title: Preparation, characterization, and complete heteronuclear NMR resonance assignments of the glutaredoxin (C14S)-ribonucleotide reductase B1 737-761 (C754S) mixed disulfide.
abstract: The first committed step in de novo DNA biosynthesis involves the conversion of ribonucleotides to the corresponding deoxyribonucleotides catalyzed by the enzyme ribonucleotide reductase. Reduction of disulfides in ribonucleotide reductase is essential and is catalyzed by the protein disulfide reductants glutaredoxin or thioredoxin. The interaction region between Escherichia coli glutaredoxin-1 and E. coli ribonucleotide reductase has been localized to the C-terminal end of the B1 subunit of ribonucleotide reductase. We have demonstrated that a 25-residue peptide corresponding to this C-terminal sequence is a very good substrate for glutaredoxin via a fluorescence assay and that this peptide binds in a specific manner via isothermal titration calorimetric measurements. By selectively mutating the two cysteines in the peptide, we have identified the electrophilic cysteine as C759 (B1 numbering) and prepared a mixed disulfide between E. coli glutaredoxin-1 (C14 --> S) and the C759 monothiol form of the peptide. The peptide and the protein have been labeled with 13C and 15N, and complete heteronuclear NMR resonance assignments have been completed for both the peptide and the protein in the complex. By using half-filtered NOESY spectra, intermolecular NOEs between the protein and the peptide have been identified and the binding site on glutaredoxin has been mapped. The electrostatic charge distribution of the protein in this region is very positive, thus providing an excellent match for the highly negatively charged peptide. In addition, the electrostatic potential of the peptide provides a rationale for the observed cysteine selectivity in the reaction between glutaredoxin and the B1 peptide.
authors.affiliation: Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Berardi
authors.forename: C L
authors.initials: CL
authors.lastname: Pendred
authors.forename: J H
authors.initials: JH
authors.lastname: Bushweller
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology; D054477:Glutaredoxins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010088:Oxidoreductases; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity
chemical_list: D004220:Disulfides; D054477:Glutaredoxins; D010446:Peptide Fragments; D011506:Proteins; D010088:Oxidoreductases; D012264:Ribonucleotide Reductases; D003545:Cysteine