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Structural insight into mechanisms for dynamic regulation of PKM2.

Wang P, Sun C, Zhu T, Xu Y - Protein Cell (2015)

Bottom Line: We determined crystal structures of human PKM2 mutants and proposed a "seesaw" model to illustrate conformational changes between an inactive T-state and an active R-state tetramers of PKM2.K422R, a patient-derived mutation of PKM2, favors a stable, inactive T-state tetramer because of strong intermolecular interactions.Our study reveals the mechanism for dynamic regulation of PKM2 by post-translational modifications and a patient-derived mutation and provides a structural basis for further investigation of other modifications and mutations of PKM2 yet to be discovered.

View Article: PubMed Central - PubMed

Affiliation: Fudan University Shanghai Cancer Center and Institute of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai, 200032, China.

ABSTRACT
Pyruvate kinase isoform M2 (PKM2) converts phosphoenolpyruvate (PEP) to pyruvate and plays an important role in cancer metabolism. Here, we show that post-translational modifications and a patient-derived mutation regulate pyruvate kinase activity of PKM2 through modulating the conformation of the PKM2 tetramer. We determined crystal structures of human PKM2 mutants and proposed a "seesaw" model to illustrate conformational changes between an inactive T-state and an active R-state tetramers of PKM2. Biochemical and structural analyses demonstrate that PKM2(Y105E) (phosphorylation mimic of Y105) decreases pyruvate kinase activity by inhibiting FBP (fructose 1,6-bisphosphate)-induced R-state formation, and PKM2(K305Q) (acetylation mimic of K305) abolishes the activity by hindering tetramer formation. K422R, a patient-derived mutation of PKM2, favors a stable, inactive T-state tetramer because of strong intermolecular interactions. Our study reveals the mechanism for dynamic regulation of PKM2 by post-translational modifications and a patient-derived mutation and provides a structural basis for further investigation of other modifications and mutations of PKM2 yet to be discovered.

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Activities and tetramer formation for wild-type PKM2 and mutants of PKM2. (A and B) Kinetic activities of PKM2 proteins in the absence (A) or presence (B) of FBP. The data were fit with the Allosteric Sigmoidal Equation (A) or the Michaelis-Menten Equation (B). (C) Normalized activities (kcat/Km) of wild-type PKM2 and mutants of PKM2 calculated according to the results from Fig. 1A and 1B with the value (kcat/Km) of PKM2WT as a standard. The error bars represent mean ± SD for triplicate experiments. (D and E). Gel filtration of wild-type and mutants of PKM2 in the absence (D) or presence (E) of FBP. Peak positions of monomer, dimer and tetramer are indicated as dashed lines. The color scheme for PKM2 proteins is indicated. Superdex 200 (GE Healthcare, 10/300 GL) was used in gel-filtration analyses
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Fig1: Activities and tetramer formation for wild-type PKM2 and mutants of PKM2. (A and B) Kinetic activities of PKM2 proteins in the absence (A) or presence (B) of FBP. The data were fit with the Allosteric Sigmoidal Equation (A) or the Michaelis-Menten Equation (B). (C) Normalized activities (kcat/Km) of wild-type PKM2 and mutants of PKM2 calculated according to the results from Fig. 1A and 1B with the value (kcat/Km) of PKM2WT as a standard. The error bars represent mean ± SD for triplicate experiments. (D and E). Gel filtration of wild-type and mutants of PKM2 in the absence (D) or presence (E) of FBP. Peak positions of monomer, dimer and tetramer are indicated as dashed lines. The color scheme for PKM2 proteins is indicated. Superdex 200 (GE Healthcare, 10/300 GL) was used in gel-filtration analyses

Mentions: To investigate how the enzymatic activity of PKM2 is regulated, we purified wild-type PKM2 (PKM2WT), PKM2R399E (a dimeric mutant) (Gao et al., 2012) and PKM2K422R (a patient-derived mutation). Given the difficulty to obtain phosphorylated or acetylated PKM2 proteins, we purified PKM2Y105E (a phosphorylation mimic of Y105) and PKM2K305Q (an acetylation mimic of K305) to mimic PKM2 containing the two modifications. The above four PKM2 mutants and PKM2WT were used for enzymatic activity assays and structural studies (Fig. S1A). We first measured the enzymatic activities for wild-type and mutants of PKM2 and calculated the Km values for PEP. The kinetic activities were calculated based on the PEP saturation curves in the absence or presence of FBP, an allosteric activator of PKM2 (Fig. 1A and 1B). We also calculated the normalized pyruvate kinase activity (kcat/Km, (mol/L)−1 s−1), as represented by the value of Vmax/Km of PKM2WT, because an equal amount of protein was used for all activity assays (Fig. 1C). We could not detect protein kinase activity for human PKM2 under our experimental conditions using histone H3 as substrate (data not shown). Therefore, only pyruvate kinase activities (enzymatic activity hereafter if not specified) were measured for wild-type and mutants of PKM2. A high enzyme concentration in the reaction would greatly decrease the accuracy of calculated reaction rate. Because of the extremely fast reaction rate (less than 1.5 min), we measured the pyruvate kinase activity in nanomolar concentrations.Figure 1


Structural insight into mechanisms for dynamic regulation of PKM2.

