Convergent allostery in ribonucleotide reductase

Audrey A Burnim,F Phil Brooks,Jason T Kaelber,James Z Chen,William C Thomas,Joanne Stubbe,John-Paul Bacik,Nozomi Ando

doi:10.1038/s41467-019-10568-4

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 distinct 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 inhibited helical 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.

Highlights

Ribonucleotide reductases (RNRs) use a conserved radical-based mechanism to catalyze the conversion of ribonucleotides to deoxyribonucleotides
We describe the unexpected emergence of a convergent form of activity regulation in the class Ib RNRs, a major subset of aerobic RNRs that lack ATP-cones[7,8,9,10,11]
To obtain conformationally pure scattering profiles, small-angle X-ray scattering (SAXS) was performed with in-line size-exclusion chromatography (SEC–SAXS), and the resultant datasets were mathematically decomposed with evolving factor analysis (EFA), a method we developed to separate dynamically exchanging species that partially co-elute[32] (Supplementary Table 2)

Summary

Introduction

Ribonucleotide reductases (RNRs) use a conserved radical-based mechanism to catalyze the conversion of ribonucleotides to deoxyribonucleotides. 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 inhibited helical 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. Once the thiyl radical is generated in α, nucleotide reduction proceeds via a conserved mechanism using two additional redox-active cysteines in the catalytic site as reducing equivalents[2]. The resulting disulfide is reduced by a cysteine pair on the flexible C-terminus of α17

Methods

Results

Conclusion