Lloyd W. Ruddock

The b′ domain provides the principal peptide‐binding site of protein disulfide isomerase but all domains contribute to binding of misfolded proteins

Protein disulfide isomerase (PDI) is a very efficient catalyst of folding of many disulfide‐bonded proteins. A great deal is known about the catalytic functions of PDI, while little is known about its substrate binding. We recently demonstrated by cross‐linking that PDI binds peptides and misfolded proteins, with high affinity but broad specificity. To characterize the substrate‐binding site of PDI, we investigated the interactions of various recombinant fragments of human PDI, expressed in Escherichia coli, with different radiolabelled model peptides. We observed that the b′ domain of human PDI is essential and sufficient for the binding of small peptides. In the case of larger peptides, specifically a 28 amino acid fragment derived from bovine pancreatic trypsin inhibitor, or misfolded proteins, the b′ domain is essential but not sufficient for efficient binding, indicating that contributions from additional domains are required. Hence we propose that the different domains of PDI all contribute to the binding site, with the b′ domain forming the essential core.

A major fraction of endoplasmic reticulum-located glutathione is present as mixed disulfides with protein.

The tripeptide glutathione is the most abundant thiol/disulfide component of the eukaryotic cell and is known to be present in the endoplasmic reticulum lumen. Accordingly, the thiol/disulfide redox status of the endoplasmic reticulum lumen is defined by the status of glutathione, and it has been assumed that reduced and oxidized glutathione form the principal redox buffer. We have determined the distribution of glutathione between different chemical states in rat liver microsomes by labeling with the thiol-specific label monobromobimane and subsequent separation by reversed phase high performance liquid chromatography. More than half of the microsomal glutathione was found to be present in mixed disulfides with protein, the remainder being distributed between the reduced and oxidized forms of glutathione in the ratio of 3:1. The high proportion of the total population of glutathione that was found to be in mixed disulfides with protein has significant implications for the redox state and buffering capacity of the endoplasmic reticulum and, hence, for the formation of disulfide bonds in vivo.

The pancreas-specific protein disulphide-isomerase PDIp interacts with a hydroxyaryl group in ligands

Using a cross-linking approach, we have recently demonstrated that radiolabelled model peptides or misfolded proteins specifically interact in vitro with two members of the protein disulphide- isomerase family, namely PDI and PDIp, in a crude extract from sheep pancreas microsomes. In addition, we have shown that tyrosine and tryptophan residues within a peptide are the recognition motifs for the binding to PDIp. Here we examine non-peptide ligands and present evidence that a hydroxyaryl group is a structural motif for the binding to PDIp; simple constructs containing this group and certain xenobiotics and phytoestrogens, which contain an unmodified hydroxyaryl group, can all efficiently inhibit peptide binding to PDIp. To our knowledge this is the first time that the recognition motif of a molecular chaperone or folding catalyst has been specified as a simple chemical structure.

Protein folding: A missing redox link in the endoplasmic reticulum

Native disulphide-bond formation during protein folding in the endoplasmic reticulum requires oxidative machinery, the components and mechanism of which are not yet fully understood. Two recent papers have identified a novel protein component that appears to play a key role in this important redox pathway.

Oxidative stress: Protein folding with a novel redox switch

A novel cellular response to oxidative stress has been discovered, in which the activity of a molecular chaperone, Hsp33, is modulated by the environmental redox potential. This provides a rapid first defence mechanism against the potentially very harmful toxic effects of oxidative stress.

Assembly of the B subunit pentamer of Escherichia coli heat-labile enterotoxin. Kinetics and molecular basis of rate-limiting steps in vitro.

The B subunits of Escherichia coli heat-labile enterotoxin (EtxB) and cholera toxin (CtxB) assemble in vivo into exceptionally stable homopentameric complexes, which maintain their quaternary structure in a range of conditions that would normally be expected to cause protein denaturation. Recently, we showed that the simultaneous protonation of two of the COOH-terminal carboxylates in pentameric EtxB was required to cause its disassembly at pH values below 2.0 (Ruddock, L., Ruston, S. P., Kelly, S. M., Price, N. C., Freedman, R. B., and Hirst, T. R. (1995) J. Biol. Chem. 270, 29953-29958). Here, we investigate the influence of environmental parameters on the kinetics of reassembly of acid-generated EtxB monomers in vitro. Such monomers were found to undergo a further acid-mediated conformational change, with an activation energy of 76 ± 2 J·mol−1·K−1, consistent with isomerization of the cis-proline residue at position 93, and which prevented subsequent EtxB reassembly. By using rapid neutralization of acid-generated monomers, a high proportion of the B-subunits adopted an assembly-competent conformation, which resulted in up to 75% of the protein reassembling into a stable pentameric complex, indistinguishable from native EtxB pentamers. The rate-limiting step in reassembly, over a concentration range of 50-200 μg/ml, was shown to be due to an intramolecular event, which exhibited a pH dependence with a pKa of 7.0. Modification of EtxB with amine-specific probes revealed that the protonation state of the NH2-terminal alanine residue was responsible for the pH dependence of reassembly. The implications of these findings for the biogenesis of Escherichia coli enterotoxin and related enterotoxins in vivo, are considered.