Structural Characterisation of Chordin Family Regulators and Their Interactions with Twisted Gastrulation

Abstract

Bone morphogenetic protein (BMP) signalling is a critical signalling pathway in embryonic development and adult tissue homeostasis. Consequently, aberrant signalling is associated with a number of pathologies. In the extracellular matrix, the chordin family of proteins antagonise BMP signalling by preventing interaction of BMPs with their cognate receptors. Twisted gastrulation (Tsg) has a dual role in the modulation of the chordin and chordin-like proteins of this family, wherein Tsg exhibits both pro- and anti-BMP behaviour. Tsg interacts with both chordin and BMPs to form a chordin/BMP/Tsg ternary complex, enhancing the interaction of chordin with BMPs, to act in an anti-BMP manner. Tsg has also been shown to increase BMP antagonism by chordin-like 1 (CHRDL1) and chordin-like 2 (CHRDL2). Additionally, Tsg enhances the cleavage of chordin by tolloid proteinases, which results in chordin inactivation, to act in a pro-BMP manner. The mechanism behind the dual roles of Tsg is not known, and a Tsg-containing complex has not been previously structurally characterised. It is hypothesised that Tsg causes a conformational change in chordin. BMP point mutants suggest differences in the binding of BMPs to chordin and CHRDL2. Therefore, there may also be subtle differences in the modulation of chordin and chordin-like proteins by Tsg. This thesis aims to contribute to the understanding of the chordin family of BMP antagonists and their modulation by Tsg. This study identifies the chordin von Willebrand Factor C (vWC) 2 and vWC3 domains as the specific Tsg-binding region of chordin. This may place Tsg at the centre of the ternary complex, facilitating interactions with both chordin and BMPs. A chordin/Tsg complex could not be isolated for structural characterisation, and further stabilisation of the complex may be required. This thesis provides the first structural characterisation of CHRDL2. Furthermore, a CHRDL2/Tsg complex has been successfully isolated and investigated. Analytical ultracentrifugation, multi-angle light scattering, and small angle X-ray scattering were used to characterise CHRDL2 and the CHRDL2/Tsg complex. These indicate CHRDL2 exists as a monomer in solution, with an elongated conformation. CHRDL2 and Tsg appear to form a 1:1 complex in solution, with Tsg binding towards the end of CHRDL2, maintaining an overall elongated shape.

Date of Award	1 Aug 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Clair Baldock (Supervisor) & Hilary Ashe (Supervisor)