Mutations That Destabilize the a′ Domain of Human Protein-disulfide Isomerase Indirectly Affect Peptide Binding

Abstract

Protein-disulfide isomerase (PDI) is a catalyst of folding of disulfide-bonded proteins and also a multifunctional polypeptide that acts as the ␤-subunit in the prolyl 4-hydroxylase ␣ 2 ␤ 2 -tetramer (P4H) and the microsomal triglyceride transfer protein ␣␤-dimer. The principal peptide-binding site of PDI is located in the b domain, but all domains contribute to the binding of misfolded proteins. Mutations in the C-terminal part of the a domain have significant effects on the assembly of the P4H tetramer and other functions of PDI. In this study we have addressed the question of whether these mutations in the C-terminal part of the a domain, which affect P4H assembly, also affect peptide binding to PDI. We observed a strong correlation between P4H assembly competence and peptide binding; mutants of PDI that failed to form a functional P4H tetramer were also inactive in peptide binding. However, there was also a correlation between inactivity in these assays and indicators of conformational disruption, such as protease sensitivity. Peptide binding activity could be restored in inactive, protease-sensitive mutants by selective proteolytic removal of the mutated a domain. Hence we propose that structural changes in the a domain indirectly affect peptide binding to the b domain. Protein-disulfide isomerase (PDI),1 an abundant protein of the eukaryotic endoplasmic reticulum, is a catalyst of disulfide bond formation and rearrangement in the course of protein folding (for review see Ref. 1). A molecular interpretation of PDI activity is made difficult by the fact that PDI is multifunctional in the cell. In addition, it has a wide range of actions in vitro and consists of multiple domains. Furthermore, there is as yet no high resolution structure of full-length PDI. Current efforts are focused on analyzing the domain structure of PDI and in establishing the roles of specific domains in specific functional activities.It is now clear (2, 3) that PDI has a structural organization based on duplicated sequence modules (Fig. 1). The full-length protein is constructed of four structural domains with homologous thioredoxin folds plus a C-terminal acidic extension. The homologous a and a sequence modules contain the active site motif -WCGHC-and show significant sequence identity to thioredoxin; a high resolution NMR analysis of the recombinant a domain confirms that it has the thioredoxin fold. The homologous b and b modules do not show significant sequence similarity to the a domain, but NMR analysis has revealed that the b domain also exhibits the thioredoxin fold (2, 4). Analysis of the properties of individual domains as catalysts of simple thiol:disulfide oxidoreduction and of more complex protein folding linked to disulfide isomerization shows that the a and a domains function effectively as simple thiol:disulfide oxidoreductases but that the remaining domains are required for full activity in catalyzing protein folding associated with the formation of native disulfide bonds (5). No specific function has yet been ascribed...

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.…”

Three Binding Sites in Protein-disulfide Isomerase Cooperate in Collagen Prolyl 4-Hydroxylase Tetramer Assembly

“…F449R) result in a decrease in ⌬-somatostatin binding by the bЈ domain (33). Accordingly, all 15 single and double a and aЈ domain mutants of PDI generated here were screened for ⌬-somatostatin binding.…”

The b’x Region of Yeast Protein Disulfide Isomerase is Not Essential for Saccharomyces cerevisiae Viability at 30 °C

“…Stability of mutant PDI toward proteinase K was conducted based on method described by Klappa et al (2000). PDI was mixed with various concentration of Proteinase K (1; 3; 10 mg mL -1 ) in PBS buffer (NaCl 140 mM, KCl 2.7 mM, Na 2 HPO 4 10 mM, KH 2 PO 4 1.8 mM, pH 8) for 30 min at 4 °C.…”

“…The a and a' domains each contain active site sequence CXXC which is required for disulphide bond formation. The b' domain of human PDI contributes significantly to protein substrate interactions (Klappa et al 2000;Denisov et al 2009). …”