The significance of the domains of protein disulfide isomerase for the different functions of the protein

Annamari Pirneskoski

Collagen Research Unit, University of Oulu
Biocenter Oulu, University of Oulu
Department of Medical Biochemistry and Molecular Biology, University of Oulu

Abstract

Protein disulfide bonds are covalent links formed between the thiol groups of cysteine residues. In many proteins, they have an important role in stabilizing the three-dimensional conformation of the polypeptide chain. Usually proteins are physiologically active and functional only when they are correctly folded. Protein folding takes place very soon after the synthesis of a new polypeptide chain. Proteins which are to be secreted from the cell fold in a specialized compartment, the endoplasmic reticulum (ER).

Folding and disulfide bond formation in the ER does not happen spontaneously, there are proteins which are specialized in assisting in these processes. Protein disulfide isomerase (PDI) is a multifunctional protein, which is capable of catalysing both of disulfide bond formation and folding of a protein. In addition, it has other functions: it is an essential part of two protein complexes: collagen prolyl 4-hydroxylase (C-P4H) and microsomal triglyceride transfer protein.

C-P4H is an enzyme essential in the formation of collagens, proteins found in connective tissue. The function of C-P4H is to catalyse the hydroxylation of prolines, which is essential for the structural stability of collagens. C-P4H is a tetramer, formed of two catalytic α subunits and two β subunits, which are identical to PDI. The function of PDI in C-P4H is apparently to keep it in a soluble, functionally active conformation.

In mammals there are several proteins similar to PDI, together forming a PDI family of proteins. They share both structural and functional similarities. One of these proteins is ERp57. It is specialized in assisting in the folding and disulfide bond formation of glycoproteins.

PDI consists of four domains, two of which contain a catalytic site for disulfide bond formation. One domain is the main site of interaction with other proteins and one domain is of unknown function. In this study, the role of these domains in the activities of PDI was investigated. The peptide-binding domain was characterized in detail. In addition, structural similarities of PDI and ERp57 were studied by formation of hybrid proteins containing domains of both and comparing the activities of these recombinant proteins to those of PDI.

Table of Contents
Acknowledgements
Abbreviations
List of original articles
1. Introduction
2. Review of the literature
2.1. Native disulfide bond formation in proteins
2.1.1. Disulfide bonds
2.1.2. Pathways of protein disulfide bond formation
2.2. Protein disulfide isomerase
2.2.1. Structure
2.2.2. Functions
2.2.3. PDI family
3. Outlines of the study
4. Materials and methods
4.1. Generation of E.coli and baculovirus expression constructs (I, II, III)
4.2. Generation of recombinant baculoviruses (I, II)
4.3. Site-directed mutagenesis (I, III)
4.4. Production, purification and analyses of recombinant proteins (I, II, III)
4.5. Enzyme activity assays ( I, III)
4.6. Binding and cross-linking of synthetic peptides (II)
4.7. Biophysical characterization and NMR analysis of proteins (I, III)
5. Results
5.1. The role of the C-terminal region of PDI in the C-P4H enzyme tetramer formation and in the chaperone and isomerase activities of the enzyme (I)
5.2. The effect of the domain constructs of PDI and of PDI/ERp57 hybrid constructs in C-P4H assembly and activity and in the binding of Δ-somatostatin and scRNase (II)
5.3. Molecular characterization of the main substrate binding site, the b’ domain of PDI
6. Discussion
6.1. Region c of PDI is not crucial for the main functions of the protein, but the end of domain a’ contains an area sensitive to mutations
6.2. C-P4H tetramer assembly requires the minimum of b’a’ domains of PDI but effective association requires longer constructs, in part substitutable by domains of ERp57
6.3. Molecular characteristics of the primary peptide binding site of PDI
References
List of Figures
1. Domain architecture of PDI. The boundaries for the domains a and b are those defined by NMR (Kemmink et al. 1996, 1999), while those for the a’ domain are defined by homology to domain a.
2. Structures of domains a and b as defined by NMR (Kemmink et al. 1996, 1999). For the a domain the active site cysteine residues are shown.
3. Mechanisms of catalysis by PDI.
4. Domain organisation of the mammalian PDI family. Catalytic domains are patterned with vertical lines, b-like domains with dots, b’-like with diagonal lines, c-like are white and regions with no homology to any of the domains of PDI are patterned with dashed diagonal lines.
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