Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.676585
Title: Structural and biophysical characterisation of Escherichia coli alpha-2-macroglobulin and its interaction with penicillin binding protein 1C
Author: Fyfe, Cameron D.
ISNI:       0000 0004 5373 0031
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2015
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Abstract:
The alpha-2-macroglobulin superfamily consists of large multi-domain proteins that are activated by protease cleavage. One arm of this family consists of protease inhibitors that undergo a large conformational change upon protease cleavage, simultaneously physically trapping the cleaving protease and covalently linking to it via a thioester bond. However, there is little mechanistic understanding of how protease cleavage activates the conformational changes that lead to protease inactivation. These protease inhibitors are found in tetrameric, dimeric and monomer forms within eukaryotic blood/lymph fluid. The recently described Escherichia coli alpha-2-macroglobulin (ECAM) is a periplasmic, inner membrane anchored protease inhibitor. The gene encoding ECAM, yfhM, is found within an operon alongside pbpC, which encodes penicillin binding protein 1C. These two proteins have been proposed to function in defence and repair against host proteases with ECAM acting to inhibit proteases that have breached the outer membrane and Pbp1C repairing damage to the peptide linkages within the peptidoglycan layer. This thesis describes the structural and biophysical characterisation of ECAM and an investigation into the role of Pbp1C in ECAM function. In order to gain insight into the mechanism through which protease cleavage activates ECAM we used a combination of X-ray crystallography, small angle X-ray scattering and analytical ultracentrifugation to characterise the conformational changes that occur on protease cleavage. The X-ray structure of protease cleaved ECAM revealed a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native inter-domain interactions that maintain ECAM in the inactivated form. Owing to the similarity in structure and domain architecture of ECAM and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group. Further to this, it was shown that ECAM is processed in vivo, existing largely as truncated forms in growing E. coli cells. Interestingly, Pbp1C plays a key role in ECAM processing, with cell lacking pbpC showing an accumulation of full-length and dimeric forms of ECAM.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.676585  DOI: Not available
Keywords: QR Microbiology
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