Dataset Information

Synergistic Allosteric Mechanism of Fructose-1,6-bisphosphate and Serine for Pyruvate Kinase M2 via Dynamics Fluctuation Network Analysis.

ABSTRACT: Pyruvate kinase M2 (PKM2) plays a key role in tumor metabolism and regulates the rate-limiting final step of glycolysis. In tumor cells, there are two allosteric effectors for PKM2: fructose-1,6-bisphosphate (FBP) and serine. However, the relationship between FBP and serine for allosteric regulation of PKM2 is unknown. Here we constructed residue/residue fluctuation correlation network based on all-atom molecular dynamics simulations to reveal the regulation mechanism. The results suggest that the correlation network in bound PKM2 is distinctly different from that in the free state, FBP/PKM2, or Ser/PKM2. The community network analysis indicates that the information can freely transfer from the allosteric sites of FBP and serine to the substrate site in bound PKM2, while there exists a bottleneck for information transfer in the network of the free state. Furthermore, the binding free energy between the substrate and PKM2 for bound PKM2 is significantly lower than either of FBP/PKM2 or Ser/PKM2. Thus, a hypothesis of "synergistic allosteric mechanism" is proposed for the allosteric regulation of FBP and serine. This hypothesis was further confirmed by the perturbational and mutational analyses of community networks and binding free energies. Finally, two possible synergistic allosteric pathways of FBP-K433-T459-R461-A109-V71-R73-MG2-OXL and Ser-I47-C49-R73-MG2-OXL were identified based on the shortest path algorithm and were confirmed by the network perturbation analysis. Interestingly, no similar pathways could be found in the free state. The process targeting on the allosteric pathways can better regulate the glycolysis of PKM2 and significantly inhibit the progression of tumor.

SUBMITTER: Yang J

PROVIDER: S-EPMC5115163 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Synergistic Allosteric Mechanism of Fructose-1,6-bisphosphate and Serine for Pyruvate Kinase M2 via Dynamics Fluctuation Network Analysis.

Yang Jingxu J Liu Hao H Liu Xiaorui X Gu Chengbo C Luo Ray R Chen Hai-Feng HF

Journal of chemical information and modeling 20160609 6

Pyruvate kinase M2 (PKM2) plays a key role in tumor metabolism and regulates the rate-limiting final step of glycolysis. In tumor cells, there are two allosteric effectors for PKM2: fructose-1,6-bisphosphate (FBP) and serine. However, the relationship between FBP and serine for allosteric regulation of PKM2 is unknown. Here we constructed residue/residue fluctuation correlation network based on all-atom molecular dynamics simulations to reveal the regulation mechanism. The results suggest that t ...[more]

PMID: 27227511

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Project description:A variant form of yeast pyruvate kinase (EC 2.7.1.40) with Ser-384 mutated to proline has been engineered in order to study the allosteric properties of this enzyme. Both the mutant and wild-type enzymes were overexpressed in a strain of yeast in which the genomic copy of the pyruvate kinase gene had been disrupted by an insertion of the Ura3 gene. Both enzymes were purified to homogeneity and their kinetic properties characterized. The wild-type enzyme displays sigmoid kinetics with respect to phosphoenolpyruvate (PEP) concentration, and is activated by the allosteric effect fructose 1,6-bisphosphate with concomitant reduction in co-operativity. In contrast, the mutant was found to be dependent on the presence of the effector for catalytic activity and was inactive in its absence. The fully activated mutant enzyme had a kcat. 1.6 times greater than that of the wild-type enzyme. The mutation introduced into the enzyme is in an intersubunit contact which is known to be critical for the allosteric properties of the enzyme, and is far removed from the active site. The major effect of the mutation seems to be to stabilize the low-affinity T state of the apoenzyme, although kcat. is also affected. The S0.5 for PEP and S0.5 for ADP of the wild-type enzyme were 0.22 +/- 0.004 and 0.15 +/- 0.01 mM respectively (means +/- S.E.M.). In the activated mutant enzyme, these kinetic parameters increased to 0.67 +/- 0.03 and 0.43 +/- 0.03 mM respectively. The cooperativity between ADP-binding sites was altered in the mutant enzyme, with the Hill coefficient (h) for ADP increasing to 1.65 +/- 0.07 in the presence of the effector, compared with a value of 0.01 +/- 0.07 for the wild-type enzyme under the same conditions. CD spectroscopy revealed the secondary structure of the mutant enzyme to be little different from that of the wild-type enzyme, indicating that the two enzymes have similar secondary structures in solution. Precise tertiary and quaternary structures such as intersubunit and interdomain interactions may be modified. An improved purification procedure has been devised that allows large quantities of enzyme to be rapidly prepared.

Project description:In the study of allosteric proteins, understanding which effector-protein interactions contribute to allosteric activation is important both for designing allosteric drugs and for understanding allosteric mechanisms. The antihyperglycemic target, human liver pyruvate kinase (hL-PYK), binds its allosteric activator, fructose 1,6-bisphosphate (Fru-1,6-BP), such that the 1'-phosphate interacts with side chains of Arg501 and Trp494 and the 6'-phosphate interacts with Thr444, Thr446, Ser449 (i.e., the 444-449 loop), and Ser531. Additionally, backbone atoms from the 527-533 loop interact with a sugar ring hydroxyl and the two effector phosphate moieties. An effector analogue series indicates that only one phosphate on the sugar is required for activation. However, singly phosphorylated sugars, including Fru-1-P and Fru-6-P, bind with a Kix in the range of 0.07-1 mM. The second phosphate of Fru-1,6-BP causes tight effector binding, because this native effector has a Kix of 0.061 μM. Glucose 1,6-bisphosphate and ribulose 1,5-bisphosphate bind in the 0.07-1 mM range. The contrast with a higher Fru-1,6-BP binding indicates specificity for the fructose sugar conformation. Site-directed random mutagenesis at each residue that contacts bound Fru-1,6-BP showed that a negative charge introduced at position 531 mimics allosteric activation, even in the absence of Fru-1,6-BP. Collectively, analogue and mutagenesis studies are consistent with the 527-533 loop playing a key role in allosteric function. Deletion mutations that shortened the 527-533 loop were expected to prevent formation of hydrogen bonds between backbone atoms on the loop and Fru-1,6-BP. Indeed, Fru-1,6-BP did not activate these loop-shortened mutant proteins. Previous structural comparisons of M1-PYK and M2-PYK indicate that the 527-533 loop makes interactions across a subunit interface when an activator is not present. Mutating the hL-PYK subunit interface interactions among Trp527, Arg528, and Asp499 mimics allosteric activation. Considered with published structures, these results are consistent with (1) the two phosphates of Fru-1,6-BP docking to Arg501/Trp494 and the 444-449 loop, respectively, and (2) the formation of hydrogen bonds among Fru-1,6-BP and backbone atoms of the 527-533 loop pulling this loop away from the subunit interface, which results in breaking of the Trp527-Arg528-Asp499 interactions to elicit an allosteric response.

Dataset Information

Synergistic Allosteric Mechanism of Fructose-1,6-bisphosphate and Serine for Pyruvate Kinase M2 via Dynamics Fluctuation Network Analysis.

Publications

Synergistic Allosteric Mechanism of Fructose-1,6-bisphosphate and Serine for Pyruvate Kinase M2 via Dynamics Fluctuation Network Analysis.

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