Recognition of Protein Substrates by Protein-disulfide Isomerase

Abstract

Refolding of partially folded mitochondrial malate dehydrogenase (mMDH) is assisted by protein-disulfide isomerase (PDI).It is shown that the fluorescence probe is covalently attached to a SH residue located in the b domain. Based on the fluorescence measurements of native and derivatized PDI, it is suggested that recognition of the unfolded substrate involves conformational changes propagated to several domains of PDI. Protein-disulfide isomerase (PDI)1 is a multifunctional enzyme that both catalyzes the formation of disulfide bonds (1, 2) and acts as a subunit of prolyl-4-hydroxylase (3). PDI has been proposed to function as a molecular chaperone by binding to unfolded protein species, thereby preventing aggregation and misfolding (4, 5). Despite these interesting studies, the situation is complicated because PDI, unlike other chaperones, catalyzes disulfide bond formation and reduction.Several laboratories have attempted to show the site of binding of small molecular weight polypeptides that compete with misfolded protein substrates. Mutated PDI with the carboxyl terminus deleted shows neither peptide binding nor chaperone activity in assisting the refolding of denatured D-glyceraldehyde-3-phosphate dehydrogenase (6). On the other hand, it has been reported that deletion of the carboxyl-terminal domain (C domain) has no inhibitory effect on the assembly of recombinant prolyl-4-hydroxylase (7). Other investigators have reported that small molecular weight peptides bind to the bЈ domain of PDI (8).The possibility exists that more than one site in the structure of PDI is involved in recognition and refolding of protein substrates. The binding and hydrolysis of ATP by PDI has been reported by Guthapfel et al. (9); strikingly, the ATPase reaction is stimulated in the presence of denatured polypeptides, whereas the disulfide oxidation of PDI is not influenced by ATP. However, the functional role played by ATP hydrolysis during the refolding of denatured proteins has not been investigated in detail.The aim of the present work is 2-fold: first, to study regions of the primary structure of PDI involved in recognition of unfolded protein substrates and, second, to investigate whether the free energy of hydrolysis of ATP is required for unfolding of misfolded protein substrates. EXPERIMENTAL PROCEDURESPurification of Proteins-PDI was purified by the method described in Ref. 10 with small modifications. Fresh porcine livers (600 g) were homogenized in 0.1 M phosphate (pH 7.5) containing 1% Triton X-100 and 5 mM EDTA. After centrifugation, the supernatant was treated with ammonium sulfate, and the fractions obtained between 55-85% saturation were suspended in 25 mM citrate buffer (pH 5.3), dialyzed against the same buffer (buffer A), applied to CM-Sephadex C-50 column and eluted with the same buffer. Fractions displaying PDI activity were pooled, dialyzed against 20 mM sodium phosphate (pH 6.3) (buffer B), and applied to a DEAE-Sepharose fast flow column, which was eluted using a linear gradient of 0 -0.7 M NaCl in buffer...

Reversal of Alpha-Synuclein Fibrillization by Protein Disulfide Isomerase

“…Moreover, PDI could not reverse the aggregation of MDH when added in 7-fold molar excess over MDH ( Figure 4C ). MDH is used as a model aggregation-prone substrate for the activity of molecular chaperones such as PDI ( Cheung and Churchich, 1999 ; Goloubinoff et al, 1999 ; Nillegoda et al, 2015 ), but it does not form amyloid structures. The aggregation of MDH is therefore detected by light scattering instead of ThT fluorescence.…”

Quercetin-3-Rutinoside Blocks the Disassembly of Cholera Toxin by Protein Disulfide Isomerase

“…The a and a’ domains allow PDI to function as an oxidoreductase, but this enzymatic activity is not required for PDI to act as a chaperone [20,21]. The b and b’ domains at the base of the U-shaped protein serve as the main sites of substrate binding [22,23,24]. The x linker is an unstructured segment that contributes to the conformational flexibility of PDI, while the c region contains a KDEL tag that retains PDI in the ER [25].…”

“…The x linker is an unstructured segment that contributes to the conformational flexibility of PDI, while the c region contains a KDEL tag that retains PDI in the ER [25]. Interactions at one domain of PDI influence the structure/function of other PDI domains [24,25,26,27,28]. PDI also undergoes redox-dependent conformational changes that can influence its interactions with select substrates [29].…”

Thermodynamics of the folding of D‐glyceraldehyde‐3‐phosphate dehydrogenase assisted by protein disulfide isomerase studied by microcalorimetry

“…The dissociation constant, K d , for the binding reaction of GAPDH folding intermediates with PDI at 25.0 °C, 30.9 n m , is six‐fold that with GroEL at the same temperature. Recently, Cheung and Churchich [38] reported that PDI binds with the malate dehydrogenase folding intermediate with a K d of 0.2 µ m , which is fivefold the dissociation constant for the interaction of GroEL with the same substrate. Generally, GroEL binds with its substrate to form a stable complex, which does not dissociate unless ATP is added.…”