Identifying and characterizing a structural domain of protein disulfide isomerase

Abstract

Protein disulfide isomerase (PDI) appears on the basis of its primary structure to be a multidomain protein, but the number and nature of the domains has been uncertain. Two of the domains, a and a', which are homologous to thioredoxin and active in catalysis of disulfide bond formation, have been identified and characterized previously. Sections of the N-terminal half of the PDI sequence have been expressed and the limits of their folded structures delineated by limited proteolysis. In addition to the a-domain, the boundaries of a domain with no activity on thiol/disulfide groups, designated b, have been identified. This domain has been produced independently; its cooperative unfolding transition and its CD and NMR spectra confirm that it is an autonomously folded structure in isolation and when part of PDI. Fusion of the b-domain to the a-domain, as occurs naturally in the first half of PDI, did not alter substantially the catalytic activity of the a-domain. It still catalyzes only a subset of the thiol/disulfide exchange reactions of intact PDI and has a reduced ability to catalyze protein disulfide rearrangements. The a- and b-domains account structurally for virtually all of the first half of the PDI polypeptide chain, and it is very unlikely that there exists a proposed third domain homologous to the estrogen receptor. The b-domain exhibits some sequence homology to calsequestrin, a calcium binding protein from the sarcoplasmic reticulum of muscle.