adenosine-5--o-(3-thiotriphosphate) and Intellectual-Disability

Tousled-like kinases (TLKs) are required for genome stability and normal development in numerous organisms and have been implicated in breast cancer and intellectual disability. In humans, the similar TLK1 and TLK2 interact with each other and TLK activity enhances ASF1 histone binding and is inhibited by the DNA damage response, although the molecular mechanisms of TLK regulation remain unclear. Here we describe the crystal structure of the TLK2 kinase domain. We show that the coiled-coil domains mediate dimerization and are essential for activation through ordered autophosphorylation that promotes higher order oligomers that locally increase TLK2 activity. We show that TLK2 mutations involved in intellectual disability impair kinase activity, and the docking of several small-molecule inhibitors of TLK activity suggest that the crystal structure will be useful for guiding the rationale design of new inhibition strategies. Together our results provide insights into the structure and molecular regulation of the TLKs.

Topics: Adenosine Triphosphate; Binding Sites; Cloning, Molecular; Crystallography, X-Ray; Escherichia coli; Gene Expression; Genetic Vectors; Humans; Indoles; Intellectual Disability; Kinetics; Molecular Docking Simulation; Mutation; Oximes; Phosphorylation; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Protein Kinase Inhibitors; Protein Kinases; Protein Multimerization; Recombinant Proteins; Substrate Specificity

Polyadenylation, performed by poly(A) polymerases (PAPs), is a ubiquitous post-transcriptional modification that plays key roles in multiple aspects of RNA metabolism. Although cytoplasmic and nuclear PAPs have been studied extensively, the mechanism by which mitochondrial PAP (mtPAP) selects adenosine triphosphate over other nucleotides is unknown. Furthermore, mtPAP is unique because it acts as a dimer. However, mtPAP's dimerization requirement remains enigmatic. Here, we show the structural basis for mtPAP's nucleotide selectivity, dimerization and catalysis. Our structures reveal an intricate dimerization interface that features an RNA-recognition module formed through strand complementation. Further, we propose the structural basis for the N478D mutation that drastically reduces the length of poly(A) tails on mitochondrial mRNAs in patients with spastic ataxia 4 (SPAX4), a severe and progressive neurodegenerative disease.

Topics: Adenosine Triphosphate; Animals; Binding Sites; Chickens; Dimerization; DNA-Directed RNA Polymerases; Humans; Intellectual Disability; Mitochondrial Proteins; Models, Molecular; Muscle Spasticity; Mutation; Nucleotides; Nucleotidyltransferases; Optic Atrophy; Phenotype; RNA; Schizosaccharomyces pombe Proteins; Spinocerebellar Ataxias