Molecular characterization of the principal substrate binding site of the ubiquitous folding catalyst protein disulfide isomerase.

Annamari Pirneskoski,Kirsi E.H Salo,Robert B Freedman,Peter Klappa,Richard A Williamson,Heli I Alanen,Lee Byrne,Lloyd W Ruddock,Mario Lobell,Kari I Kivirikko

doi:10.1074/jbc.m312193200

Annamari Pirneskoski, Kirsi E.H Salo + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m312193200

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Abstract

Disulfide bond formation in the endoplasmic reticulum of eukaryotes is catalyzed by the ubiquitously expressed enzyme protein disulfide isomerase (PDI). The effectiveness of PDI as a catalyst of native disulfide bond formation in folding polypeptides depends on the ability to catalyze disulfide-dithiol exchange, to bind non-native proteins, and to trigger conformational changes in the bound substrate, allowing access to buried cysteine residues. It is known that the b' domain of PDI provides the principal peptide binding site of PDI and that this domain is critical for catalysis of isomerization but not oxidation reactions in protein substrates. Here we use homology modeling to define more precisely the boundaries of the b' domain and show the existence of an intradomain linker between the b' and a' domains. We have expressed the recombinant b' domain thus defined; the stability and conformational properties of the recombinant product confirm the validity of the domain boundaries. We have modeled the tertiary structure of the b' domain and identified the primary substrate binding site within it. Mutations within this site, expressed both in the isolated domain and in full-length PDI, greatly reduce the binding affinity for small peptide substrates, with the greatest effect being I272W, a mutation that appears to have no structural effect.

Highlights

Disulfide bond formation in the endoplasmic reticulum of eukaryotes is catalyzed by the ubiquitously expressed enzyme protein disulfide isomerase (PDI)
It is known that the b؅ domain of PDI provides the principal peptide binding site of PDI and that this domain is critical for catalysis of isomerization but not oxidation reactions in protein substrates
What is clear is that the rate-limiting step for native disulfide bond formation in proteins that contain multiple disulfides is latestage isomerization reactions, where disulfide bond formation is linked to conformational changes in protein substrates with substantial regular secondary structure

Summary

EXPERIMENTAL PROCEDURES

Secondary Structure Prediction—The sequence of mature human PDI was submitted to the PredictProtein server [16]. PredictProtein uses the subprogram MaxHom [17] to perform a data base search for homologous sequences and to align those sequences into a multiple alignment file. The MaxHom-derived multiple alignment file contained 53 sequences, and this was used as an input to PredictProtein. The sequence data from the MaxHom-derived multiple alignment file was used as an input for the secondary structure prediction program PREDATOR [18]. The secondary structure assignments for the b and b؅ domains of human PDI were predicted with PREDATOR using a different input file that was specially created using the sequences of just the b and b؅ domains as input for the generation of a multiple alignment file, which contained 35 sequence fragments. This parameter was chosen at it is independent of concentration and less dependent on the direct effects of guanidinium chloride on tryptophan fluorescence

RESULTS

Protein produced

DISCUSSION