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Crystal structure of dATP bound E. coli class Ia ribonucleotide reductase alpha construct fused with the C-terminal tail of E. coli class Ia beta subunit
Structural highlights
FunctionRIR1_ECOLI Provides the precursors necessary for DNA synthesis. Catalyzes the biosynthesis of deoxyribonucleotides from the corresponding ribonucleotides. R1 contains the binding sites for both substrates and allosteric effectors and carries out the actual reduction of the ribonucleotide. It also provides redox-active cysteines.RIR2_ECOLI Provides the precursors necessary for DNA synthesis. Catalyzes the biosynthesis of deoxyribonucleotides from the corresponding ribonucleotides. R2 contains the tyrosyl radical required for catalysis. Publication Abstract from PubMedClass Ia ribonucleotide reductases (RNRs) are allosterically regulated by ATP and dATP to maintain the appropriate deoxyribonucleotide levels inside the cell for DNA biosynthesis and repair. RNR activity requires precise positioning of the beta(2) and alpha(2) subunits for the transfer of a catalytically essential radical species. Excess dATP inhibits RNR through the creation of an alpha-beta interface that restricts the ability of beta(2) to obtain a position that is capable of radical transfer. ATP breaks the alpha-beta interface, freeing beta(2) and restoring enzyme activity. Here, we investigate the molecular basis for allosteric activity regulation in the well-studied Escherichia coli class Ia RNR through the determination of six crystal structures and accompanying biochemical and mutagenesis studies. We find that when dATP is bound to the N-terminal regulatory cone domain in alpha, a helix unwinds, creating a binding surface for beta. When ATP displaces dATP, the helix rewinds, dismantling the alpha-beta interface. This reversal of enzyme inhibition requires that two ATP molecules are bound in the cone domain: one in the canonical nucleotide-binding site (site 1) and one in a site (site 2) that is blocked by phenylalanine-87 and tryptophan-28 unless ATP is bound in site 1. When ATP binds to site 1, histidine-59 rearranges, prompting the movement of phenylalanine-87 and trytophan-28, and creating site 2. dATP hydrogen bonds to histidine-59, preventing its movement. The importance of site 2 in the restoration of RNR activity by ATP is confirmed by mutagenesis. These findings have implications for the design of bacterial RNR inhibitors. How ATP and dATP Act as Molecular Switches to Regulate Enzymatic Activity in the Prototypical Bacterial Class Ia Ribonucleotide Reductase.,Funk MA, Zimanyi CM, Andree GA, Hamilos AE, Drennan CL Biochemistry. 2024 Aug 20. doi: 10.1021/acs.biochem.4c00329. PMID:39164005[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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