Biology subjects

Yadav, L. R.

Publications and source records attributed to Yadav, L. R..

3 recordsLinked to original sources

bioRxiv2026

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

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.

biochemistry↗

bioRxiv2025

Structural basis of half-site reactivity in Class Ib ribonucleotide reductases

Ribonucleotide reductases (RNRs) employ radical chemistry to generate deoxyribonucleotides required for DNA synthesis and repair. A notable feature of RNRs is half-site reactivity, where, despite the enzyme being a symmetric 2 dimer, only one active site is catalytically active at a time while the other remains in a "poised" state for substrate binding. This phenomenon is tightly linked to the asymmetric 2{beta}2 interaction required for radical transfer. Here, we determined cryo-EM structures of the -subunit in the apo and holo states, i.e., the complex bound to TTP (effector) and GDP (substrate). The structures reveal asymmetric binding of the effector TTP and the substrate GDP across the dimer, with concomitant stabilization of loops surrounding the ligand-binding site. Interestingly, this asymmetry leads to well-resolved N-terminal density for [~]150 residues in the substrate-bound subunit, but weak density for this region in the effector-bound monomer. N-terminal domains are unresolved in both monomers of the apo structure. Isothermal titration calorimetry supports asymmetric binding of pyrimidine effectors with micromolar affinities. Molecular dynamics simulations and three-dimensional variability analysis reveal synchronous motions of loop 2, which together with the N-terminal domain drive alternate opening and closing of the active sites in the two monomers. These conformational dynamics provide key insights into the mechanistic basis of half-site reactivity. Together, these findings provide new insights into the structural dynamics and thermodynamic principles governing regulation and half-site activity in Class Ib RNRs. Significance statementRibonucleotide reductases (RNRs) are essential enzymes that supply the building blocks required for DNA synthesis and repair, yet the structural basis of their half-site reactivity has remained unclear. Using cryo-electron microscopy, calorimetry, molecular dynamics simulations, and conformational variability analysis, we show that the catalytic -subunit of a Class Ib RNR exhibits asymmetric nucleotide binding and coordinated conformational dynamics between the two monomers. These motions drive alternating opening and closing of the active sites and are linked to differential stabilization of the N-terminal region. Our findings suggest that asymmetric conformational gating and N-terminal sampling regulate productive interaction with the radical-generating {beta}-subunit, providing a mechanistic framework for understanding half-site reactivity and allosteric regulation in RNRs.

biophysics↗

bioRxiv2023

Structural insights into the initiation of free radical formation in the Class Ib ribonucleotide reductases

Class I ribonucleotide reductases consisting of R1 and R2 subunits convert ribonucleoside diphosphates to deoxyribonucleoside diphosphates involving an intricate free radical mechanism. The generation of free radicals in the Class Ib ribonucleotide reductases is mediated by reaction involving di-manganese ions in the R2 subunits and is externally assisted by flavodoxin-like NrdI subunit. This is unlike Class Ia ribonucleotide reductases, where the free radical generation is initiated at its di-iron centre in the R2 subunits with no external support from another subunit. Despite much work on the R2 subunits of Class Ib ribonucleotide reductases, also referred as NrdF, and its partner NrdI, the structural details of free radical generation remain largely unknown. In this study we have determined the crystal structures of Mycobacterial NrdI in oxidized and reduced forms, and similarly those of NrdF2: NrdI complex (NrdF2I). These structures provide the first atomic view of the mechanism of free radical generation in the R2 subunit. We propose that oxygen molecule accesses FMN through a well-formed channel in NrdI, seen clearly in the crystal structure, and upon electron transfer is converted to a superoxide ion. Similarly, a path for superoxide radical transfer between NrdI and NrdF2 is also observed. A delocalised Mn ion in the R2 subunit is seen in the electron density, which attacks Tyr 110 to produce a Tyr* free radical. Finally, a solvent channel to the dimanganese-binding site is observed to complete the cycle. The study therefore provides important structural clues on the initiation of free radical generation in the R2 subunit of the ribonucleotide reductase complex. Significance statementRibonucleotide reductases generate the deoxyribonucleotide pool in the cell for DNA replication and repair. The enzymes utilise free radical mechanism, where the mechanism of radical formation defines different classes of ribonucleotide reductases. Class Ib ribonucleotide reductases generate the free radical though di-manganese chemistry, assisted externally by NrdI. We describe here structural features required to achieve this mechanism. The structures clearly show a tunnel for oxygen access to the FMN site, tunnel to transport the consequent superoxide radical that is formed, a dislocated activated Mn, which appears to coordinate with a Tyrosine residue to form Tyr* radical and a water channel to complete the reaction cycle, thus enhancing our understanding of the steps of free radical generation.

biochemistry↗