Structural and biophysical characterisation of Escherichia coli alpha-2-macroglobulin and its interaction with penicillin binding protein 1C

Fyfe, Cameron D. (2015) Structural and biophysical characterisation of Escherichia coli alpha-2-macroglobulin and its interaction with penicillin binding protein 1C. PhD thesis, University of Glasgow.

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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.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Subjects: Q Science > QR Microbiology
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Bacteriology
Supervisor's Name: Walker, Dr Daniel
Date of Award: 2015
Depositing User: Dr Cameron D. Fyfe
Unique ID: glathesis:2015-6971
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 22 Dec 2015 11:09
Last Modified: 19 Dec 2018 15:03
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