Rational design of a conformation-switchable Ca2+- and Tb3+-binding protein without the use of multiple coupled metal-binding sites.
Ca2+, as a messenger of signal transduction, regulates numerous target molecules via Ca2+-induced conformational changes. Investigation into the determinants for Ca2+-induced conformational change is often impeded by cooperativity between multiple metal-binding sites or protein oligomerization in naturally occurring proteins. To dissect the relative contributions of key determinants for Ca2+-dependent conformational changes, we report the design of a single-site Ca2+-binding protein (CD2.trigger) created by altering charged residues at an electrostatically sensitive location on the surface of the host protein rat Cluster of Differentiation 2 (CD2).CD2.trigger binds to Tb3+ and Ca2+ with dissociation constants of 0.3 +/- 0.1 and 90 +/- 25 microM, respectively. This protein is largely unfolded in the absence of metal ions at physiological pH, but Tb3+ or Ca2+ binding results in folding of the native-like conformation. Neutralization of the charged coordination residues, either by mutation or protonation, similarly induces folding of the protein. The control of a major conformational change by a single Ca2+ ion, achieved on a protein designed without reliance on sequence similarity to known Ca2+-dependent proteins and coupled metal-binding sites, represents an important step in the design of trigger proteins.
Li
Shunyi
S
Department of Chemistry, Center of Drug Design and Biotechnology, Georgia State University, Atlanta, GA, USA.
Yang
Wei
W
Maniccia
Anna W
AW
Barrow
Doyle
D
Tjong
Harianto
H
Zhou
Huan-Xiang
HX
Yang
Jenny J
JJ
D019943:Amino Acid Substitution; D000818:Animals; D018801:CD2 Antigens / Q000737:chemistry* / Q000235:genetics; D002118:Calcium / Q000737:chemistry* / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation / Q000187:drug effects; D015202:Protein Engineering; D051381:Rats; D055672:Static Electricity; D013725:Terbium / Q000737:chemistry*
D018801:CD2 Antigens; D002135:Calcium-Binding Proteins; D013725:Terbium; D002118:Calcium
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