Design of a calcium-binding protein with desired structure in a cell adhesion molecule.
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+ (Kd 5.0 microM) and Tb3+ (Kd 6.6 microM) bind to the designed protein somewhat more tightly than does Ca2+ (Kd 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.
Yang
Wei
W
Department of Chemistry, Center for Drug Design and Biotechnology, Georgia State University, Atlanta, Georgia 30303, USA.
Wilkins
Anna L
AL
Ye
Yiming
Y
Liu
Zhi-ren
ZR
Li
Shun-yi
SY
Urbauer
Jeffrey L
JL
Hellinga
Homme W
HW
Kearney
Alice
A
van der Merwe
P Anton
PA
Yang
Jenny J
JJ
D000818:Animals; D001665:Binding Sites; D018801:CD2 Antigens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D051381:Rats; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance; D013725:Terbium / Q000737:chemistry / Q000378:metabolism
D018801:CD2 Antigens; D002135:Calcium-Binding Proteins; D015815:Cell Adhesion Molecules; D013725:Terbium; D002118:Calcium