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Title: Signalling mechanisms of the Toll ectodomain and its extracellular ligand Spätzle
Author: Arnot, C. J.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2010
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The Drosophila Toll transmembrane receptor functions in both embryonic development and the innate immune response against Gram-positive bacteria and fungi. Proteolytic cleavage of Spätzle, its pro-protein ligand, results in an active cysteine knot-containing dimer (C-106) that binds to and activates Toll. This pathway is considered to be the evolutionary precursor of the Toll-like Receptor (TLR) family, which serve as sentinels of the mammalian innate immune system. This study describes how the pro-domain masks the putative receptor-binding site, located at the ‘head-like’ region of C-106. Spectroscopic and binding analysis reveals that the sole Trp29 residue, located within the Trp-loop in the same region, is not only increasingly exposed as the pro-domain is reoriented prior to receptor interaction, but is also involved in pro-domain binding. mutational analysis of Spätzle indicates that a combination of electrostatic and hydrophobic forces govern the structure of C-106 as well as its interaction with the pro-domain and Toll. Furthermore, low resolution structural analysis of the Toll ectodomain, combined with homology modelling and biochemical data, shows that C-106 binds to the N-terminal tip of Toll, inducing the formation of a 2:2 complex with two sites of interaction between the ectodomain chains; one near the N-terminus and the other at the juxtamembrane position. This data therefore suggests that Toll activation is an allosterically controlled mechanism induced by the end-on binding of C-106. Supplementary analysis of TLR2 activation by agonists of non-microbial origin, specifically the lectins KM+ and Galectin-3, provided a limited insight into the non-specific activation of Toll-like Receptors, thereby implying a more flexible model of receptor activation may exist.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available