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Kaira, Bubacarr (2019) Structural Investigation of Zn2+ ions, FXII and Complement receptor gC1q-R interaction: Triggering the Contact activation System. PhD thesis, University of Nottingham.

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Abstract

The contact activation system or CAS, is an important process that contributes in immunity, inflammation and thrombosis. Contact activation is triggered when coagulation FXII (FXII) and High molecular weight kininogen (HK) bound to Prekallikrein (PK) dock on the surface of complement receptor gC1q-R. Binding of FXII and HK to gC1q-R is mediated by Zn2+ ions, and can trigger contact activation which ultimately leads to inflammation and plasma coagulation.

The aim of this PhD project is to mainly elucidate the binding of full-length FXII and Fibronectine type II (FnII) domain binding to gC1q-R and structure determination of FXII FnII binding to Zn2+ ions and also structure of FXII FnII bound to gC1q-R. Several FXII and gC1q-R constructs have been successfully subcloned into plasmid vectors for bacterial and insect (S2) cell expression. Expression and purification of FXII and gC1q-R constructs were successfully achieved.

Solution studies (size exclusion chromatography or gel filtration), Surface Plasmon Resonance (SPR) and X-ray crystallography successfully characterise FXII FnII interaction with gC1q-R. Furthermore, interaction of FXII and HK to gC1q-R in the presence of Zn2+ ions was investigated using gel filtration. The structural data reveals interesting molecular insight into how Zn2+ ions play a role in 3D structures of FXII FnII in isolation and FXII FnII bound to gC1q-R. The FXII FnII bound gC1q-R structure reports novel binding sites, in which FXII FnII bridges two gC1q-R trimers using Arg36 and Arg65 of FXII FnII to interact with G1 and G2 pockets of gC1q-R, respectively. Finally, recombinant gC1q-R was shown to shorten clotting time in plasma in a dose-dependent fashion. In summary, our structural, binding and functional assay are the first reported data shedding light into how Zn2+ induced conformational changes in FXII FnII and broadens our understanding of how contact factors (FXII and HK bound PK) assemble on gC1q-R to trigger contact activation.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Emsley, Jonas Dreveny, Ingrid
Keywords:	contact activation system, CAS, inflammation, binding sites
Subjects:	Q Science > QP Physiology > QP501 Animal biochemistry R Medicine > RB Pathology
Faculties/Schools:	UK Campuses > Faculty of Science > School of Pharmacy
Item ID:	57187
Depositing User:	Kaira, Bubacarr
Date Deposited:	04 Oct 2023 09:58
Last Modified:	04 Oct 2023 09:58
URI:	https://eprints.nottingham.ac.uk/id/eprint/57187

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