*EASTBIO* Structure and function of histidine-rich glycoprotein – a modulator of coagulation, vascularisation and immunity

Histidine-rich glycoprotein (HRG) is a glycosylated ~70 kDa protein present in mammalian blood plasma at relatively high concentrations (~1.5 µM). It has numerous binding partners, such as heparin, divalent metal ions, or heme, and is involved in various regulatory biological processes, such as blood coagulation, angiogenesis, cell migration, proliferation, and adhesion, as well as immune complex clearance. HRG has therefore been referred to as the “Swiss Army knife of mammalian plasma”. The protein exhibits a multi-domain structure with two N-terminal and one C-terminal domain and a central histidine-rich region (HRR) that is flanked by proline-rich-regions (PRRs) on either side. The HRR binds divalent metal ions, and binding of Zn(II) was demonstrated to regulate HRG activity by modifying its affinities to other targets and ligands. Apart from the multi-domain arrangement, there is very limited structural information available on the HRG protein, with only one high-resolution structure of its N2 domain (reported by Stewart group) available to date [1]. The HRR and both PRRs are predicted to be intrinsically disordered, which might explain the lack of available high-resolution structures for the full-length protein. Work in our labs has shown that HRG possesses up to 10 Zn(II) sites [2]. However, to date there is little structural information relating to these sites. We have previously utilised Cu(II) as a proxy for Zn(II) and employ EPR (electron paramagnetic resonance)-based approaches (continuous wave, pulse dipolar and hyperfine methods) to probe the metal ion binding sites on HRG and establish their properties.
The project will initially establish the recombinant production of HRG wild-type and modified HRG constructs. We will aim to establish the structures and topologies of HRG domains ultimately seeking to understand structure and function of full length HRG. Based on our previous work we will investigate metal ion and effector binding and their functional relevance.
Once expression of the protein is established we will seek spin and fluorophore labelling in presence of essential cysteines by incorporation of artificial amino acids.
This project will exploit the combined expertise of the Bode lab, driving methodology for biological EPR spectroscopy, and the Stewart lab investigating metal ions in medicine. The prospective student will receive training in a wide range of skills: molecular biology and mutagenesis, protein purification, spin-labelling, hands-on EPR on advanced instruments and data analysis.
This is an exciting opportunity to expand your skills in both, biomedical sciences and physical sciences, and bring them to bear tackling an elusive target of high medical relevance.
The successful candidate will have experience in molecular biology and have enthusiasm for working with cutting edge instrumentation and advanced structural and functional models.

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