6.3. Molecular characteristics of the primary peptide binding site of PDI

Protein-protein interaction mediated by efficient binding of substrates is an essential prerequisite for all of the functions of PDI. The b’ domain is known to be the primary peptide binding domain of PDI and sufficient in itself for binding of small substrates, whereas for longer peptides and non-native proteins the presence of domains a or a’ is necessary (Klappa et al. 1998). For C-P4H tetramer assembly the minimum requirement are both of domains b’and a’.

In this work the precise boundaries for domain b’ were defined and the localization of the primary substrate binding site identified. A linker region of 18 amino acids, termed x, was identified between domains b’ and a’ which has previously been thought to be part of the structure of the domain b’.

The binding site was identified based on a molecular model and validated by mutational analysis. Amino acids L242, L244, F258 and I272 define a small hydrophobic pocket in b’ and mutations introduced into these residues greatly diminish or abolish the ability of the protein to bind peptides. The biggest effects were seen for residue I272, as even a conservative mutation of isoleucine to leucine has a negative effect on the peptide binding ability of the protein. Further validation was given by NMR. While the NMR spectra of b’ can be improved by stabilization of the protein into a single state by addition of a hexa-peptide ligand, I272W mutant shows no improvement upon the addition of the ligand.

The only previously reported specificity of substrate binding for any member of the PDI family is for the pancreas specific PDIp, in which the recognition motif is a single amino acid tyrosine or tryptophan with no adjacent negative charge (Ruddock et al. 2000). Therefore it is not surprising that the binding site identified in PDI is also small in size. While the specificity of PDI was not solved here, it seems according to the preliminary studies that a single amino acid may provide the recognition motif for PDI as per PDIp. It is likely the interaction between PDI and misfolded protein substrate may require interaction by hydrogen bonding to the substrate backbone in addition to the binding of a hydrophobic side chain into the binding site in b’ .

NMR and fluorescence studies revealed that there is a dynamic structural exchange occurring in b’ which may be important in the binding processes and in elucidating the complexities of how PDI acts as a folding catalyst. In addition, it was shown that mutating the binding site in b’ domain of PDI has a negative effect on the isomerase activity, but not the oxidase activity of the enzyme. This is consistent with previous observations, that for simple oxidation the isolated catalytic domains are sufficient, but for complex reactions such as isomerisation of disulfides the presence of b’ domain is needed, most probably due to its binding site (Darby et al. 1998).