Wang P, Sun C, Zhu T, Xu Y - Protein Cell (2015)

Activities and tetramer formation for wild-type PKM2 and mutants of PKM2. (A and B) Kinetic activities of PKM2 proteins in the absence (A) or presence (B) of FBP. The data were fit with the Allosteric Sigmoidal Equation (A) or the Michaelis-Menten Equation (B). (C) Normalized activities (kcat/Km) of wild-type PKM2 and mutants of PKM2 calculated according to the results from Fig. 1A and 1B with the value (kcat/Km) of PKM2WT as a standard. The error bars represent mean ± SD for triplicate experiments. (D and E). Gel filtration of wild-type and mutants of PKM2 in the absence (D) or presence (E) of FBP. Peak positions of monomer, dimer and tetramer are indicated as dashed lines. The color scheme for PKM2 proteins is indicated. Superdex 200 (GE Healthcare, 10/300 GL) was used in gel-filtration analyses
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig1: Activities and tetramer formation for wild-type PKM2 and mutants of PKM2. (A and B) Kinetic activities of PKM2 proteins in the absence (A) or presence (B) of FBP. The data were fit with the Allosteric Sigmoidal Equation (A) or the Michaelis-Menten Equation (B). (C) Normalized activities (kcat/Km) of wild-type PKM2 and mutants of PKM2 calculated according to the results from Fig. 1A and 1B with the value (kcat/Km) of PKM2WT as a standard. The error bars represent mean ± SD for triplicate experiments. (D and E). Gel filtration of wild-type and mutants of PKM2 in the absence (D) or presence (E) of FBP. Peak positions of monomer, dimer and tetramer are indicated as dashed lines. The color scheme for PKM2 proteins is indicated. Superdex 200 (GE Healthcare, 10/300 GL) was used in gel-filtration analyses
Mentions: To investigate how the enzymatic activity of PKM2 is regulated, we purified wild-type PKM2 (PKM2WT), PKM2R399E (a dimeric mutant) (Gao et al., 2012) and PKM2K422R (a patient-derived mutation). Given the difficulty to obtain phosphorylated or acetylated PKM2 proteins, we purified PKM2Y105E (a phosphorylation mimic of Y105) and PKM2K305Q (an acetylation mimic of K305) to mimic PKM2 containing the two modifications. The above four PKM2 mutants and PKM2WT were used for enzymatic activity assays and structural studies (Fig. S1A). We first measured the enzymatic activities for wild-type and mutants of PKM2 and calculated the Km values for PEP. The kinetic activities were calculated based on the PEP saturation curves in the absence or presence of FBP, an allosteric activator of PKM2 (Fig. 1A and 1B). We also calculated the normalized pyruvate kinase activity (kcat/Km, (mol/L)−1 s−1), as represented by the value of Vmax/Km of PKM2WT, because an equal amount of protein was used for all activity assays (Fig. 1C). We could not detect protein kinase activity for human PKM2 under our experimental conditions using histone H3 as substrate (data not shown). Therefore, only pyruvate kinase activities (enzymatic activity hereafter if not specified) were measured for wild-type and mutants of PKM2. A high enzyme concentration in the reaction would greatly decrease the accuracy of calculated reaction rate. Because of the extremely fast reaction rate (less than 1.5 min), we measured the pyruvate kinase activity in nanomolar concentrations.Figure 1

Bottom Line: We determined crystal structures of human PKM2 mutants and proposed a "seesaw" model to illustrate conformational changes between an inactive T-state and an active R-state tetramers of PKM2.K422R, a patient-derived mutation of PKM2, favors a stable, inactive T-state tetramer because of strong intermolecular interactions.Our study reveals the mechanism for dynamic regulation of PKM2 by post-translational modifications and a patient-derived mutation and provides a structural basis for further investigation of other modifications and mutations of PKM2 yet to be discovered.

View Article: PubMed Central - PubMed

Affiliation: Fudan University Shanghai Cancer Center and Institute of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai, 200032, China.

ABSTRACT
Pyruvate kinase isoform M2 (PKM2) converts phosphoenolpyruvate (PEP) to pyruvate and plays an important role in cancer metabolism. Here, we show that post-translational modifications and a patient-derived mutation regulate pyruvate kinase activity of PKM2 through modulating the conformation of the PKM2 tetramer. We determined crystal structures of human PKM2 mutants and proposed a "seesaw" model to illustrate conformational changes between an inactive T-state and an active R-state tetramers of PKM2. Biochemical and structural analyses demonstrate that PKM2(Y105E) (phosphorylation mimic of Y105) decreases pyruvate kinase activity by inhibiting FBP (fructose 1,6-bisphosphate)-induced R-state formation, and PKM2(K305Q) (acetylation mimic of K305) abolishes the activity by hindering tetramer formation. K422R, a patient-derived mutation of PKM2, favors a stable, inactive T-state tetramer because of strong intermolecular interactions. Our study reveals the mechanism for dynamic regulation of PKM2 by post-translational modifications and a patient-derived mutation and provides a structural basis for further investigation of other modifications and mutations of PKM2 yet to be discovered.

Show MeSH
Related in: MedlinePlus