Structural, Ligand Binding and Redox Studies of hPDI fragments using NMR spectroscopy

Human Protein Disulphide Isomerase (hPDI) is an important protein folding catalyst involved in the reduction, oxidation and isomerisation of disulphide bonds, a major rate-limiting step in the formation of many proteins. hPDI also exhibits molecular chaperone activity, selectively binding small, unfolded peptides and a large range of mis-folded protein intermediates. hPDI is the founding member of a family of 21 structurally related proteins, and consists of domain subunits a and a' (which are catalytically active) and b and b' (which are not). b domain is thought to confer structural stability to the protein, and b' contains the primary ligand binding site. There is also an x-linker region, and a C-terminal acidic tail c, in the complete domain structure abb'xa'c.

Despite a recently solved crystal structure of hPDI abb'xa' (4EKZ.pdb) and over 50 years of research, we still have limited knowledge of how hPDI works, and more specifically how it interacts with ligands. Obtaining more detailed information is of significant importance; not only is hPDI associated with several human disease states, but it shows great potential as a therapeutic treatment, and is also used industrially to improve recombinant protein yields in mammalian and bacterial cell expression systems. Nonetheless, physiological ligand specificity of hPDI is still poorly understood.

This study focuses on characterising the ligand binding ability and specificity of hPDI using a model binding peptide, ?-somatostatin (?-som), and two recombinantly expressed fragments of hPDI: b'x, the smallest monomeric fragment of hPDI capable of ligand binding, and abb'x, a larger fragment previously uncharacterised by NMR. Backbone and side-chain resonance assignments were carried out, and allowed hPDI binding affinity to be quantified using both protein-observed and ligand-observed NMR methods. Peptide affinity was in agreement with the previously estimated 0.1-1 mM affinity, and was found to be independent of neighbouring domains and protein redox state. Fluorination of the peptide at key aromatic residues and subsequent Ala substitutions showed that the three Phe residues of the peptide all contributed in some part to hPDI binding, with substitution of the C-terminal Phe having the largest negative effect. Substitution of the three Phe residues to Ala abolished binding to hPDI, suggesting that unlike the family member PDIp (pancreas-specific Protein Disulphide Isomerase), hPDI ligand specificity may be mediated by Phe residues.

Investigation of abb'x redox potential by NMR supported recently published values of full- length protein as determined by mass spectrometry, but also highlighted how the redox state of a domain becomes progressively more reducing upon addition of subsequent domains to the protein, further confirming cross-talk among the four domains of hPDI.

Recombinant fluoroindole incorporation into abb'x showed the incorporation rate was not as extensive as previously reported for b'x fragment. Preliminary work showed the fluorine probe did not significantly alter ligand binding affinity, but in contrast reported a markedly more reducing redox potential, suggesting care must be taken to consider the potential impact protein fluorine modification can infer.