Mj. Berardi et Jh. Bushweller, Binding specificity and mechanistic insight into glutaredoxin-catalyzed protein disulfide reduction, J MOL BIOL, 292(1), 1999, pp. 151-161
The reduction equivalents necessary for the ribonucleotide reductase (RNR)-
catalyzed production of deoxyribonucleotides are provided by glutaredoxin (
Grx) or thioredoxin (Trx). The initial location for transfer of reducing eq
uivalents to RNR is located at the C terminus of the B1 subunit and involve
s the reduction of a disulfide between Cys754 and Cys759. We have used a 25
-mer peptide corresponding to residues 737-761 of RNR B1 (C754-->S) to synt
hesize a stable mixed disulfide with Escherichia coli Grx-1 (C14 --> S) res
embling the structure of an intermediate in the reaction. The high-resoluti
on solution structure of the mixed disulfide has been obtained by NMR with
an RMSD of 0.56 Angstrom for all the backbone atoms of the protein and the
well-defined portion of the peptide. The binding interactions responsible f
or specificity have been identified demonstrating the importance of electro
static interactions in this system and providing a rationale for the specif
icity of the Grx-RNR interaction. The disulfide is buried in this complex,
implying a solely intra-molecular mechanism of reduction in contrast to the
previously determined structure of the glutathione complex where the disul
fide was exposed; mutagenesis studies have shown the relevance of intermole
cular reduction processes. Substantial conformational changes in the helice
s of the protein are associated with peptide binding which have significant
mechanistic implications for protein disulfide reduction by glutaredoxins.
(C) 1999 Academic Press.