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

• Published in: Journal of the American Chemical Society 2005-02, Vol.127 (7), p.2085-2093
• Main Authors: Yang, Wei, Wilkins, Anna L, Ye, Yiming, Liu, Zhi-ren, Li, Shun-yi, Urbauer, Jeffrey L, Hellinga, Homme W, Kearney, Alice, van der Merwe, P. Anton, Yang, Jenny J
• Format: Article
• Language: English
• Subjects: Animals; Binding Sites; Biological and medical sciences; Calcium - chemistry; Calcium - metabolism; Calcium-Binding Proteins - chemistry; Calcium-Binding Proteins - genetics; Calcium-Binding Proteins - metabolism; CD2 Antigens - chemistry; CD2 Antigens - genetics; CD2 Antigens - metabolism; Cell Adhesion Molecules - chemistry; Cell Adhesion Molecules - genetics; Cell Adhesion Molecules - metabolism; Fundamental and applied biological sciences. Psychology; Interactions. Associations; Intermolecular phenomena; Models, Molecular; Molecular biophysics; Nuclear Magnetic Resonance, Biomolecular; Protein Engineering; Protein Structure, Tertiary; Rats; Spectrometry, Fluorescence; Surface Plasmon Resonance; Terbium - chemistry; Terbium - metabolism
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja0431307

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.