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<p>Molecular recognition within the crowded cellular milieu requires biomolecules to navigate complex and dynamic energy landscapes. Such a challenge is particularly acute during biogenesis of bacterial ribosomes, where the erythromycin resistance methyltransferase (Erm) modifies nascent ribosomal RNA to confer antibiotic resistance. Recently, it has been shown that the head domain of Erm binds to a helical cleft of the precursor (immature) ribosome, which allows the catalytic domain to access the&#xa0;target helix where the methylation substrate (adenine) is located. While Cryo-EM structures of the enzyme-substrate complex suggest an ‘anchor-and-sway’ mechanism for Erm, static reconstructions are unable to capture the stochastic dynamics that the enzyme has to undergo to locate the target site on a flexible ribosomal precursor. Here, we resolve the real-time conformational landscape of the Erm-precursor complex using variability analysis of individual single-molecule FRET (smFRET) efficiency trajectories. Our analyses reveal that the enzyme's C-terminal anchor domain is not very rigidly fixed to a transient helical cleft of the immature ribosome; rather, its motion acts as a sensor for the intrinsic ‘breathing’ dynamics of the precursor binding cleft. In contrast, the catalytic N-terminal domain vacillates between catalytically poised states and a distinct, high-entropy conformation characterised by anomalous spatial flexibility (&gt; 3 Å). Our results support a 'tethered search' model wherein the anchoring domain prevents dissociation, enabling the&#xa0;catalytic domain to sample the local free-energy landscape,&#xa0;thereby preventing kinetic trapping. This study reaffirms site-specific recognition not as a deterministic lock-and-key event, but as a dynamic search process, offering a quantitative framework to resolve the rigid-to-fluid continuum in bio-macromolecular assemblies.</p> Graphical abstract <p>Variability analyses of Single-molecule FRET demonstrate that Erm employs a 'tethered search' mechanism to navigate the dynamic surface of the precursor ribosome. While its C-terminal head domain anchors to a transient cleft to maintain a high local concentration, the catalytic domain retains the conformational freedom necessary to scan for the target methylation site, preventing kinetic trapping</p>

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Dynamic basis for substrate recognition in a precursor ribosome by erythromycin resistance methyltransferase

  • 期刊论文

  • 发表日期：

    2026年7月24日

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  • Rajat Mukherjee,
  • Sombuddha Sengupta,
  • Advait Risbud,
  • Ruchi Anand,
  • Arindam Chowdhury

摘要

Molecular recognition within the crowded cellular milieu requires biomolecules to navigate complex and dynamic energy landscapes. Such a challenge is particularly acute during biogenesis of bacterial ribosomes, where the erythromycin resistance methyltransferase (Erm) modifies nascent ribosomal RNA to confer antibiotic resistance. Recently, it has been shown that the head domain of Erm binds to a helical cleft of the precursor (immature) ribosome, which allows the catalytic domain to access the target helix where the methylation substrate (adenine) is located. While Cryo-EM structures of the enzyme-substrate complex suggest an ‘anchor-and-sway’ mechanism for Erm, static reconstructions are unable to capture the stochastic dynamics that the enzyme has to undergo to locate the target site on a flexible ribosomal precursor. Here, we resolve the real-time conformational landscape of the Erm-precursor complex using variability analysis of individual single-molecule FRET (smFRET) efficiency trajectories. Our analyses reveal that the enzyme's C-terminal anchor domain is not very rigidly fixed to a transient helical cleft of the immature ribosome; rather, its motion acts as a sensor for the intrinsic ‘breathing’ dynamics of the precursor binding cleft. In contrast, the catalytic N-terminal domain vacillates between catalytically poised states and a distinct, high-entropy conformation characterised by anomalous spatial flexibility (> 3 Å). Our results support a 'tethered search' model wherein the anchoring domain prevents dissociation, enabling the catalytic domain to sample the local free-energy landscape, thereby preventing kinetic trapping. This study reaffirms site-specific recognition not as a deterministic lock-and-key event, but as a dynamic search process, offering a quantitative framework to resolve the rigid-to-fluid continuum in bio-macromolecular assemblies.

Graphical abstract

Variability analyses of Single-molecule FRET demonstrate that Erm employs a 'tethered search' mechanism to navigate the dynamic surface of the precursor ribosome. While its C-terminal head domain anchors to a transient cleft to maintain a high local concentration, the catalytic domain retains the conformational freedom necessary to scan for the target methylation site, preventing kinetic trapping