Rational design of a calcium-binding protein.
Calcium ions play key roles as structural components in biomineralization and as a second messenger in signaling pathways. We have introduced a de novo designed calcium-binding site into the framework of a non-calcium-binding protein, domain 1 of CD2. The resulting protein selectively binds calcium over magnesium with calcium-binding affinity comparable to that of natural extracellular calcium-binding proteins (K(d) of 50 microM). This experiment is the first successful metalloprotein design that has a high coordination number (seven) metal-binding site constructed into a beta-sheet protein. Our results demonstrate the feasibility of designing a single calcium-binding site into a host protein, taking into account only local properties of a calcium-binding site obtained by a survey of natural calcium-binding proteins and chelators. The resulting site exhibits strong metal selectivity, suggesting that it should now be feasible to understand and manipulate signaling processes by designing novel calcium-modulated proteins with specifically desired functions and to affect their stability.
Yang
Wei
W
Department of Chemistry, Center of Drug Design, Georgia State University, Atlanta 30303, USA.
Jones
Lisa M
LM
Isley
Leanne
L
Ye
Yiming
Y
Lee
Hsiau-Wei
HW
Wilkins
Anna
A
Liu
Zhi-ren
ZR
Hellinga
Homme W
HW
Malchow
Russell
R
Ghazi
Mohammed
M
Yang
Jenny J
JJ
D001665:Binding Sites; D018801:CD2 Antigens / Q000737:chemistry* / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D015195:Drug Design; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008670:Metals / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary; D013725:Terbium / Q000737:chemistry
D018801:CD2 Antigens; D002135:Calcium-Binding Proteins; D008670:Metals; D013725:Terbium