Greeshma, S. P. | Search bioRxiv

bioRxiv2026

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

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.

biophysics↗

bioRxiv2026

Evolutionary plasticity of regulatory--catalytic coupling in mono- and poly-ADP-ribosyl transferases

The evolution of modular proteins enables biological systems to diversify regulatory functions while preserving conserved catalytic activities. ADP-ribosyltransferases (ARTs), including mono-ADP-ribosyltransferases (MARylating enzymes) and poly-ADP-ribosyltransferases (PARylating enzymes), provide an ideal model for investigating how regulatory and catalytic domains co-evolve. Although ART catalytic domains are highly conserved, their regulatory architectures vary extensively, raising the question of whether regulatory domains determine catalytic activation, allosteric communication, and enzymatic output. Here, we systematically examined regulatory--catalytic interoperability by reconstituting evolutionarily distant ARTs from mammals, plants, and bacteria. We show that the regulatory domain of the MARylating enzyme human PARP3 (hPARP3) functionally complements the catalytic domains from the PARylating enzymes human PARP1 (hPARP1), human PARP2 (hPARP2), and Arabidopsis PARP2 (atPARP2), restoring robust DNA-break-dependent allosteric coupling for catalytic activation in response to both phosphorylated double-strand and single-strand DNA breaks. Complementation stimulates NAD+ consumption, promotes cis- and trans-poly(ADP-ribosyl)ation, and is accompanied by direct physical interactions between heterologous regulatory and catalytic domains. Unexpectedly, the strongest activation was observed between the hPARP3 regulatory domain and the atPARP2 catalytic domain, demonstrating conservation of productive allosteric communication across both enzymatic classes and species boundaries. In contrast, regulatory domains from PARylating ARTs failed to activate the catalytic domain of MARylating hPARP3, revealing a striking asymmetry in regulatory--catalytic compatibility. These findings identify conserved and specialised modes of allosteric communication within the ART superfamily and establish the concepts of general and specific combining ability as a framework for understanding modular protein evolution. More broadly, our results demonstrate that conserved regulatory--catalytic signalling networks can be functionally transferred between divergent enzymes, providing a foundation for mapping allosteric communication across ARTs and for engineering synthetic ADP-ribosylation systems with programmable regulatory inputs and catalytic outputs.

biochemistry↗