The Crystal Structure of Cancer Osaka Thyroid Kinase Reveals an Unexpected Kinase Domain Fold

• Published in: The Journal of biological chemistry Vol. 290; no. 24; pp. 15210 - 15218
• Main Authors: Gutmann, Sascha, Hinniger, Alexandra, Fendrich, Gabriele, Drückes, Peter, Antz, Sylvie, Mattes, Henri, Möbitz, Henrik, Ofner, Silvio, Schmiedeberg, Niko, Stojanovic, Aleksandar, Rieffel, Sebastien, Strauss, André, Troxler, Thomas, Glatthar, Ralf, Sparrer, Helmut
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 12.06.2015; American Society for Biochemistry and Molecular Biology
• Subjects: cancer; Crystallography, X-Ray; drug discovery; Humans; immunology; MAP Kinase Kinase Kinases - chemistry; mitogen-activated protein kinase (MAPK); Models, Molecular; Protein Conformation; Protein Folding; Protein Structure and Folding; Proto-Oncogene Proteins - chemistry; Recombinant Proteins - chemistry; structural biology; mitogen-activated protein kinase (MAPK); cancer; structural biology; drug discovery; immunology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M115.648097

Macrophages are important cellular effectors in innate immune responses and play a major role in autoimmune diseases such as rheumatoid arthritis. Cancer Osaka thyroid (COT) kinase, also known as mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and tumor progression locus 2 (Tpl-2), is a serine-threonine (ST) kinase and is a key regulator in the production of pro-inflammatory cytokines in macrophages. Due to its pivotal role in immune biology, COT kinase has been identified as an attractive target for pharmaceutical research that is directed at the discovery of orally available, selective, and potent inhibitors for the treatment of autoimmune disorders and cancer. The production of monomeric, recombinant COT kinase has proven to be very difficult, and issues with solubility and stability of the enzyme have hampered the discovery and optimization of potent and selective inhibitors. We developed a protocol for the production of recombinant human COT kinase that yields pure and highly active enzyme in sufficient yields for biochemical and structural studies. The quality of the enzyme allowed us to establish a robust in vitro phosphorylation assay for the efficient biochemical characterization of COT kinase inhibitors and to determine the x-ray co-crystal structures of the COT kinase domain in complex with two ATP-binding site inhibitors. The structures presented in this study reveal two distinct ligand binding modes and a unique kinase domain architecture that has not been observed previously. The structurally versatile active site significantly impacts the design of potent, low molecular weight COT kinase inhibitors. Background: Cancer Osaka thyroid (COT) kinase plays a crucial role in inflammatory diseases and cancer. Results: Production of catalytically competent COT kinase yielded protein suitable for structure guided drug discovery. Conclusion: COT kinase has a unique and structurally versatile active site. Significance: The discovery of a novel variation of the protein kinase fold will impact drug discovery for COT kinase.