Rational design of a novel calcium‐binding site adjacent to the ligand‐binding site on CD2 increases its CD48 affinity

Abstract

Electrostatic interactions are important for molecular recognition processes including Ca 2+ ‐binding and cell adhesion. To understand these processes, we have successfully introduced a novel Ca 2+ ‐binding site into the non‐Ca 2+ ‐dependent cell adhesion protein CD2 using our criteria that are specifically tailored to the structural and functional properties of the protein environment and charged adhesion surface. This designed site with ligand residues exclusively from the β‐sheets selectively binds to Ca 2+ and Ln 3+ over other mono‐ and divalent cations. While Ca 2+ and Ln 3+ binding specifically alters the local environment of the designed Ca 2+ ‐binding site, the designed protein undergoes a significantly smaller conformation change compared with those observed in naturally occurring Ca 2+ ‐binding sites that are composed of at least part of the flexible loop and helical regions. In addition, the CD2–CD48‐binding affinity increased approximately threefold after protein engineering, suggesting that the cell adhesion of CD2 can be modulated by altering the local electrostatic environment. The study provides site‐specific information for regulating cell adhesion within CD2 and gives insight into the structural factors required for Ca 2+ ‐modulated biological processes.