Molecular Characterization of the Principal Substrate Binding Site of the Ubiquitous Folding Catalyst Protein Disulfide Isomerase

Abstract

Disulfide bond formation in the endoplasmic reticulum of eukaryotes is catalyzed by the ubiquitously expressed enzyme protein disulfide isomerase (PDI). The effectiveness of PDI as a catalyst of native disulfide bond formation in folding polypeptides depends on the ability to catalyze disulfide-dithiol exchange, to bind non-native proteins, and to trigger conformational changes in the bound substrate, allowing access to buried cysteine residues. It is known that the b domain of PDI provides the principal peptide binding site of PDI and that this domain is critical for catalysis of isomerization but not oxidation reactions in protein substrates. Here we use homology modeling to define more precisely the boundaries of the b domain and show the existence of an intradomain linker between the b and a domains. We have expressed the recombinant b domain thus defined; the stability and conformational properties of the recombinant product confirm the validity of the domain boundaries. We have modeled the tertiary structure of the b domain and identified the primary substrate binding site within it. Mutations within this site, expressed both in the isolated domain and in fulllength PDI, greatly reduce the binding affinity for small peptide substrates, with the greatest effect being I272W, a mutation that appears to have no structural effect.Native disulfide bond formation in the endoplasmic reticulum is a complex process that is rate-limiting in the biogenesis of many outer membrane and secreted proteins. Native disulfide bond formation can occur via multiple parallel pathways, and there is evidence that a large number of different gene families and redox carriers may play a role in the supply of redox equivalents for protein disulfide bond formation. What is clear is that the rate-limiting step for native disulfide bond formation in proteins that contain multiple disulfides is latestage isomerization reactions, where disulfide bond formation is linked to conformational changes in protein substrates with substantial regular secondary structure. These steps are thought to be catalyzed only by proteins belonging to the protein disulfide isomerase (PDI) family. PDI 1 was the first catalyst of protein folding identified over 40 years ago (1), but despite probably being the most widely studied protein folding catalyst, significant details of the mechanisms of action of this critical enzyme are still unclear. In all eukaryotes, there exists a species-dependent PDI family of enzymes; for example, in humans (2), ERp72, ERp57, P5, PDIp, PDIr, ERp44 (3), ERp28/29 (4), ERdj5 (5), and ERp18 (6) have been reported to date. Functional characterization and differentiation between these family members is far from complete. PDI is a multifunctional, multidomain enzyme. The domain structure of PDI has been determined by theoretical (7) and experimental (8 -11) procedures and comprises two catalytic domains, a and a, separated by two homologous non-catalytic domains, b and b, plus a C-terminal region designated as c. In addition, it has bee...

Truncation of a Protein Disulfide Isomerase, PDIL2-1, Delays Embryo Sac Maturation and Disrupts Pollen Tube Guidance inArabidopsis thaliana

“…In lines 4 and 5, the insertions caused truncations of the protein, with only the first thioredoxin domain remaining intact. For typical PDI proteins, the noncatalytic b and b9 domains are considered to have high affinity for the substrate, while the active thioredoxin domains a and a9 have weak affinity (Darby et al, 1998;Klappa et al, 2000;Pirneskoski et al, 2004). However, PDI-D family proteins do not have b and b9 domains, so it is unknown how PDI-D proteins define their substrates.…”

“…The noncatalytic domains b and b9 are similar in sequence to each other but not to thioredoxin, and domain c is acidic. The b9 domain contains the high affinity substrate binding site, but the a and a9 domains might contain low affinity binding sites (Darby et al, 1998;Klappa et al, 2000;Pirneskoski et al, 2004). Most PDIs also have a KDEL sequence at the C terminus, which serves as an endoplasmic reticulum (ER) retention signal.…”

Domain a’ of protein disulfide isomerase plays key role in inhibiting α-synuclein fibril formation

“…The b' domain of PDI has been identified as the primary site for binding short peptides to PDI and essential for binding more complex substrates to PDI while the a and a' domains of PDI contribute to binding large substrates to PDI (Klappa et al 1998;Pirneskoski et al 2004).…”

Redox-regulated Export of the Major Histocompatibility Complex Class I-Peptide Complexes from the Endoplasmic Reticulum

“…In the bЈ domain, PDI has a hydrophobic binding pocket that recognizes small peptides (Pirneskoski et al, 2004) and the mutation of bЈ domain inhibits peptide binding by causing a conformational change in PDI (Nguyen et al, 2008). Moreover, the interaction between the peptides and PDI is specific and can be saturated, reversed, and abolished by the denaturation of PDI (Klappa et al, 1997).…”

“…The bЈ domain of PDI provides the principal peptide-binding site (Klappa et al, 1998). Mutations within this site, in particular, the I272A and F258W point mutations, greatly reduce the binding affinity for small peptide substrates (Pirneskoski et al, 2004). We examined which of these two functions dictates the tapasin-ERp57 interaction.…”