CryoEM reveals the dynamic conformational landscape and C -terminal gating mechanism of Mycobacterial Class Ib ribonucleotide reductase

Abstract

Ribonucleotide reductases (RNRs) are essential enzymes that catalyze the reduction of ribonucleoside diphosphates to deoxyribonucleoside diphosphates and function as multi-subunit, asymmetric heterotetrameric (2{beta}2) complexes. Despite their central role in DNA synthesis, the mechanism of subunit association and regulation within this asymmetric assembly has remained incompletely understood. Here, we employed cryo-electron microscopy (cryo-EM) to investigate the apo state assembly and dynamics of a class Ib RNR complex from Mycobacterium thermoresistibile. The 3.8 [A] structure provides a detailed architecture of the complex, including order-disorder transition in the C-terminal tail of the {beta} subunit, which is critical for long-range radical transfer. The alternative {beta}-hairpin conformation in the subunit suggests its role in stabilizing the 2{beta}2 interaction. Structural analysis of the 2{beta}2 complex identified seven distinct conformational states, highlighting substantial heterogeneity and variability in -{beta} subunit association. The observed structural heterogeneity supports a model for apo-state dynamics and suggests {beta}-subunit movements toward the subunit that may facilitate productive {beta} and potential '{beta}' interactions. Complementary thermodynamic analyses further support a model in which conformational flexibility is central to -{beta} subunit recognition and stabilization. Together, these findings illuminate the dynamic conformational landscape that governs subunit association and radical transfer in class Ib RNRs. Given the importance of mycobacteria as pathogens of significant importance in humans and animals, this first structural characterization of a mycobacterial class Ib RNR provides a foundation for future structure-based inhibitor design. Significance StatementRNRs are essential for DNA synthesis in all life forms. Here, we present cryo-EM structures of the ligand-free mycobacterial 2{beta}2 complex, capturing multiple modes of asymmetric subunit association. These structures show that the enzyme samples multiple conformations that likely correspond to different activity states, potentially associated with processes such as DNA repair or replication. Notably, this flexibility arises intrinsically from subunit interactions, even in the absence of substrates or regulatory nucleotides. A 3.8 [A] structure reveals ordering in the {beta}-subunit C-terminal tail critical for long-range radical transfer, while an alternative {beta}-hairpin in the subunit may stabilize the complex. These results provide a structural framework for RNR function and inform future drug design.