Combinations of protein-disulfide isomerase domains show that there is little correlation between isomerase activity and wild-type growth

Protein-disulfide isomerase (PDI) has five domains: a, b, b', a' and c, all of which except c have a thioredoxin fold. A single catalytic domain (a or a') is effective in catalyzing oxidation of a reduced protein but not isom erization of disulfides (Darby, N. J., and Creighton, T. E. (1995) Biochemi stry 34, 11725-11735). 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', ab'a' display low disulfide isomerase activity, but all show significant reactivity with mammalian thio redoxin reductase, suggesting that the structure is not seriously compromis ed. The only domain order that retains significant isomerase activity has t he b' domain coupled to the N terminus of the a' domain. This b'a'c 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 (Walke r, K. W., Lyles, M. M., and Gilbert, H. F. (1996) Biochemistry 35, 1972-198 0). Individual a and a' domains, despite their very low isomerase activitie s in vitro, support wild-type growth of a pdi1 Delta Saccharomyces cerevisi ae strain yeast. Thus, most of the PDI structure is dispensable for its ess ential function in yeast, and high-level isomerase activity appears not req uired for viability or rapid growth.