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Protein disulfide isomerase (PDI) is a multifunctional protein of the endoplasmic reticulum, which catalyzes the formation, breakage and rearrangement of disulfide bonds during protein folding. It consists of four domains designated a, b, b′ and a′. Both a and a′ domains contain an active site with the sequence motif -Cys-Gly-His-Cys- involved directly in thiol-disulfide exchange reactions. As expected these domains have structures very similar to the ubiquitous redox protein thioredoxin. A low-resolution NMR structure of the b domain revealed that this domain adopts a fold similar to the PDI a domain and thioredoxin [Kemmink, J., Darby, N.J., Dijkstra, K., Nilges, M. and Creighton, T.E. (1997) Curr. Biol., 7, 239–245]. A refined ensemble of solution structures based on the input of 1865 structural restraints shows that the structure of PDI b is well defined throughout the complete protein except for about 10 residues at the C-terminus of the sequence. 15N relaxation data show that these residues are disordered and not part of this structural domain. Therefore the domain boundaries of PDI can now be fixed with reasonable precision. Structural comparison of the PDI b domain with thioredoxin and PDI a reveals several features important for thiol-disulfide exchange activity.

Recent protein engineering studies have confirmed the multidomain nature of protein disulfide isomerase previously suggested on the basis of analysis of its amino acid sequence. The boundaries of three domains, denoted a, a′ and b, have been determined, and each domain has been expressed as an individual soluble folded protein. In this report, the boundaries of the final structural domain, b′, are defined by a combination of restricted proteolysis and protein engineering approaches to complete our understanding of the domain organization of PDI. Using these data an optimized polypeptide construct has been prepared and characterized with a view to further structural and functional studies.

Protein disulfide isomerase (PDI) catalyzes protein folding linked to disulfide bond formation in secreted proteins. It consists of four major domains, denoted a, b, b′ and a′. The a and a′ domains each contain an active site motif, -CGHC-, which is directly involved in thiol-disulfide exchange reactions during catalysis. The roles of the b and b′ domains and the functional necessity for the multi-domain structure of PDI are unknown. We now demonstrate that full catalytic activity requires the involvement of multiple PDI domains and that the b′ domain has a particularly important role in catalysis. Reconstruction of the PDI molecule from the isolated a and a′ domains results in a progressive increase in catalytic efficiency as further domains are added. These effects are especially significant in the catalysis of disulfide bond rearrangements in folded substrates, for which all the domains of the protein are required for maximum catalytic efficiency. It is likely that all of the domains of PDI participate in substrate binding interactions and that PDI has evolved its multidomain structure as an adaptation that allows it to catalyze transformations involving difficult conformational changes.