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Title: Structural and biochemical characterisation of the regulatory principles of twitchin kinase, a member of the titin-like family of muscle kinases
Author: Williams, R. M.
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2016
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The titin-like kinases are mechanotransducers of stretch-activated signalling pathways in the muscle sarcomere, thought to undergo mechanoactivation in response to stretch in the myofibril. This work explores the mechanistic principles of twitchin kinase, a titin-like kinase from Caenorhabditis elegans. We show that twitchin kinase undergoes extensive autophosphorylation of its catalytic domain, identifying four modification sites in key mechanistic regions. We find that twitchin kinase is not activated by autophosphorylation as is common in protein kinases, but uses autophosphorylation to inhibit catalysis. We conclude that modification of the twitchin kinase substrate-binding region is likely to be a major contributor to inhibition, with phosphorylation at multiple sites having a cumulative effect to silence catalysis. We present the crystal structure of the active twitchin kinase conformation, showing that conformational changes in twitchin kinase are limited to closure of the glycine-rich loop, with the aC-helix adopting an unusually open conformation while still maintaining catalytically essential active site interactions. Our structure supports the hypothesis of mechanical activation, in which significant rearrangement of the catalytic domain would not be possible under mechanical stress. Finally, we establish the use of DNA molecular springs to study the mechanoenzymatic properties of twitchin kinase. Twitchin kinase:DNA chimeras have been constructed to apply mechanical stress while monitoring catalysis. We show that twitchin kinase is catalytically active under mechanical deformation, supporting the hypothesis of mechanoactivation and providing a reliable method for future work. Together, these results show that twitchin kinase is likely to employ a triple mechanism for regulating catalysis, combining biochemical regulation by autophosphorylation with intrasteric inhibition by N- and C-terminal regulatory tails, indicative of the need for strict regulation of twitchin kinase activity.
Supervisor: Mayans, O. ; Rigden, D. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral