The <i>Drosophila </i>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 Trp<sup>29</sup> 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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:596167 |
| Date | January 2010 |
| Creators | Arnot, C. J. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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