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Title: Using chemical and structural biology to dissect the Plasmodium invasion motor complex
Author: Douse, Christopher Henry
ISNI:       0000 0004 5918 3585
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2014
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Motility is a key feature of the complex life cycle of Plasmodium falciparum, the apicomplexan parasite that causes human malaria. In particular, the invasion of erythrocytes by blood stages known as merozoites represents the gateway to symptomatic disease. The motive force required for this process is thought to be provided by a conserved actomyosin motor consisting of an unusual myosin (myoA) that is part of a multi-protein assembly making up the biomolecular invasion machinery. One of these proteins is Myosin A Tail Domain Interacting Protein (MTIP), which acts as an anchor for the myoA motor at the inner membrane of the merozoite. The MTIP-myoA complex can be reconstituted in vitro using peptides mimicking the C-terminal tail of myoA, and since inhibition of the interaction in vivo should stall invasion and disrupt the parasitic life cycle, it has been identified as a target for the development of novel antimalarials and chemical genetic tools. This thesis describes a detailed analysis of the structure and dynamics of the MTIP-myoA tail complex, and the subsequent development of molecules intended to disrupt this interaction. Initially, the crystal structure of the complex from P. falciparum is solved and validated as a reasonable model for the solution-state structure using a range of NMR spectroscopic techniques. MTIP forms a rigid clamp around the myoA tail, which adopts a helical conformation and is completely buried by the protein. NMR spectroscopy is also used as a primary tool to investigate MTIP in the absence of a ligand, a crystal structure of which is lacking. The backbone motions of this 'unliganded' protein on a range of timescales are examined, and these data are complemented by other biophysical analyses. Structure-function relationships are extracted and the specificity of MTIP as a binding partner for myoA is investigated. By comparison to 'wild-type' data, the effect of a recently-reported MTIP phosphorylation event is explored, via phospho-mimetic mutation of the site. This analysis provides a structural basis for a possible mode of post-translational regulation of myoA-based motility, since modification appears to break the rigid MTIP-myoA clamp. The structural data are also used to design constrained myoA tail helices, with the aim of improving the cell permeability and stability of wild-type sequences. Three distinct helix constraint technologies are compared using a suite of biophysical experiments, and co-crystal structures with MTIP are solved to validate the design strategy. Further, these structures provide novel insights into the targeting of buried protein-helix interactions. Finally, fragment-based approaches to MTIP-myoA inhibition are investigated. A screen of a 500-member library is conducted, with the aim of identifying small molecules that target hotspots of the interaction and/or stabilise non-productive conformations of MTIP.
Supervisor: Tate, Edward ; Cota, Ernesto Sponsor: Institute of Chemical Biology
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral