Experimental and Computational Studies on the Dynamics and Flexibility of Protein Disulfide-Isomerase (PDI)

PDI is an abundant folding catalyst and chaperone, required for formation of native disulfide bonds in secretory proteins. PDI interacts with many redox partners in the oxidative protein folding pathway and acts on an immense range of unfolded and part-folded protein substrates. It is a multi-domain protein (a-b-b'-x-a'-c) comprising two redox-active thioredoxin-like domains (a, a'), two other domains structurally homologous to thioredoxin (b, b'), a linker (x) and an inessential C-terminal tail (c). Key sites are the redox-active motifs (-WCGHCK-) in a and a' domains, and a promiscuous non-covalent ligand-binding site in b' which can be gated by x. X-ray structures of PDI and its homologues provide static information, but evidence from NMR and intrinsic fluorescence suggests PDI is highly dynamic.We have explored PDI flexibility using FRET and computational techniques. Using yeast PDI, a very rapid method for predicting protein dynamics, based on FRODA and normal mode analysis, has been validated by a comparison of predictions with those from a full MD analysis. Both methods highlight inter-domain motion and show that the character of inter-domain motion at each domain boundary is different, with the x linker providing great freedom for twisting and tilting at the b'-a' interface. Two crystal structures show distinctive twist motion at the a-b interface which is predicted by the rapid method, but not by MD. Similar analyses are now underway on members of the human PDI family.Both theoretical analyses predict that PDI flexibility permits very significant change in the distances between functional sites. We are analysing this experimentally by single-molecule FRET techniques, with FRET probes attached to the redox-active sites and elsewhere. The data indicate that human PDI in solution can access a variety of conformations.