Abstract The correct three dimensional structures of proteins are essential for their ability to function properly. Proteins start to fold as soon as they are synthesized in the ribosomes from activated amino acids. Many secreted, cell-surface, secretory pathway and endoplasmic reticulum (ER) lumenal proteins have in their amino acid sequence cysteine residues which form intra- and intermolecular disulfide bridges that stabilize the overall fold of the proteins and protein complexes. The formation of correct disulfide bonds is a complex process which takes place within the ER. Protein disulfide isomerase (PDI) is the key enzyme in the formation and rearrangement of correct disulfide bonds in the ER. It is an archetypal and the best studied member of the PDI family, i.e. a group of ER proteins that resemble thioredoxin (TRX), a protein reductase, in their structure. PDI has a four domain a-b-b'-a' structure the a and a' domains having the catalytic activity and amino acid sequence similarity to TRX. In addition to its function as a thiol-disulfide oxidoreductase, PDI acts as the β subunit in two protein complexes: collagen prolyl 4-hydroxylase (C-P4H) and microsomal triglyceride transfer protein (MTP). The closest homologue of PDI is the multifunctional enzyme and chaperone ERp57 that functions in concert with two lectins, calnexin (CNX) and calreticulin (CRT) specifically in the folding of proteins that have sugar moieties linked to them. ERp57 is 56% similar to PDI in its amino acid sequence and has also the four-domain architecture. Despite the high similarity in their structures ERp57 cannot substitute for PDI as the β subunit of C-P4H. The minimum requirement for the C-P4H tetramer assembly is fulfilled by domains b' and a' of PDI, while domains a and b enhance this function and can be substituted in part by those of ERp57. Until very recently the structural information of any of the PDI family members, which contains the TRX active site was limited to solution structures of human PDI domains a and b. In this research the domain boundaries of the full length ERp57 were defined and the individual domains characterized. Furthermore the solution structures of the catalytically active domains a and a' of ERp57 were studied by nuclear magnetic resonance (NMR).

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