An endogenous dAMP ligand in Bacillus subtilis class Ib RNR promotes assembly of a noncanonical dimer for regulation by dATP

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 115; no. 20; pp. E4594 - E4603
• Main Authors: Parker, Mackenzie J., Maggiolo, Ailiena O., Thomas, William C., Kim, Albert, Meisburger, Steve P., Ando, Nozomi, Boal, Amie K., Stubbe, JoAnne
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 15.05.2018
• Series: PNAS Plus
• Subjects: Allosteric properties; Allosteric Regulation; allostery; ATP; ATP-cone; Bacillus subtilis; Bacillus subtilis - enzymology; Bacillus subtilis - genetics; Bacillus subtilis - growth & development; Bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; BASIC BIOLOGICAL SCIENCES; Binding; Biochemistry; Biological Sciences; Crystallography; dAMP; Deoxyadenine Nucleotides - chemistry; Deoxyadenine Nucleotides - metabolism; Deoxyadenosine; Deoxyribonucleic acid; Dimers; DNA; DNA biosynthesis; DNA repair; Domains; Enzymes; Inhibition; Ligands; nucleotide metabolism; Oligomers; PNAS Plus; Protein Binding; Protein Conformation; Protein structure; Quaternary structure; Reductases; Ribonucleotide reductase; Ribonucleotide Reductases - chemistry; Ribonucleotide Reductases - genetics; Ribonucleotide Reductases - metabolism; Scattering, Small Angle; Small angle X ray scattering; Substrate Specificity; X-ray crystallography; X-ray scattering; ribonucleotide reductase; nucleotide metabolism; dAMP; ATP-cone; allostery
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1800356115

The high fidelity of DNA replication and repair is attributable, in part, to the allosteric regulation of ribonucleotide reductases (RNRs) that maintains proper deoxynucleotide pool sizes and ratios in vivo. In class Ia RNRs, ATP (stimulatory) and dATP (inhibitory) regulate activity by binding to the ATP-cone domain at the N terminus of the large α subunit and altering the enzyme’s quaternary structure. Class Ib RNRs, in contrast, have a partial cone domain and have generally been found to be insensitive to dATP inhibition. An exception is the Bacillus subtilis Ib RNR, which we recently reported to be inhibited by physiological concentrations of dATP. Here, we demonstrate that the α subunit of this RNR contains tightly bound deoxyadenosine 5′-monophosphate (dAMP) in its N-terminal domain and that dATP inhibition of CDP reduction is enhanced by its presence. X-ray crystallography reveals a previously unobserved (noncanonical) α₂ dimer with its entire interface composed of the partial N-terminal cone domains, each binding a dAMP molecule. Using small-angle X-ray scattering (SAXS), we show that this noncanonical α₂ dimer is the predominant form of the dAMP-bound α in solution and further show that addition of dATP leads to the formation of larger oligomers. Based on this information, we propose a model to describe the mechanism by which the noncanonical α₂ inhibits the activity of the B. subtilis Ib RNR in a dATP- and dAMP-dependent manner.