Beyond photosynthesis, chloroplasts function as stress sensors to activate acclimation responses via chloroplast-to-nucleus signalling. One such signalling pathway involves the SAL1 phosphatase enzyme and its catabolic substrate, 3'-phosphoadenosine 5'-phosphate (PAP). As the SAL1-PAP pathway is evolutionarily conserved across land plants, modulation of SAL1 activity could be a strategy for enhancing crop resilience. However, genetic ablation of SAL1 causes complex pleiotropic effects. Thus, there is a need for detailed insights into the molecular mechanisms of SAL1 activity, and for tools allowing refined control of SAL1 activity.
Our in-silico simulations of SAL1 revealed crucial amino acids required for recognition of its substrate PAP, as well as protein conformation dynamics upon PAP binding to the active site. Significantly, we discovered a previously unknown inactivated state of the SAL1 enzyme that occurs through PAP stably binding to a region adjacent to the catalytic site.
We then performed a high-throughput in vitro screen of 13,000 small molecules against SAL1 to identify novel inhibitors. By simulating binding of these inhibitory ligands to SAL1, we identified novel regulatory regions, distinct from the active site, that are essential for the catalysis of PAP. This data was validated via protein mutagenesis and chemical modification of the lead inhibitors. Significantly, some of the identified inhibitors were also potent in vivo and enhanced stress tolerance of plants.
Taken together, we propose that the identified mechanisms provide potential targets for engineering the SAL1 enzyme. More broadly, our strategy can be applied to other plant proteins for engineering of other traits of interest.