Convergent Allostery in Ribonucleotide Reductase

Abstract

Ribonucleotide reductases (RNRs) use a conserved radical-based mechanism to catalyze the conversion of ribonucleotides to deoxyribonucleotides. Within the RNR family, class Ib RNRs are notable for being largely restricted to bacteria, including many pathogens, and for lacking an evolutionarily mobile ATP-cone domain that allosterically controls overall activity. In this study, we report the emergence of a new and unexpected mechanism of activity regulation in the sole RNR of the model organism Bacillus subtilis. Using a hypothesis-driven structural approach that combines the strengths of small-angle X-ray scattering (SAXS), crystallography, and cryo-electron microscopy (cryo-EM), we describe the reversible interconversion of six unique structures, including a flexible, active tetramer and two novel, inhibited filaments. These structures reveal the conformational gymnastics necessary for RNR activity and the molecular basis for its control via an evolutionarily convergent form of allostery.Convergent Allostery in Ribonucleotide Reductase W. C. Thomas, et al.

Structures of Class Id Ribonucleotide Reductase Catalytic Subunits Reveal a Minimal Architecture for Deoxynucleotide Biosynthesis

“…Our work on class Id RNR α subunits shows that they share in common with other class I enzymes a propensity to interconvert between different oligomeric forms. , This feature of class I enzymes presents challenges in structural study, often requiring a suite of characterization techniques to discern the composition of complex mixtures and probe quaternary structure within different concentration regimes. , For example, our current understanding of the properties of the aforementioned dATP-inhibited human α 6 oligomer required X-ray crystallography, SAXS, and electron microscopy analysis. ,, Importantly, there are few experimental approaches to probing the role of dynamics in RNR allostery, even though the relative stability of quaternary forms is known to govern the transition to the active holoenzyme complex in the human enzyme . Recently, a similar combined approach was used to delineate six distinct oligomeric forms of B. subtilis class Ib RNR. , Initial detection of filaments in SAXS studies of dATP-inhibited Bs α required subsequent cryo-EM analysis to determine the precise structure. Observation of multiple oligomeric forms in the Bs Ib enzyme, including filamentous helical structures, resembles our findings from SAXS and crystallographic analysis of Fj class Id α.…”

“…Bs NrdE uses an endogenously bound dAMP in a binding site that involves a partial ATP cone and a region of an extended connector domain to form a noncanonical α 2 dimer. Upon dATP binding, the enzyme is proposed to form extended higher-order inhibited oligomers, recently validated by cryo-EM analysis . There are several possible explanations for the regulatory differences within the class Ib subclass.…”