Dissertation

Crystal structure of mammalian casein kinase I exhibits basis for phosphate recognition by a protein kinase family

Longenecker, Kenton Lamar

1997

Overview

Casein kinase I (CKI) enzymes phosphorylate multiple proteins with diverse functions in eukaryotic organisms and may play an important regulatory role in a variety of biological processes. These protein kinases utilize ATP in a reaction forming a phosphate mono-ester that typically can be hydrolyzed by a protein phosphatase. Common features of certain substrates show that CKI activity has an unusual specificity for proteins that are already phosphorylated on an amino acid N-terminal to the site phosphorylated by CKI. Recently, cDNAs encoding several CKI enzymes have been cloned. Sequence analysis reveals that these enzymes constitute a unique family of protein kinases. While their catalytic domains have many similarities, regions outside of this domain vary in length and sequence. To understand the molecular basis for protein phosphorylation by CKI enzymes, experiments were designed to study the three-dimensional structure of a recombinant mammalian isoform of CKI expressed in Escherichia coli. Crystals of a truncation mutant of CKI$\\delta$ lacking a C-terminal autoinhibitory domain diffracted X-rays to 2.3 A and the structure of the catalytic domain was determined by X-ray crystallography. Like other protein kinases, the catalytic domain is composed of two lobes with a cleft between them for binding ATP. Comparison with the recent crystal structure of a CKI homolog from Schizosaccharomyces pombe suggests that a rotation of the N-terminal lobe occurs upon ATP binding. Furthermore, binding of a phosphate analog, tungstate, in a derivative structure reveals an anion binding site that likely contributes to the unique substrate specificity of CKI enzymes. Additional experiments resulted in crystallization of another truncation mutant of CKI$\\delta$, a mutant containing the autoinhibitory domain, and X-ray diffraction data were collected to 2.4 A. Structural analysis suggests that the inhibitory domain is disordered in these crystals, but a conserved intermolecular contact suggests formation of a dimer that would inhibit enzymatic activity. Relating the primary sequences of other CKI enzymes to the three-dimensional architecture of CKI$\\delta$ reveals a catalytic face that is especially conserved among a subset of CKI family members associated with the regulation of DNA repair.

• Publisher: ProQuest Dissertations & Theses
• Subject: Biochemistry / Biophysics
• ISBN: 0591371448, 9780591371444