Redox properties and cross-linking of the dithiol/disulphide active sites of mammalian protein disulphide-isomerase

121

Covalent attachment of heme to apocytochromes c in bacteria occurs on the outside of the cytoplasmic membrane and requires two reduced cysteinyls at the heme binding site. A constructed ResA-deficient Bacillus subtilis strain was found to lack c-type cytochromes. Cytochrome c synthesis was restored in the mutant by: (i) in trans expression of resA; (ii) deficiency in BdbD, a thioldisulfide oxidoreductase that catalyzes formation of an intramolecular disulfide bond in apocytochrome c after transfer of the polypeptide across the cytoplasmic membrane; or (iii) by addition of the reductant dithiothreitol to the growth medium. In vivo studies of ResA showed that it is membrane-associated with its thioredoxin-like domain on the outside of the cytoplasmic membrane. Analysis of a soluble form of the protein revealed two redox reactive cysteine residues with a midpoint potential of about ؊340 mV at pH 7. We conclude that ResA, probably together with another thiol-disulfide oxidoreductase, CcdA, is required for the reduction of the cysteinyls in the heme binding site of apocytochrome c.

Section: Discussionmentioning

confidence: 99%

Bacillus subtilis ResA Is a Thiol-Disulfide Oxidoreductase involved in Cytochrome c Synthesis

Erlendsson

Acheson

Hederstedt

et al. 2003

121

“…Since SDS-PAGE and crystallography revealed that roGFPs form only intramolecular disulfide bonds, R can be related to K eq by Equation 2 (42).…”

Section: Methodsmentioning

confidence: 99%

Investigating Mitochondrial Redox Potential with Redox-sensitive Green Fluorescent Protein Indicators

Hanson

Aggeler

Oglesbee

et al. 2004

901

957

Current methods for determining ambient redox potential in cells are labor-intensive and generally require destruction of tissue. This precludes single cell or real time studies of changes in redox poise that result from metabolic processes or environmental influences. By substitution of surface-exposed residues on the Aequorea victoria green fluorescent protein (GFP) with cysteines in appropriate positions to form disulfide bonds, reduction-oxidation-sensitive GFPs (roGFPs) have been created. roGFPs have two fluorescence excitation maxima at about 400 and 490 nm and display rapid and reversible ratiometric changes in fluorescence in response to changes in ambient redox potential in vitro and in vivo. Crystal structure analyses of reduced and oxidized crystals of roGFP2 at 2.0-and 1.9-Å resolution, respectively, reveal in the oxidized state a highly strained disulfide and localized main chain structural changes that presumably account for the state-dependent spectral changes. roGFP1 has been targeted to the mitochondria in HeLa cells. Fluorometric measurements on these cells using a fluorescence microscope or in cell suspension using a fluorometer reveal that the roGFP1 probe is in dynamic equilibrium with the mitochondrial redox status and responds to membrane-permeable reductants and oxidants. The roGFP1 probe reports that the matrix space in HeLa cell mitochondria is highly reducing, with a midpoint potential near ؊360 mV (assuming mitochondrial pH ϳ8.0 at 37°C). In other work (C. T. Dooley, T. M. Dore, G. Hanson, W. C. Jackson, S. J. Remington, and R. Y. Tsien, submitted for publication), it is shown that the cytosol of HeLa cells is also unusually reducing but somewhat less so than the mitochondrial matrix.

“…The entire PDI molecule, with such an annular domain arrangement, can catalyze those protein folding reactions through cooperative action not only between the a and aЈ domains but also involving the bЈ domain, which does not participate directly in thiol-disulfide exchange, because of lack of a -CXXC-active site, but provides the principal specific peptide-binding site (1,2). In the present model the a and aЈ domains could be very close to each other, consistent with the cross-linking results (20), so that they can function synergistically; in contrast, the two domains would be at least 60 Å apart if the four domains were arranged in a linear fashion. Because the cavity observed in this model appears not to be sufficiently large to allow all possible PDI substrates to fit, the inter-domain motion is likely required for the full range of PDI activities.…”

Section: Pdi Exists As a Short And Roughly Ellipticalmentioning

confidence: 99%

Annular Arrangement and Collaborative Actions of Four Domains of Protein-disulfide Isomerase

Hong

Shi

et al. 2006

We presented for the first time a small angle x-ray scattering study of intact protein-disulfide isomerase (PDI) in solution. The restored model revealed that PDI is a short and roughly elliptical cylinder with a molecular mass of 69 kDa and dimensions of 105 ؋ 65 ؋ 40 Å , and the four thioredoxin-fold domains in the order a-bb-a are arranged in an annular fashion. Atomic force microscope imaging also supported the finding that PDI appears as an approximately flat elliptical cylinder. A PDI species with apparent molecular mass of 116 kDa measured by using size-exclusion chromatography, previously assumed to be a dimer, was determined to exist mainly as a monomer by using analytical ultracentrifugation. The C-terminal fragment 441-491 contributed to the anomalous molecular mass determination of PDI by size-exclusion chromatography. The annular model of PDI accounted for the cooperative properties of the four domains in both the isomerase and chaperone functions of PDI.Protein-disulfide isomerase (PDI, 3 EC 5.3.4.1) is an abundant multifunctional protein within the lumen of the endoplasmic reticulum (1, 2). It plays important roles in many physiological processes as both an enzyme and a molecular chaperone (3-5) by assisting in folding, unfolding, and translocation of many disulfide-containing proteins (6) and even some disulfide-free proteins (7). In addition, PDI serves as an obligatory ␤ subunit of hetero-oligomeric prolyl 4-hydroxylase (8) and microsomal triglyceride transfer protein (9) to maintain highly insoluble ␣ subunits in an active, nonaggregated conformation (10).The four-domain architecture of PDI in the order a-b-bЈ-aЈ was proposed when the PDI cDNA was first sequenced, according to the internal sequence homology within the molecule (11). Later, the domain boundaries were defined (12) and further refined as shown in Scheme 1 by combined use of protein engineering, limited proteolysis, and bioinformatics (13-16). The a and aЈ domains, each with a -CGHC-motif as the active site, share 47% sequence identity, whereas the b and bЈ domains, with no such active site motif, share 28% sequence identity (11). The C-terminal 29 residues, 463-491, constitute an acidic c extension (2), in which over half of the residues are Glu/Asp, and represent a putative Ca 2ϩ -binding region (12). The three-dimensional structure determined by using NMR of the a domain, which shares 27% identity to thiroredoxin (Trx), shows a Trx-fold (15), and the b domain, which shows no significant sequence similarity to the a and Trx, also has a Trx-fold (14). PDI has thus been suggested to consist of four Trx-fold domains (14). Recently, a 19-amino acid linker region, 333-351, was identified between the bЈ and aЈ domains (16), which was suggested to potentially allow more flexibility between these domains than between the other domains. The three-dimensional structure of the entire PDI molecule, or of any other catalytically active eukaryotic PDI family member, has not yet been determined even though PDI was discovered over 40 years ago....