Characterization of the Human Atypical Kinase, aarf Domain-Containing Kinase 3, and its Substrates Required for Coenzyme Q biosynthesis
ADCK3 , Coq8 , Protein Expression , Protein Purification , X-Ray Crystallography , Coenzyme Q , Coq5 , Coq7 , Methyltransferase , ATPase Activity Assay , Kinase Activity Assay , Protein Kinase , Atypical Kinase , Co-Immunoprecipitation
Aarf domain-containing kinase 3, ADCK3, is a human atypical protein kinase located in the inner membrane of the mitochondria that is required for the biosynthesis of coenzyme Q (CoQ). Stable supply of CoQ is essential to human health as it plays a critical role as an electron transporter in the electron transport chain which is required for proper ATP production. Although the exact role of ADCK3 is unknown, patients possessing mutations within this gene are often diagnosed with autosomal recessive cerebellar ataxia type 2, which results from CoQ deficiency. To better understand the involvement of ADCK3 and other proteins in CoQ biosynthesis, their structure, function, and their interactions with each other were investigated. ADCK3 is hypothesized to regulate the CoQ biosynthesis using its kinase activity to activate several proteins found in the CoQ biosynthetic complex; however, previous work has failed to show direct evidence of its kinase activity and its functional interaction with the other Coq proteins. In this work, using a soluble construct of ADCK3, we solved its structure by X-ray crystallography. Various constructs of ADCK3 were transfected into Human Embryonic Kidney cells in an attempt to extract and identify its substrates. Further, the genes of the potential substrates, Coq3, Coq5, and Coq7 were cloned into various expression vectors and transformed into Escherichia coli expression cell lines. After the extensive expression trials, the combination of N-terminal fusions with maltose-binding proteins and the RIPL E. coli cell expression line was found to facilitate the expression of Coq5 and Coq7. However, using radiometric kinase assays, we show that ADCK3 exhibited no kinase activity towards Coq5 and Coq7. Finally, this work also shows the methyltransferase activity of Coq5 using its substrate analog. It is anticipated that our results will serve as a foundation for further characterization of the proteins involved in the human CoQ10 biosynthesis.