Structural Insights Into The Regulation Of PKM2 Structure And Function By The Post-translational Modifications And Dominant Negative Mutation

Aerobic glycolysis, known as "Warburg effect", is the main metabolic pathway utilized by cancer cells and is one of the main characteristics of cancer. With reduced generation of ATP, cancer cell tends to accumulate a large amount of metabolic intermediates which are essential for the synthesis of building blocks, such as 3PG, GA3P, G6P and so on, to meet the demand of cell proliferation. PKM2, functioning as a homo-tetramer, catalyzes the final step and the rate-limiting step of glycolysis. Recently, many research demonstrated that PKM2, a key enzyme in glycolysis and cancer metabolism, plays a significant role in cancer metabolism. Many cancer cell lines exclusively express PKM2 but not other isoforms. The regulation of PKM2 through the post-translational modifications, such as phosphorylation, acetylation and oxidation, has been widely investigated, while it has been reported there is dominant negative mutation affecting the enzymatic activity and function of PKM2. It is speculated that the post-translational modifications and dominant negative mutation might affect the overall structure of PKM2 to regulate its function. The protein function is based on its three dimentional structure, hence due to the lack of the information of the relationship between the structure and functional regulation of PKM2, it is valuable to elucidate whether and how the post-translational modifications and dominant negative mutation affect the overall structure of PKM2.In order to study the effect of post-translational modifications and dominant negative mutation on the structure of PKM2, we constructed four mutations to mimic the post-translational modification states and the dominant negative mutation, including Y105 phosphorylation mimic mutation Y105E, K305 acetylation mimic mutation K305Q, dominant negative mutation K422R and artificial mutation R399E. Comprehensive in vitro biochemical assays demonstrated that the mutations showed different behavior compared with the wild type. We solved the crystal structures of all these mutations. According to the intergrated structural analysis, a "See-Saw effect" on the interface of two dimers to control the structural transitions between T-state tetramer and R-state tetramer and to regulate the activity and function of PKM2 was proposed. Mutant Y105E, K422R and R399E regulate the enzymatic activity and function of PKM2 through affecting its tetramer assembly, while mutant K305Q leading to the disruption of tetramer greatly impairs its activity.Taken into consideration of all our results, we elucidated the effect of all these post-translational modifications and mutation to the overall structure of PKM2, proposed a working model for the regulation of PKM2 by post-translational modifications and dominant negative mutation and clarified the comformational change induced by different post-translational modifications and dominant negative mutation. Due to the tight connection between the different post-translational modification states of PKM2 and cancer development and the strong correlation between dominant negative mutation of PKM2 and cancer, our structures provide the structure basis for the structure based drug design against the different post-translational modifications and dominant negative mutation.