The origins of binding specificity of a lanthanide ion binding peptide

Lanthanide ions (Ln³⁺) show similar physicochemical properties in aqueous solutions, wherein they exist as + 3 cations and exhibit ionic radii differences of less than 0.26 Å. A flexible linear peptide lanthanide binding tag (LBT), which recognizes a series of 15 Ln³⁺, shows an interesting characteristic in binding specificity, i.e., binding affinity biphasically changes with an increase in the atomic number, and shows a greater than 60-fold affinity difference between the highest and lowest values. Herein, by combining experimental and computational investigations, we gain deep insight into the reaction mechanism underlying the specificity of LBT3, an LBT mutant, toward Ln³⁺. Our results clearly show that LBT3-Ln³⁺ binding can be divided into three, and the large affinity difference is based on the ability of Ln³⁺ in a complex to be directly coordinated with a water molecule. When the LBT3 recognizes a Ln³⁺ with a larger ionic radius (La³⁺ to  Sm³⁺), a water molecule can interact with Ln³⁺ directly. This extra water molecule infiltrates the complex and induces dissociation of the Asn5 sidechain (one of the coordinates) from Ln³⁺, resulting in a destabilizing complex and low affinity. Conversely, with recognition of smaller Ln³⁺ (Sm³⁺ to Yb³⁺), the LBT3 completely surrounds the ions and constructs a stable high affinity complex. Moreover, when the LBT3 recognizes the smallest Ln³⁺, namely Lu³⁺, although it completely surrounds Lu³⁺, an entropically unfavorable phenomenon specifically occurs, resulting in lower affinity than that of Yb³⁺. Our findings will be useful for the design of molecules that enable the distinction of sub-angstrom size differences.