Finding the Missing Link - Modeling the Conformational Space of the Active and Inactive forms of E. Coli Ribonucleotide Reductase

Abstract

Ribonucleotide Reductase (RNR) is an enzyme that catalyzes the production of deoxyribonucletides from ribonucleotides. The active form of E. coli RNR is comprised of two homodimeric subunits, α2 and β2, and catalysis involves radical transfer between Y122 on the β2 subunit and C439 on the α2 subunit. By contrast, recent structural data suggest that the dATP-inhibited (inactive) form of the enzyme corresponds to an α4β4 ring. In this structure Y122 on the β2 subunit and C439 on the α2 subunit are separated by more than 60 angstroms - a finding that explains, in part, why this structure is inactive. Interestingly, in crystal structures of the inactive complex, ∼27 residues near the C-terminus the β2 subunit do not have electron density, suggesting that these residues are disordered in the inactive form of the enzyme. This missing “linker region” contains a tyrosine residue (Y356) that plays a crucial role in radical transfer in the active complex. In this work we use molecular simulations to explore the conformational space of these disordered residues in both active and inactive models of E. coli RNR. Our data suggest that Y356 adopts states that are inconsistent with radical transfer in the inactive complex. These observations argue that inactivity is explained both by the increased distance required for radical transfer, and by the relative position of crucial residues in the flexible linker region in the inactive state.