Protein disulfide isomerase : function and mechanism in oxidative protein folding

University dissertation from Stockholm : Karolinska Institutet, Department of Medical Biochemistry and Biophysics

Abstract: The formation of native intramolecular disulfide bonds is critical for the folding and stability of many secreted proteins. This process involves oxidation of protein thiols to form disulfide bonds as well as rearrangement of any non-native disulfide bonds that might form. Protein disulfide isomerase (PDI) is an abundant catalyst for native disulfide bond formation in the lumen of endoplasmic reticulum (ER). PDI is composed of four domains (termed a, b, b' and a') and an anionic tail (c).The a and a' domains of PDI are homologous to thioredoxin and each contains an active site with the sequence CGHC. In this study, we examined the structural basis for the redox activity of PDI. A number of PDI mutants with shuffled domain order were generated using a novel method that should be generally applicable to multidomain proteins. Activity measurement on PDI mutants reveals that the multidomain structure of PDI is essential for high isomerase activity. Except for the b'-a'-c construct, which has ~ 38% of the isomerase activity of wild-type PDI all new PDI constructs with one or two domains have less than 10% activity of the wild-type protein. Unlike for the isomerase activity, the multidomain structure of PDI does not contribute to its function as an oxidase. The single catalytic domains of PDI (a or a') possess less than 5% of the isomerase activity but - 50% of the oxidase activity. The redox potential of ratPDI is -150inV; glutaredoxin 1 (Grx1) from E.coli, has a redox potential of -233 mV. Thermodynamically, ratPDI is a 600-fold better oxidizing agent than Grx 1. Despite that, Grx1 is surprisingly good protein oxidant. It catalyzes the formation of protein disulfides in a redox buffer with an initial velocity that is 30-fold faster than the PDI-catalyzed oxidation. To investigate the basis of the unusual versatility of Grx 1 as a protein oxidant and reductant, we have compared the kinetics of disulfide formation and reduction catalyzed by Grx 1 and PDI. Mutations in the active site cysteines reveal that PDI introduces disulfides into reduced substrates through a dithiol mechanism, where both active site cysteines are required, while Grx 1 can utilize a single active site cysteine to catalyze protein oxidation through the formation of a Grx-SG mixed disulfide. As an oxidase, Grx 1 provides oxidizing equivalents to its substrates through a reactive mixed disulfide with glutathione. As reductants of protein disulfides, both Grx 1 and PDI require two active site cysteines. For PDI, its multi-domain structure is needed to catalyze protein disulfide reduction. It is suggested that both Grx1 and PDI have developed specialized mechanisms to enhance catalysis of reactions that would normally be difficult because of the stability of the active site thiols and disulfides. In the yeast Saccharomyces cerevisiae, deletion of the PDII gene is lethal. There has been some uncertainty about which part of this catalytic activity, the oxidase or isomerase activity, represents the essential in vivo function of PDI. The single catalytic a' domain of yeastPDI (yeastPDla') has 50% of the oxidase activity of yeast PDI, but less than 5% of the isomerase activity. However, this isomerasedeficient mutant of PDI supports wild-type, growth even in a strain in which all of the PDI homologues of the yeast ER (Mpd1p, Mpd2p, Eug1p, Eps1p) have been deleted. Thus, sulfhydryl oxidation, not disulfide isomerization, is the essential function of PDI. Despite the importance of its oxidase activity, pulse-chase experiments monitoring the maturation of caboxypeptidase Y (CPY) suggest that PDI does display its isomerase activity in vivo. Using a more sensitive gel-shift assay to measure the redox state of PDI in the ER, we find that a significant fraction of the PDI active sites are in the reduced state (32±8%), capable of catalyzing disulfide isomerization. ER homologues of PDI also provide oxidase and isomerase activity to sustain relatively efficient processing of native disulfide bond formation when the catalytic functions of PDI are compromised. Surprisingly, yeast strains that have all the homologues of Pdi 1p deleted still show wild-type growth rates even when isomerase-deficient Pdi 1p provides the only source of PDI function. High levels of disulfide isomerization are evidently not essential to the survival and growth of yeast, suggesting an evolutionary process in yeast that may select against essential proteins that require disulfide isomerization.

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