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

Self Cite

To examine the structural basis for this oxidase and isomerase activity of PDI, shuffled domain mutants were generated using a method that should be generally applicable to multidomain proteins. Domains a and a along with constructs ab, aa, aba, aba display low disulfide isomerase activity, but all show significant reactivity with mammalian thioredoxin reductase, suggesting that the structure is not seriously compromised. The only domain order that retains significant isomerase activity has the b domain coupled to the N terminus of the a domain. This bac has 38% of the isomerase activity of wild-type PDI, equivalent to the activity of full-length PDI with one of the active sites inactivated by mutation (Walker, K. W., Lyles, M. M., and Gilbert, H. F. (1996) Biochemistry 35,[1972][1973][1974][1975][1976][1977][1978][1979][1980]. Individual a and a domains, despite their very low isomerase activities in vitro, support wild-type growth of a pdi1⌬ Saccharomyces cerevisiae strain yeast. Thus, most of the PDI structure is dispensable for its essential function in yeast, and highlevel isomerase activity appears not required for viability or rapid growth.The correct formation of disulfides is essential for proper folding and export of secreted proteins from the endoplasmic reticulum. In vitro, chemical steps involving the formation and rearrangement of disulfides are usually rate-limiting during uncatalyzed oxidative folding (1, 2). Protein-disulfide isomerase (PDI), 1 a major 55-kDa protein of the endoplasmic reticulum, catalyzes thiol-disulfide exchange reactions during protein folding that introduce disulfides and rearrange those that are incorrectly paired (1). Structurally, PDI consists of five discrete domains, each of which is structurally related to thioredoxin (3). The catalytically active domains, like thioredoxin, also contain a CXXC motif that is responsible for the active site chemistry.The multidomain nature of PDI was initially recognized from an analysis of its amino acid sequence in which four domains, denoted a, b, b, and a, were identified. These four structural domains are followed by a stretch of acidic residues at the C terminus (designated c) (4). The domain boundaries were confirmed and refined by proteolytic mapping and heterologous expression of individual domains (3). The a and a domains are homologous both to each other (47% identity) and to thioredoxin (27% identity). Each of these domains contains an active site CGHC motif that can be reduced by mammalian thioredoxin reductase, which further supports a close structural relationship to thioredoxin (5). The b and b domains have similar sequences (28% identical), but show no or very low sequence relatedness to thioredoxin.NMR structures of the isolated a and b domains provided the first detailed structural information about PDI. As expected, the structure of the a domain resembles that of thioredoxin (6), but surprisingly, the b domain also has a thioredoxin-like fold (7). Sequence homology between the b and b and a and a imply that PDI consis...

Section: Expression Of Pdi Domains and Multidomain Constructs-supporting

confidence: 59%

“…Puig et al reported that "weePDI" (residues 307-491) has 16% of wild-type PDI isomerase activity (25) (Fig. 3).…”

Section: Combinations Of Pdi Domainsmentioning

confidence: 99%

Combinations of Protein-disulfide Isomerase Domains Show That There Is Little Correlation between Isomerase Activity and Wild-type Growth

Xiao¹,

Solovyov²,

Gilbert³

et al. 2001

Self Cite

“…The 700-kDa species is presumably a disulfide-bonded high order oligomer. Another C-terminal fragment 308 -491 of PDI has also been reported to behave as a dimer on SEC (39). Therefore, it seemed to us that the species containing the C-terminal 441-491 sequence consistently elutes with an apparent molecular mass equivalent to a dimer on SEC, whereas PDI (1-440) behaves as a monomer.…”

Section: Saxs Parameters-as Shown Inmentioning

confidence: 99%

“…As PDI (19) and PDI (308 -491) (39) have been characterized to be monomeric by analytical ultracentrifugation, and also by SAXS for PDI, the C-terminal 441-491 sequence is likely a contributor to the anomalous behavior of these molecules on SEC.…”

Section: Saxs Parameters-as Shown Inmentioning

confidence: 99%

Annular Arrangement and Collaborative Actions of Four Domains of Protein-disulfide Isomerase

