Structural Investigation of Allosteric Regulation in Class III Ribonucleotide Reductases

Abstract

Ribonucleotide reductases (RNRs) are essential enzymes that use radical-based chemistry to catalyze the reduction of ribonucleotides to deoxyribonucleotides. Each RNR class uses a different cofactor to generate a catalytically essential thiyl radical in the active site. Anaerobic (class III) RNRs employ an oxygen-sensitive glycyl radical cofactor installed within the adjacent glycyl radical domain. To maintain intracellular nucleotide pool balance, RNRs are allosterically regulated. For the prototypical class Ia RNR from Escherichia coli, this regulation involves the Nterminus of the catalytic subunit in a region known as the ‘cone domain’. ATP or dATP binding to the cone domain results in an association between it and the radical-generating subunit that either allows or prevents, respectively, radical transfer. Most class III RNRs have a cone domain and are allosterically regulated, but the mechanism of how this regulation proceeds is not well understood. Allosteric activity regulation for such a class III RNR, the class III RNR from Streptococcus thermophilus (StNrdD) is the focus of this thesis. We have developed a universal liquid chromatography tandem mass spectrometry based activity assay, which was adapted for use in class III RNR, and used the assay to show that ATP is an allosteric activity enhancer and dATP is an allosteric activity inhibitor of StNrdD. We used a combination of cryogenic electron microscopy (cryo-EM) and X-ray crystallography to show that ATP and dATP bind to the StNrdD cone domain and that the cone domains adopt exceptionally different conformations in the presence of either allosteric effector. Mutagenesis assays and hydrogendeuterium exchange mass spectrometry data show that the flexible region between the cone domain and the core is important for catalysis. Changes between ATP- and dATP-binding in the cone domains underlie the observed conformational and allosteric changes. This work answers some long-standing questions surrounding class III allosteric regulation and lays the groundwork to continue improving our molecular-level understanding of this complex enzyme system.