Flexibility-Induced Collective Behavior Drives Symmetry Breaking in Discrimination of Undesired Ions

Structure flexibility is essential for the biological function of proteins. At the same time, many proteins need to discriminate ligands with subtle differences, with one example being ion selectivity. Investigating the mechanisms by which flexible proteins achieve such precise discrimination is crucial for advancing our understanding of their functions. In this work, we study transporter KCC4, which undergoes continuous conformation changes during ion transport and can realize K⁺ over Na⁺ selectivity. Our findings reveal that the center of the binding site no longer represents a stable equilibrium for the undesired Na⁺, and its binding mode exhibits bifurcation. Interestingly, protein conformation fluctuation can induce collective behavior throughout the entire binding region, which contributes to this bifurcation. Thus, the symmetry of the binding mode decreases from the inherent T _d symmetry to a C_2v symmetry, and the binding stability of Na⁺ is largely reduced. A similar phenomenon is observed in a GPCR, β₂-AR, where a less favored ligand forms a biased binding mode with reduced stability. The mechanism underlying the selectivity in such flexible regions could be interpreted as spontaneous symmetry breaking, which may represent a general mechanism by which flexible proteins achieve efficient ligand discrimination.