASFKCCSGAIIVLTKSGRSAHQVARYRPRAPIIAVTRNPQTARQA
HLYRGIFPVLCKDPVQEAWAEDVDLRVNFAMNVGKARGFFKKGDVVIVLTGWRPGSGFTNTMRVVPVP
Vector p28a-LIC
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 g/mL of kanamycin at 37C 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 20C in a SGC LEX bubbling system.
Extraction procedure Cultures were centrifuged and the cell pellets were suspended in 100 mL of the binding buffer (10 mM Tris pH 7.5, 0.5 M NaCl, 5% glycerol, 5 mM imidazole) 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 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 (10mM Tris pH 7.5, 0.5 M NaCl, 5% glycerol, 30 mM imidazole) and the protein was eluted with 15 mL of the elution buffer (10mM Tris pH 7.5, 0.5 M NaCl, 5% glycerol, 250 mM imidazole). The eluate was dialyzed overnight against a buffer containing 10 mM Tris pH 7.5, 0.5 M NaCl, 5% glycerol. 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.
Crystallization 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 (10mM Tris pH 7.5, 0.5 M NaCl, 5% glycerol, 30 mM imidazole) and the protein was eluted with 15 mL of the elution buffer (10mM Tris pH 7.5, 0.5 M NaCl, 5% glycerol, 250 mM imidazole). The eluate was dialyzed overnight against a buffer containing 10 mM Tris pH 7.5, 0.5 M NaCl, 5% glycerol. 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.
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