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Structural Insights into Allosteric Regulation of Anaerobic Ribonucleotide Reductase by Nucleotide Binding


Core Concepts
Binding of dATP to the ATP-cone domain of anaerobic ribonucleotide reductase induces increased flexibility of the glycyl radical domain and a flap over the active site, preventing substrate binding and radical mobilization, thereby inhibiting enzyme activity.
Abstract
The content provides structural, biochemical, and biophysical insights into the allosteric regulation of the anaerobic ribonucleotide reductase (RNR) from Prevotella copri (PcNrdD) by the binding of ATP and dATP to the ATP-cone domain. Key highlights: PcNrdD exists in a dimer-tetramer equilibrium, with ATP favoring the dimer and dATP favoring the tetramer. Binding of ATP to the ATP-cone activates the enzyme by promoting a fully ordered glycyl radical domain (GRD) and allowing substrate binding. Binding of dATP to the ATP-cone inhibits the enzyme by increasing the flexibility of the GRD and a flap over the active site, preventing substrate binding and radical mobilization. Cryo-EM structures reveal the structural basis for this allosteric regulation: In the ATP-bound form, the ATP-cones are flexible but the NxN flap and GRD are ordered, allowing substrate binding. In the dATP-bound form, the ATP-cones, NxN flap, and GRD are disordered, preventing substrate binding. In the dATP-bound tetramer, the ATP-cones mediate interactions between the dimers, further inhibiting enzyme activity. HDX-MS experiments validate the structural observations, showing increased dynamics in the ATP-cone, linker, and NxN flap regions upon dATP binding. The results suggest that dATP inhibition in anaerobic RNRs acts by increasing the flexibility of the flap and GRD, thereby preventing both substrate binding and radical mobilization, in contrast to the oligomerization-based inhibition mechanism observed in aerobic RNRs.
Stats
The enzyme activity of PcNrdD is stimulated by ATP with a KL of 0.67±0.12 mM and inhibited by dATP with a Ki of 74±24 μM. Binding of dATP to PcNrdD occurs with a KD of 6 μM, while binding of ATP is 4-fold weaker with a KD of 26 μM.
Quotes
"Binding of dATP to the ATP-cone results in loss of activity and increased dynamics of the GRD, such that it can not be detected in the cryo-EM structures." "The structures implicate a complex network of interactions in activity regulation that involve the GRD more than 30 Å away from the dATP molecules, the allosteric substrate specificity site and a conserved but previously unseen flap over the active site."

Deeper Inquiries

How do the structural changes induced by dATP binding in anaerobic RNRs compare to the oligomerization-based inhibition mechanisms observed in aerobic RNRs

In anaerobic RNRs, the structural changes induced by dATP binding differ from the oligomerization-based inhibition mechanisms observed in aerobic RNRs. While aerobic RNRs typically undergo oligomerization upon dATP binding, leading to the formation of higher-order complexes that disrupt radical transfer between subunits, anaerobic RNRs exhibit a different mechanism. In anaerobic RNRs, dATP binding induces increased flexibility of key structural elements, such as the flap and glycyl radical domain, preventing substrate binding and radical transfer. This modulation of flexibility inhibits the enzyme's activity by blocking substrate binding and radical mobilization, rather than through oligomerization as seen in aerobic RNRs.

What are the potential physiological implications of the different inhibition mechanisms between aerobic and anaerobic RNRs

The different inhibition mechanisms between aerobic and anaerobic RNRs have significant physiological implications. In aerobic RNRs, dATP-induced oligomerization disrupts radical transfer between subunits, leading to enzyme inactivation. This mechanism serves as a regulatory checkpoint to prevent unnecessary DNA synthesis when dATP levels are high. On the other hand, in anaerobic RNRs, dATP-induced flexibility changes in key structural elements prevent substrate binding and radical transfer, effectively shutting down enzyme activity. This mechanism allows anaerobic RNRs to regulate their activity in response to dATP levels without the need for oligomerization. Understanding these differences in inhibition mechanisms provides insights into the diverse strategies employed by different RNR classes to regulate DNA synthesis under varying cellular conditions.

How might the insights into allosteric regulation of anaerobic RNRs inform the development of targeted inhibitors for therapeutic applications

Insights into the allosteric regulation of anaerobic RNRs can inform the development of targeted inhibitors for therapeutic applications. By understanding the structural changes and dynamics induced by dATP binding in anaerobic RNRs, researchers can design specific inhibitors that target key regions involved in the inhibition mechanism. For example, targeting the flexible flap or glycyl radical domain that are affected by dATP binding could lead to the development of selective inhibitors that block substrate binding and radical transfer in anaerobic RNRs. These targeted inhibitors could potentially be used to modulate DNA synthesis in specific cell types or pathogens, offering new avenues for therapeutic intervention in diseases where RNR activity plays a crucial role.
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