From Interfaces to Networks: Energetic Control of Specificity in Bacterial Two-Component Systems

Bacterial two-component systems (TCSs) mediate environmental sensing and adaptive responses through signal transduction between histidine kinases (HKs) and response regulators (RRs), thereby regulating biochemical processes essential for survival and, in pathogenic species, infection. How signaling specificity and insulation are maintained in organisms encoding multiple paralogous two-component systems remains an open question. Here, we investigate specificity in the Actinobacillus pleuropneumoniae TCS signaling network using an integrated computational framework that combines coevolutionary analysis, structural modeling, molecular dynamics simulations, and free-energy calculations. We show that cognate HK-RR recognition is established locally through clusters of coevolving interface residues, termed the orthologue interface specificity core (OISC), which mediate symmetric molecular recognition at individual interaction interfaces. However, interface-level recognition alone is insufficient to explain signaling fidelity across the network. Instead, system-wide specificity and pathway insulation emerge in this network from asymmetric energetic discrimination among cognate and non-cognate interactions across the ensemble of paralogous interfaces. Graded free-energy profiles reveal that broadly compatible interfaces can coexist with robust signaling insulation, reconciling interface promiscuity with stable network organization. Together, these findings support a two-tiered model for the TCS network analyzed here, in which symmetric interface constraints enable cognate recognition, while asymmetric network-level energetics govern signaling specificity. This framework may extend to other paralogous TCS networks.