Hong

Shi

et al. 2006

We presented for the first time a small angle x-ray scattering study of intact protein-disulfide isomerase (PDI) in solution. The restored model revealed that PDI is a short and roughly elliptical cylinder with a molecular mass of 69 kDa and dimensions of 105 ؋ 65 ؋ 40 Å , and the four thioredoxin-fold domains in the order a-bb-a are arranged in an annular fashion. Atomic force microscope imaging also supported the finding that PDI appears as an approximately flat elliptical cylinder. A PDI species with apparent molecular mass of 116 kDa measured by using size-exclusion chromatography, previously assumed to be a dimer, was determined to exist mainly as a monomer by using analytical ultracentrifugation. The C-terminal fragment 441-491 contributed to the anomalous molecular mass determination of PDI by size-exclusion chromatography. The annular model of PDI accounted for the cooperative properties of the four domains in both the isomerase and chaperone functions of PDI.Protein-disulfide isomerase (PDI, 3 EC 5.3.4.1) is an abundant multifunctional protein within the lumen of the endoplasmic reticulum (1, 2). It plays important roles in many physiological processes as both an enzyme and a molecular chaperone (3-5) by assisting in folding, unfolding, and translocation of many disulfide-containing proteins (6) and even some disulfide-free proteins (7). In addition, PDI serves as an obligatory ␤ subunit of hetero-oligomeric prolyl 4-hydroxylase (8) and microsomal triglyceride transfer protein (9) to maintain highly insoluble ␣ subunits in an active, nonaggregated conformation (10).The four-domain architecture of PDI in the order a-b-bЈ-aЈ was proposed when the PDI cDNA was first sequenced, according to the internal sequence homology within the molecule (11). Later, the domain boundaries were defined (12) and further refined as shown in Scheme 1 by combined use of protein engineering, limited proteolysis, and bioinformatics (13-16). The a and aЈ domains, each with a -CGHC-motif as the active site, share 47% sequence identity, whereas the b and bЈ domains, with no such active site motif, share 28% sequence identity (11). The C-terminal 29 residues, 463-491, constitute an acidic c extension (2), in which over half of the residues are Glu/Asp, and represent a putative Ca 2ϩ -binding region (12). The three-dimensional structure determined by using NMR of the a domain, which shares 27% identity to thiroredoxin (Trx), shows a Trx-fold (15), and the b domain, which shows no significant sequence similarity to the a and Trx, also has a Trx-fold (14). PDI has thus been suggested to consist of four Trx-fold domains (14). Recently, a 19-amino acid linker region, 333-351, was identified between the bЈ and aЈ domains (16), which was suggested to potentially allow more flexibility between these domains than between the other domains. The three-dimensional structure of the entire PDI molecule, or of any other catalytically active eukaryotic PDI family member, has not yet been determined even though PDI was discovered over 40 years ago....

“…Several properties of mammalian PDI domains have been studied. Recent investigations on recombinant fragments have elucidated domains involved in folding (18,19,34), catalysis (19, 34 -36), chaperone activity (35,37), and peptide/protein recognition (38).…”

mentioning

confidence: 99%

The Role of Calcium on the Activity of ERcalcistorin/Protein-disulfide Isomerase and the Significance of the C-terminal and Its Calcium Binding

Lucero

Kaminer

1999

The endoplasmic reticulum (ER) 1 is a multifunctional organelle involved in post-translational modification of nascent proteins, in lipid synthesis and regulation of intracellular Ca 2ϩ . Protein folding in the ER associated with disulfide bond formation is likely to be catalyzed by protein-disulfide isomerase (PDI), the identification of which stemmed from the early findings of a factor which catalyzed the renaturation of ribonuclease in vitro (1, 2).The properties and functions of mammalian PDI have been extensively studied and characterized (3-6). In vitro, PDI catalyzes the formation and cleavage of protein disulfide bonds (7), it catalyzes the glutathione-dependent reduction of dehydro-Lascorbic acid to ascorbic acid (8), it binds the hormones, triiodothyronine (9) in a non-stoichiometric manner (10) and estrogen (11), both of which inhibit PDI activity. It displays chaperone (12-14) or anti-chaperone activity (13). PDI has also been identified as a structural subunit of other enzymes, e.g. prolyl-4-hydroxylase (15) and the triglyceride transfer complex (16).On the basis of sequence homology in the primary structure of PDI, Edman et al. (17) identified multiple domains, a, b, b and a. The a domain (at the N-terminal) and the a (a portion of the C-terminal) are homologous with thioredoxin in their sequence and folded structure and each contains the CGHC sequence, the active sites. b and b are two homologous central domains with folded structures similar to thioredoxin but with no thioredoxin sequence homology (18). Interestingly, domain b shares a limited sequence identity with calsequestrin (19). These four domains therefore resemble thioredoxin folded structures (18). A domain, between the a domain and b domain was designated e because of its homology to the ligand-binding site of the estrogen receptor (11). Whether this should be regarded as a separate domain is now being questioned (19).A sixth domain in the C-terminal was recently labeled c as a putative Ca 2ϩ -binding domain (4). This speculation is in keeping with our findings and hypothesis on calcium binding. We found quantitatively, for the first time, that sea urchin (20) and mammalian PDI (21) are high capacity low affinity Ca 2ϩ -binding proteins. And it should be recognized that calcium binding by mammalian PDI was not quantitated previously (22). Having determined the number of calcium ions bound, we related them to the number of paired acidic residues and hypothesized that paired acidic residues constituted low affinity Ca 2ϩ -binding sites, a cluster of which resides in the tail end of the C-terminal of mammalian and sea urchin PDI (23). The Cterminal of PDI ends with a KDEL sequence characteristic of ER-resident proteins (24). However, PDI has also been detected on the cell surface by immunocytology (25) and other techniques (26), in different cell types (27, 28) and platelets (29).Mammalian PDI has been regarded for many years to behave as a homodimer on gel filtration (30, 31) and to form a tetramer under certain conditions (32). However,...