A long-standing question in allostery is how alternative ligands binding to the same allosteric pocket can drive opposite regulatory outcomes. The Escherichia coli lactose repressor protein (LacI) provides a striking example: IPTG acts as an inducer, reducing DNA-binding affinity by >1,000-fold, whereas ONPF functions as an anti-inducer, increasing affinity by ∼10-fold, despite both ligands occupying the same site. Although experimental studies have revealed distinct signatures of IPTG- and ONPF-bound LacI, the molecular basis of these divergent effects has remained unresolved, in part because critical DNA-binding domains and linkers undergo dynamics too fast to capture with traditional structural methods and because potential asymmetric responses in this homodimer remain underexplored. Here, we investigate how alternative ligands reprogram LacI’s dynamic flexibilities to produce opposite regulatory outcomes. Hypotheses generated from molecular dynamics simulations and network-based analyses were confirmed with high-throughput mutational assays of LacI variants in the presence of IPTG and ONPF. By comparing Dynamic Flexibility Index (DFI) profiles and collective modes of motion across Apo, IPTG-, and ONPF-bound states, we show that ligand binding reshapes fluctuation patterns and redistributes intrinsic asymmetry between monomers. To further capture long-distance effects, we applied the Dynamic Coupling Index (DCI), which quantifies how perturbations at one site alter flexibility at distant positions. Because this coupling is often directional, we used DCI asymmetry (DCI asym ) as a sensitive measure of influence between residues. Distinct DCI and DCI asym profiles were observed for IPTG- and ONPF-bound states relative to Apo LacI, highlighting ligand-specific rewiring of communication pathways. Consistently, deep mutational scanning revealed that the same substitutions can yield neutral outcomes with one ligand but disruptive effects with the other. Together, these results provide a mechanistic framework for how LacI encodes ligand-specific allosteric signals through dynamic flexibility, directional coupling, and intrinsic asymmetry.
Ausbeck et al. (Sun,) studied this question.