A 21-kDa C-terminal Fragment of Protein-disulfide Isomerase has Isomerase, Chaperone, and Anti-chaperone Activities

Alberto Puig,Todd P Primm,Rajendran Surendran,J Ching Lee,Kevin D Ballard,Ralph S Orkiszewski,Vladimir Makarov,Hiram F Gilbert

doi:10.1074/jbc.272.52.32988

Abstract

A catalyst of disulfide formation and isomerization during protein folding, protein-disulfide isomerase (PDI) has two catalytic sites housed in two domains homologous to thioredoxin, one near the N terminus and the other near the C terminus. The thioredoxin domains, by themselves, can catalyze disulfide formation, but they are unable to catalyze disulfide isomerizations (Darby, N. J. and Creighton, T. E. (1995) Biochemistry 34, 11725-11735). A 21-kDa, C-terminal fragment of PDI (amino acids 308-491), termed weePDI, comprises the C-terminal third of the molecule. The kcat for ribonuclease oxidative folding by weePDI is 0.26 +/- 0.02 min-1, 3-fold lower than the wild-type enzyme but indistinguishable from the activity of a full-length mutant of PDI in which both active site cysteines of the N-terminal thioredoxin domain have been mutated to serine. Eliminating the ability of weePDI to escape easily from covalent complexes with substrate by mutating the active site cysteine nearer the C terminus to serine has a large effect on the isomerase activity of weePDI compared with its effect on the full-length enzyme. weePDI also displays chaperone and anti-chaperone activity characteristic of the full-length molecule. As isolated, weePDI is a disulfide-linked dimer in which the single cysteine (Cys-326) outside active site cross-links two weePDI monomers. The presence of the intermolecular disulfide decreases the activity by more than 2-fold. The results imply that the functions of the core thioredoxin domains of PDI and other members of the thioredoxin superfamily might be modified quite easily by the addition of relatively small accessory domains.

Highlights

Protein-disulfide isomerase (PDI)1 is an abundant, 55-kDa resident of the endoplasmic reticulum (ER) [1,2,3]
The cysteine residues of the two CXXC centers of the thioredoxin domains are responsible for the disulfide isomerase activity of PDI; a mutant PDI with all four active-site cysteines changed to serines has Յ0.5% of the activity of the wild-type enzyme in catalyzing ribonuclease oxidative folding [17]
An overloaded SDS-PAGE gel (Fig. 1) of purified weePDI shows that there is less than 1% wild-type PDI in the preparation

Summary

Introduction

Protein-disulfide isomerase (PDI) is an abundant, 55-kDa resident of the endoplasmic reticulum (ER) [1,2,3]. Thioredoxin domains, including active-site CXXC sequences, have been found in a number of redox-active proteins, suggesting that these proteins constitute a thioredoxin superfamily with versatile thiol/disulfide exchange activities including oxidation, reduction, and isomerization [5]. Other members of the thioredoxin superfamily such as thioredoxin itself, glutaredoxin [8], or the Escherichia coli periplasmic protein, DsbA [9], have only one thioredoxin homology domain All of these single domain proteins are poor catalysts of disulfide rearrangements, suggesting that domains other than the thioredoxin domains contribute significantly to substrate interactions that are important for disulfide rearrangements. At each thioredoxin domain active site, the cysteine that is closer to the N terminus interacts with the substrate to form a covalent intermediate [18]. The more C-terminal cysteine at each active site provides a mechanism to reduce substrate disulfides and enables PDI to escape from slowly rearranging complexes [18]

Methods

Results

Conclusion