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

Abstract

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...

Sulfhydryl Oxidation, Not Disulfide Isomerization, Is the Principal Function of Protein Disulfide Isomerase in Yeast Saccharomyces cerevisiae

“…In this mutant, the ratio of oxidase/isomerase activity is 1:2.5. A single catalytic domain of PDI (a or aЈ) has approximately 50% of the oxidase activity of wild-type PDI in vitro but only 3-5% of the isomerase activity (5). Here, the ratio of oxidase to isomerase activity is greater than 10:1, which is a much more effective separation of activities than has previously been available.…”

“…Although catalysis of thiol-disulfide exchange is essential, there has been some uncertainty about which part of this catalytic activity represents the essential in vivo function of PDI. PDI mutants with defects in the oxidase activity can complement the PDI1 deletion (3,4), but surprisingly, expression of a single PDI catalytic domain (a or aЈ), which is defective in catalyzing isomerization, can also rescue the PDI1 deletion (5).…”

“…Here, the ratio of oxidase to isomerase activity is greater than 10:1, which is a much more effective separation of activities than has previously been available. The second problem is the absence of expression level measurements and the use of different PDI proteins (yeast and mammalian) during the complementation experiments (3)(4)(5).…”

Catalysis of Thiol/Disulfide Exchange

“…Cys 14 of E. coli Grx1 was mutated to Ser using the QuikChange® site-directed mutagenesis kit from Stratagene to create Grx1C14S. The method of Xiao et al (24) was employed to create the Grx1-PDI chimera, Grx-b-bЈ-aЈ(SGHS)c. The vector combined an N-terminal Grx1 domain with the b-bЈ-aЈc domains of PDI using a GlySer linker. The cysteines in the C-terminal, aЈ, catalytic domain of PDI were both mutated to serine so that the only catalytic contribution must come from the Grx1 domain.…”

“…For PDI, Grx1, and their mutants, proteins were expressed from pET15rx with an N-terminal His 6 tag (24). Proteins were purified using a HiTrap Chelating column (Amersham Biosciences) as described (24) and were greater than 90% pure by SDS-PAGE.…”