Direct engagement of mycobacterial RadA by c-di-AMP at a non-catalytic site suppresses its DNA-stimulated ATPase activity

Abstract

Cyclic di-adenosine monophosphate couples DNA damage sensing to genome maintenance in mycobacteria, but how it regulates homologous recombination machinery beyond RecA remains unclear. Using Mycobacterium smegmatis RadA (MsRadA) as a model, we show by isothermal titration calorimetry that c-di-AMP binds to MsRadA, providing biophysical evidence for an interaction previously inferred only from genetic studies. C-di-AMP binding is independent of magnesium ions and is not affected by prior occupancy of the ATP-binding site by the ATP analogue {beta},{gamma}-methylene adenosine 5'-triphosphate, indicating that c-di-AMP binds at a site distinct from the canonical ATPase pocket. Functionally, c-di-AMP reduces MsRadA DNA-dependent ATPase activity. Structural modelling and molecular dynamics (MD) simulations show that c-di-AMP is stably bound in the central channel of the RadA hexamer, non-covalently engaged with the DNA-binding residues Arg197, Lys226, and Arg234, which are contributed by two adjacent subunit interfaces of the hexamer. In the modelled RadA-ATP-c-di-AMP-DNA quaternary complex, c-di-AMP was predicted at a peripheral site near, but distinct from, the ATP-binding pocket. Together, these findings identify RadA as a direct physical target of c-di-AMP within the homologous recombination pathway and provide a framework for testing how c-di-AMP regulates RadA during DNA damage.