Abstract:The C-terminus of protein R2 is important for the formation of the enzymatically active complex between proteins R1 and R2 of ribonucleotide reductase from Escherichia coil Some residues in this part of R2 may also be involved in intramolecular electron transfer. We now demonstrate that 26 amino acid residues at C-terminus of protein R2 are mobile in the free protein, and can be studied by aH NMR. Spectral assignment of narrow resonances was made by comparison of TOCSY and NOESY spectra from wild-type R2 with … Show more
“…These residues are crucial for interaction with the R1 subunit ( 14 , 30 , 31 ). For the mouse and E. coli proteins, the C termini only become structured upon complex formation between the two subunits ( , ). 1 Stereoviews of (a) the heterodimeric complex (PDB accession code1JK0) between Rnr2 (pink) and Rnr4 (green), (b) the Rnr2 homodimer, and (c) the Rnr4 homodimer.…”
Class I ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides. Eukaryotic RNRs comprise two subunits, the R1 subunit, which contains substrate and allosteric effector binding sites, and the R2 subunit, which houses a catalytically essential diiron-tyrosyl radical cofactor. In Saccharomyces cerevisiae, there are two variants of the R2 subunit, called Rnr2 and Rnr4. Rnr4 is unique in that it lacks three iron-binding residues conserved in all other R2s. Nevertheless, Rnr4 is required to activate Rnr2, and the functional species in vivo is believed to be a heterodimeric complex between the two proteins. The crystal structures of the Rnr2 and Rnr4 homodimers have been determined and are compared to that of the heterodimer. The homodimers are very similar to the heterodimer and to mouse R2 in overall fold, but there are several key differences. In the Rnr2 homodimer, one of the iron-binding helices, helix alphaB, is not well-ordered. In the heterodimer, interactions with a loop region connecting Rnr4 helices alphaA and alpha3 stabilize this Rnr2 helix, which donates iron ligand Asp 145. Sequence differences between Rnr2 and Rnr4 prevent the same interactions from occurring in the Rnr2 homodimer. These findings provide a structural rationale for why the heterodimer is the preferred complex in vivo. The active-site region in the Rnr4 homodimer reveals interactions not apparent in the heterodimer, supporting previous conclusions that this subunit does not bind iron. When taken together, these results support a model in which Rnr4 stabilizes Rnr2 for cofactor assembly and activity.
“…These residues are crucial for interaction with the R1 subunit ( 14 , 30 , 31 ). For the mouse and E. coli proteins, the C termini only become structured upon complex formation between the two subunits ( , ). 1 Stereoviews of (a) the heterodimeric complex (PDB accession code1JK0) between Rnr2 (pink) and Rnr4 (green), (b) the Rnr2 homodimer, and (c) the Rnr4 homodimer.…”
Class I ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides. Eukaryotic RNRs comprise two subunits, the R1 subunit, which contains substrate and allosteric effector binding sites, and the R2 subunit, which houses a catalytically essential diiron-tyrosyl radical cofactor. In Saccharomyces cerevisiae, there are two variants of the R2 subunit, called Rnr2 and Rnr4. Rnr4 is unique in that it lacks three iron-binding residues conserved in all other R2s. Nevertheless, Rnr4 is required to activate Rnr2, and the functional species in vivo is believed to be a heterodimeric complex between the two proteins. The crystal structures of the Rnr2 and Rnr4 homodimers have been determined and are compared to that of the heterodimer. The homodimers are very similar to the heterodimer and to mouse R2 in overall fold, but there are several key differences. In the Rnr2 homodimer, one of the iron-binding helices, helix alphaB, is not well-ordered. In the heterodimer, interactions with a loop region connecting Rnr4 helices alphaA and alpha3 stabilize this Rnr2 helix, which donates iron ligand Asp 145. Sequence differences between Rnr2 and Rnr4 prevent the same interactions from occurring in the Rnr2 homodimer. These findings provide a structural rationale for why the heterodimer is the preferred complex in vivo. The active-site region in the Rnr4 homodimer reveals interactions not apparent in the heterodimer, supporting previous conclusions that this subunit does not bind iron. When taken together, these results support a model in which Rnr4 stabilizes Rnr2 for cofactor assembly and activity.
“…The use of enzyme, at natural abundance, limits the sensitivity of conventional triple resonance heteronuclear NMR methods for residue assignment. 1 H- 1 H TOCSY and 1 H- 1 H NOESY have been used in view of the segmental mobility of the loop but remain of limited use (data not shown). In order to assign the methyl residues of the loop, several truncated fragments of the peptide were used to systematically identify the methyl cross-peaks belonging to each residue.…”
L-asparaginase II (MW 135 kDa) from E. coli is an FDAapproved protein drug used for the treatment of childhood leukemia. Despite its long history as a chemotherapeutic, the structural basis of enzyme action, in solution, remains widely contested. In this work, methylbased 2D [ 1 H-13 C]-heteronuclear single-quantum correlation (HSQC) NMR, at natural abundance, has been used to profile the enzymatic activity of the commercially available enzyme drug. The [ 1 H-13 C]-HSQC NMR spectra of the protein reveal the role of a flexible loop segment in the activity of the enzyme, in solution. Addition of asparagine to the protein results in distinct conformational changes of the loop that could be signatures of intermediates formed in the catalytic reaction. To this end, an isothermal titration calorimetry (ITC)-based assay has been developed to measure the enzymatic reaction enthalpy, as a marker for its activity. Combining both ITC and NMR, it was shown that the disruption of the protein conformation can result in the loss of function. The scope, robustness, and validity of the loop fingerprints in relation to enzyme activity have been tested under different solution conditions. Overall, our results indicate that 2D NMR can be used reliably to gauge the structure−function of this enzyme, bypassing the need to label the protein. Such natural abundant NMR methods can be potentially extended to probe the structure− function aspects of high-molecular-weight protein therapeutics (glycosylated protein drugs, enzymes, therapeutic monoclonal antibodies, antibody−drug conjugates, and Fc-fusion proteins), where (a) flexible loops are required for their function and (b) isotope labeling may not be straightforward.
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