Project 2. Structure and Function of Mitochondrial Protein Kinase

Mitochondrial protein kinases (mPKs), comprising the highly conserved branched-chain alpha-ketoacid dehydrogenase (BCKD) kinase (abbreviated as BCK) and pyruvate dehydrogenase kinases (PDKs), are molecular switches that down-regulate the oxidation of branched-chain alpha-ketoacids (BCKs) and pyruvate in mitochondria. Elevated levels of the alpha-ketoacid are implicated in disease states including maple syrup urine disease (MSUD), primary lactic acidosis and insulin-resistant type II diabetes. The long-term goal of this research is to understand structure and function of the mPKs in the context of their macromolecular multienzyme complexes. This information will provide the basis for rational designs of kinase inhibitors against the above-mentioned human diseases.

Our laboratory has recently solved the crystal structure of rat BCK (The Rat BCK Structure), which serves as a prototype for all mPKs. The BCK structure features a characteristic nucleotide-binding domain and a four-helix bundle domain. These two domains are reminiscent of the modules found in protein histidine kinases (PHKs) in the prokaryotic signal transduction systems. The BCK structure shows the presence of a novel K+-ion binding site (The K+ ion Binding Pocket), which along with the Mg2+ ion mediates nucleotide binding to the kinase. The structure also reveals nucleotide-induced domain communication in BCK, which is likely to be important for the kinase-catalyzed phosphorylation of the E1b component of the BCKD complex. Based on this newly available structural information, we propose to use BCK as a model to provide insight into the mode of action and protein-protein interactions for mPKs. The Specific Aims are:

  1. To identify and characterize the domains/regions in BCK that interact with E1b (the substrate) and E2b (the regulator) components of the BCKD complex

  2. To decipher the functional significance of nucleotide-induced domain communication in BCK (Nucleotide-induced Domain Communication)

  3. To elucidate reaction mechanisms for ATP hydrolysis and phosphotransfer in BCK

Supported by NIH grant DK-62306 in year 2