Titin role in muscle homeostasis: the kinase domain

Bogomolovas, Julijus
Titin role in muscle homeostasis: the kinase domain. Doctor of Philosophy thesis, University of Liverpool.

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The giant muscle protein titin is a central player in cardiovascular health and disease. Titin molecules spanning half of the sarcomere form a filament system in striated muscles. The titin filament is not only an essential structural component of sarcomere, but also plays a central role in myofibril signaling through its kinase domain (TK) and numerous protein ligands. Thus, not surprisingly, mutations in this molecule might have detrimental effects. In this work a structure-driven approach was taken to re-evaluate titin kinase catalytic activity and the pathogenicity of two cardiomyopathy-associated titin mutations. Comparison of recombinant preparations from E.coli and insect cells revealed intrinsic inactivity of TK. It was demonstrated that previously reported phosphotransfer activity towards Tcap (a small Z-disc protein) is due to contaminant kinase activity from insect cells but not TK itself. Next, it was established that the eukaryotic host produces structurally indistinguishable TK from that produced in insect cells, albeit inactive towards Tcap, classical kinase substrates or extracts from mature or differentiating muscles. Structural analysis identified evolutionary conserved residue substitutions converting vertebrate TKs to pseudokinases. The structural context of dilated cardiomyopathy associated mutation was revealed in the crystal structure of TK enclosing neighboring domains A170 and M1. The mutation site resides in a conserved helix located in the linker region between TK and the binding site of ubiquitin E3 ligase MuRF1. Aspartate to valine substitution causes disruption of a conserved hydrogen bond and detachment of the affected helix from TK. In the context of the titin filament, this causes dissociation of the binding site from TK and increases interdomain flexibility. Structural alterations translate into increased MuRF1-mediated degradation of mutant titin fragments through the ubiquitin-proteasome pathway. Speculatively, haploinsufficiency of mutant titin could be a possible pathomechanism leading to dilated cardiomyopathy associated with the mutation under study. Comprehensive analysis of arrhythmogenic left ventricular cardiomyopathy associated titin mutation generated a novel model of pathogenesis. In contrast to previous reports, we demonstrate that mutation does not cause affected domain I10 unfolding, and is structurally compatible. Observed destabilization of the domain was attributed to a disrupted hydrogen bond, causing increased flexibility. A crystal structure of the affected domain flanked by adjacent domains I9-I11 demonstrated that threonine to isoleucine substitution might have detrimental effects on interdomain arrangement, resulting in exposure of a hydrophobic patch. Functionally, differential localization of mutant protein was observed in transgenic muscles. Speculatively, mutation could result in impaired folding of mutant protein and lead to accumulation of degradation-resistant aggregates or cause an increased stickiness to thin filaments as a novel pathomechanism. Results presented in this work demonstrate that TK is a catalytically inactive pseudokinase acting as a molecular scaffold. It was demonstrated that TK and MuRF1 signaling modules are structurally interconnected and genetic perturbation of this link might lead to dilated cardiomyopathy. In similar fashion, genetic alteration of interaction between immunoglobulin domains might cause arrhythmogenic left ventricular cardiomyopathy.

Item Type: Thesis (Doctor of Philosophy)
Additional Information: Date: 2014-02 (completed)
Uncontrolled Keywords: titin filament, sarcomere, cardiomyopathy, X-ray crystallography, Biomolecular NMR, protein kinase
Subjects: ?? QH301 ??
Divisions: Faculty of Health and Life Sciences
Depositing User: Symplectic Admin
Date Deposited: 06 Aug 2014 09:00
Last Modified: 16 Dec 2022 04:42
DOI: 10.17638/00018355
  • Mayans, Olga
  • Wilkinson, Mark
URI: https://livrepository.liverpool.ac.uk/id/eprint/18355