Preparation, Characterization, and Complete Heteronuclear NMR Resonance Assignments of the Glutaredoxin (C14S)-Ribonucleotide Reductase B1 737-761 (C754S) Mixed Disulfide
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- 作者：Marcelo J. Berardi ; Cynthia L. Pendred ; and John H. Bushweller
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：April 28, 1998
- 年：1998
- 卷：37
- 期：17
- 页码：5849 - 5857
- 全文大小：214K
- 年卷期：v.37,no.17(April 28, 1998)
- ISSN：1520-4995
文摘
The first committed step in de novo DNA biosynthesis involves theconversion ofribonucleotides to the corresponding deoxyribonucleotides catalyzed bythe enzyme ribonucleotidereductase. Reduction of disulfides in ribonucleotide reductase isessential and is catalyzed by the proteindisulfide reductants glutaredoxin or thioredoxin. The interactionregion between Escherichia coliglutaredoxin-1 and E. coli ribonucleotide reductase has beenlocalized to the C-terminal end of the B1subunit of ribonucleotide reductase. We have demonstrated that a25-residue peptide corresponding tothis C-terminal sequence is a very good substrate for glutaredoxin viaa fluorescence assay and that thispeptide binds in a specific manner via isothermal titrationcalorimetric measurements. By selectivelymutating the two cysteines in the peptide, we have identified theelectrophilic cysteine as C759 (B1numbering) and prepared a mixed disulfide between E. coliglutaredoxin-1 (C14 
S) and the C759monothiol form of the peptide. The peptide and the protein havebeen labeled with 13C and 15N, andcomplete heteronuclear NMR resonance assignments have been completedfor both the peptide and theprotein in the complex. By using half-filtered NOESY spectra,intermolecular NOEs between the proteinand the peptide have been identified and the binding site onglutaredoxin has been mapped. The electrostaticcharge distribution of the protein in this region is very positive,thus providing an excellent match for thehighly negatively charged peptide. In addition, the electrostaticpotential of the peptide provides a rationalefor the observed cysteine selectivity in the reaction betweenglutaredoxin and the B1 peptide.
S) and the C759monothiol form of the peptide. The peptide and the protein havebeen labeled with 13C and 15N, andcomplete heteronuclear NMR resonance assignments have been completedfor both the peptide and theprotein in the complex. By using half-filtered NOESY spectra,intermolecular NOEs between the proteinand the peptide have been identified and the binding site onglutaredoxin has been mapped. The electrostaticcharge distribution of the protein in this region is very positive,thus providing an excellent match for thehighly negatively charged peptide. In addition, the electrostaticpotential of the peptide provides a rationalefor the observed cysteine selectivity in the reaction betweenglutaredoxin and the B1 peptide.