Structural characterization and antibiotic development for the Neisseria gonorrhoeae class Ia ribonucleotide reductase

Abstract

Ribonucleotide reductases (RNRs) are essential enzymes that catalyze the reduction of ribonucleotides to deoxyribonucleotides, a critical step in DNA biosynthesis and repair. Class Ia RNRs, found in eukaryotes and many aerobic bacteria including Escherichia coli (E. coli) and Neisseria gonorrhoeae (N. gonorrhoeae), are regulated by complex allosteric mechanisms that control enzymatic activity and substrate specificity. These enzymes function as α₂β₂ complexes, with the α₂ subunit housing regulatory sites and the β₂ subunit providing a catalytic radical. Proper regulation of RNR is vital for maintaining balanced dNTP pools and genomic integrity, making RNRs attractive targets for therapeutic intervention in both infectious disease and cancer. This thesis examines the structural basis of specificity regulation in N. gonorrhoeae class Ia RNR using cryogenic electron microscopy (cryo-EM). Near atomic-resolution structures of the enzyme bound to four canonical substrate/specificity-effector pairs (CDP/dATP, UDP/dATP, GDP/TTP, and ADP/dGTP) reveal that effectors induce conformational changes in loop 2 of the α₂ subunit, which alter hydrogen bonding contacts in the active site, leading to preferential substrate binding. This mechanism is also conserved in the E. coli class Ia RNR, a close homolog. Cryo-EM maps also show weak, non-specific binding of a second nucleotide in the cone domain, likely reflecting artifacts of high nucleotide concentrations used in the cryo-EM experiments rather than physiological relevance. Building on these findings, Chapter III investigates the potential interaction of the cyclic dinucleotide, c-diAMP, with the cone domains of E. coli and N. gonorrhoeae RNRs. We find that c-diAMP binds both enzymes with low micromolar affinity but does not alter the activity of either RNR to a large extent over the no-effector control. Although similar, the behavior of E. coli and N. gonorrhoeae RNRs are not identical, highlighting the potential of targeting the cone domain for species-specific RNR inhibition. This approach could enable the development of novel antibiotics, particularly needed for combatting antibiotic-resistant N. gonorrhoeae.