The CXXC motif: imperatives for the formation of native disulfide bonds in the cell.

Abstract

The rapid formation of native disulfide bonds in cellular proteins is necessary for the efficient use of cellular resources. This process is catalyzed in vitro by protein disulfide isomerase (PDI), with the PDI1 gene being essential for the viability of Saccharomyces cerevisiae. PDI is a member of the thioredoxin (Trx) family of proteins, which have the active‐site motif CXXC. PDI contains two Trx domains as well as two domains unrelated to the Trx family. We find that the gene encoding Escherichia coli Trx is unable to complement PDI1 null mutants of S.cerevisiae. Yet, Trx can replace PDI if it is mutated to have a CXXC motif with a disulfide bond of high reduction potential and a thiol group of low pKa. Thus, an enzymic thiolate is both necessary and sufficient for the formation of native disulfide bonds in the cell.

The protein disulphide-isomerase family: unravelling a string of folds

“…At neutral pH the reactive sulphur atom of Cys-32 may share a hydrogen bond to the -SH hydrogen of Cys-35 [37]. The pK a of Cys-32 is thought to be decreased by a nearby buried partial charge on Asp-26 [36,38], which may serve as a general acid\base in thioredoxin-catalysed redox reactions [39]. The Cys-32 thiolate can make a nucleophilic attack on disulphides, generating a mixed disulphide that is then disrupted by Cys-35 to produce a reduced substrate protein.…”

“…The redox\isomerase activities of PDI, as in thioredoxin, are due to the reactivity of the N-terminal Cys residue in two thioredoxin-like boxes (-Cys-Gly-His-Cys-) within the a and ah domains of the protein [38,[48][49][50][51], which can function independently of each other. The N-terminal Cys residue of the first active site of PDI has a pK a of 4.5, and is stabilized partly by the nearby histidine imidazole group (within the -Cys-Gly-His-Cyssequence) and partly by partial positive charges at the Nterminus of an α-helix occurring just after this cysteine.…”

“…PDI is about 50-fold more active than thioredoxin at catalysing the isomerization of disulphide bonds in scrambled RNase [58]. Studies on bacterial thioredoxin in which the thioredoxin-box -Cys-Gly-Pro-Cys-was mutated to -Cys-Gly-His-Cys-resulted in a protein with 10-fold higher oxidizing and disulphide-isomerase activity [8,62] and which had gained the ability to complement S. cere isiae PDI1 null mutants (see below) [38], highlighting the importance of these residues.…”

“…The second Cys residue is required for net oxidation of substrate proteins in itro, but its chief role in i o may be to facilitate rapid disruption of intermolecular disulphides, allowing efficient scanning of several disulphide isomers and preventing trapping of PDI in disulphide-linked complexes [16,38,67,68].…”

AATYK: A novel tyrosine kinase induced during growth arrest and apoptosis of myeloid cells

“…The existence of these domains and the tyrosine kinase domain suggest that AATYK could function as a signal transducing protein. In addition, the protein sequence showed the presence of a CXXC motif at position 504 ± 507, characteristic of the thioredoxin (Trx) family of proteins which play a critical role in the formation of native disul®de bonds in the cell (Chivers et al, 1996).…”