Design of a Calcium-Binding Protein with Desired Structure in a Cell Adhesion Molecule

Abstract

Ca²⁺, “a signal of life and death”, controls numerous cellular processes through interactions with proteins. An effective approach to understanding the role of Ca²⁺ is the design of a Ca²⁺-binding protein with predicted structural and functional properties. To design de novo Ca²⁺-binding sites in proteins is challenging due to the high coordination numbers and the incorporation of charged ligand residues, in addition to Ca²⁺-induced conformational change. Here, we demonstrate the successful design of a Ca²⁺-binding site in the non-Ca²⁺-binding cell adhesion protein CD2. This designed protein, Ca·CD2, exhibits selectivity for Ca²⁺ versus other di- and monovalent cations. In addition, La³⁺ (K_d 5.0 μM) and Tb³⁺ (K_d 6.6 μM) bind to the designed protein somewhat more tightly than does Ca²⁺ (K_d 1.4 mM). More interestingly, Ca·CD2 retains the native ability to associate with the natural target molecule. The solution structure reveals that Ca·CD2 binds Ca²⁺ at the intended site with the designed arrangement, which validates our general strategy for designing de novo Ca²⁺-binding proteins. The structural information also provides a close view of structural determinants that are necessary for a functional protein to accommodate the metal-binding site. This first success in designing Ca²⁺-binding proteins with desired structural and functional properties opens a new avenue in unveiling key determinants to Ca²⁺ binding, the mechanism of Ca²⁺ signaling, and Ca²⁺-dependent cell adhesion, while avoiding the complexities of the global conformational changes and cooperativity in natural Ca²⁺-binding proteins. It also represents a major achievement toward designing functional proteins controlled by Ca²⁺ binding.