Ion-selective conformational stabilization of a disordered repeats-in-toxin protein domain.

Ion-binding intrinsically disordered proteins (IDPs) recruit and bind to specific metal ions to perform critical biological functions. In proteins where ion binding and structural transitions are coupled, interactions with off-target toxic metals can dramatically disrupt protein structure and function, exemplified by lead and mercury poisoning. Understanding the complex mechanisms underlying how IDPs exclude or allow binding to different ionic species is crucial for addressing the origins of metal toxicity in biological systems. Here, we elucidate mechanisms of ion selectivity in an IDP that adopts a structure upon Ca2+ binding. We probed ion-induced conformational changes of a repeats-in-toxin (RTX) protein domain in the presence of different ion ligands-Mg2+, Ca2+, Sr2+, and Ba2+-with chemical similarities but drastically different ionic radii. RTX adopts ion-selective conformations measured by x-ray crystallography, small-angle x-ray scattering, and circular dichroism. High-resolution x-ray structures reveal that Sr2+ induces a nearly identical RTX structure as natively binding Ca2+, enabled by the intrinsic flexibility and disorder of the protein. Small-angle x-ray scattering and circular dichroism indicate that smaller Mg2+ does not induce a significant conformational change in RTX, whereas larger Ba2+ induces a partially folded structure. These results highlight the importance of geometric constraints imposed by protein structure in determining metal ion selectivity, yielding insights into how off-target ion binding may result in protein misfolding and malfunction.