Experimental and Theoretical Analyses of the Domain Architecture of Mammalian Protein Disulphide-Isomerase

Abstract

The high resolution structure of full-length protein disulphide-isomerase (PDI) has not been determined, but the polypeptide is generally assumed to comprise a series of consecutive domains. Models of its domain organisation have been proposed on the basis of various sequence-based criteria and, more recently, from structural studies on recombinant fragments corresponding to putative domains. We here describe direct studies of the domain architecture of full-length mammalian PDI based on limited proteolysis of the native enzyme. The results are consistent with an emerging model based on the existence of 4 consecutive domains each with the thioredoxin fold. The model was further tested by expressing recombinant fragments corresponding to alternative domain models and to truncated domains; the observed properties of these purified fragments supported the 4-domain model. A multiple alignment of many PDI-like sequences was generated to test whether domain boundaries could be predicted from any features of the alignment, such as sequence variability or hydrophilicity; neither of these parameters reliably predicted the domain boundaries determined by experiment.

Alternative Conformations of the x Region of Human Protein Disulphide-Isomerase Modulate Exposure of the Substrate Binding b’ Domain

“…Previously we showed that limited proteolysis of full length bovine PDI results in the formation of fragments in which the x region was associated either with the b' domain or with the a' domain, but that cleavage at an internal site in the x region was never observed 23 . This implies that the x region is structured and that this structure can be associated with either of the adjacent domains.…”

Oxidative Protein Folding in the Mitochondrial Intermembrane Space

“…PDI is an essential protein that is organized structurally in four thioredoxin domains and catalyzes the transfer of disulfide bonds to substrate proteins via a CX2C active center (37,53,115). The redox state of PDI is determined by the essential protein Ero1, a flavin bound membrane associated protein (36,43,92).…”

Biological Functions of Protein Disulfide Isomerase as a Target of Phenolic Endocrine-disrupting Chemicals

“…3). 55) Domains a and a' have a catalytically active Cys-Gly-His-Cys motif, while b and b' domain do not have catalytic activity. The order of the domains is a-b-b'-x-a'-c, where x is a linker region between b' and a' 56) and c is a Cterminal acidic α-helix containing the ER retention signal.…”