The role of the thiol/disulfide centers and peptide binding site in the chaperone and anti-chaperone activities of protein disulfide isomerase.

Abstract

The complexity of protein folding is often aggravated by the low solubility of the denatured state. The inefficiency of the oxidative refolding of reduced, denatured lysozyme results from a kinetic partitioning of the unfolded protein between pathways leading to aggregation and pathways leading to the native structure. Protein disulfide isomerase (PDI), a resident foldase of the endoplasmic reticulum, catalyzes the in vitro oxidative refolding of reduced, disulfide-containing proteins, including denatured lysozyme. Depending on the concentrations of foldase and denatured substrate and the order in which they are added to initiate folding, PDI can exhibit either a chaperone activity or an anti-chaperone activity (Puig, A., and Gilbert, H. F. (1994) J. Biol. Chem 269, 7764-7771). PDI's chaperone activity leads to quantitative recovery of native lysozyme. Its anti-chaperone activity diverts substrate away from productive folding and facilitates disulfide cross-linking of lysozyme into large, inactive aggregates that specifically incorporate PDI. A mutant PDI (NmCm-PDI), in which both the N- and C-terminal active site cysteines have been changed to serines, loses all chaperone activity and behaves as an anti-chaperone at all substrate and PDI concentrations tested. The dithiol/disulfide sites of PDI are essential for the chaperone activity observed at high PDI concentrations, but they are not required for the anti-chaperone activity found at low PDI concentrations. Inactivation of PDI's peptide/protein binding site by a specific photoaffinity label (Noiva, R., Freedman, R. B., and Lennarz, W. J. (1993) J. Biol. Chem. 268, 19210-19217) inhibits the disulfide isomerase and chaperone activity, but the protein still retains its anti-chaperone activity. In a glutathione redox buffer, lysozyme-PDI aggregates are disulfide cross-linked; however, disulfide cross-linking is not required for aggregate formation or for the incorporation of PDI into the aggregates. Although both the peptide binding site and the catalytic active sites of PDI are required for chaperone and disulfide isomerase activity, neither of these sites are involved in PDI's anti-chaperone activity. PDI's anti-chaperone activity could serve as a quality control device by providing an efficient mechanism to retain misfolded proteins in the endoplasmic reticulum (Marquardt, T., and Helenius, A. (1992) J. Cell. Biol. 117, 505-513).