Probing site-specific calmodulin calcium and lanthanide affinity by grafting.
Ca2+ binding is essential for the biological functions of calmodulin (CaM) as a trigger/sensor protein to regulate many biological processes in the Ca2+ -signaling cascade. A challenge in understanding the mechanism of Ca2+ signaling is to obtain site-specific information about the Ca2+ binding properties of individual Ca2+ -binding sites of EF-hand proteins, especially for CaM. In this paper, we report the first estimation of the intrinsic Ca2+ affinities of the four EF-hand loops of calmoduin (I-IV) by individually grafting into the domain 1 of CD2. Taking advantage of the Trp residues in the host protein, we first determined metal-binding affinities for Tb3+, Ca2+, and La3+ for all four grafted EF-loops using Tb3+ aromatic resonance energy transfer. EF-loop I exhibits the strongest binding affinity for Ca2+, La3+, and Tb3+, while EF-loop IV has the weakest metal-binding affinity. EF-loops I-IV of CaM have dissociation constants for Ca2+ of 34, 245, 185, and 814 microM, respectively, with the order I > III approximately equal to II > IV. These findings support a charge-ligand-balanced model in which both the number of negatively charged ligand residues and the balanced electrostatic dentate-dentate repulsion by the adjacent charged residues are two major determinants for the relative Ca2+ -binding affinities of EF-loops in CaM. Our grafting method provides a new strategy to obtain site-specific Ca2+ binding properties and a better estimation of the cooperativity and conformational change contributions of coupled EF-hand proteins.
Ye
Yiming
Y
Department of Chemistry, Center for Drug Design and Advanced Biotechnology, Georgia State University, Atlanta, Georgia 30303, USA.
Lee
Hsiau-Wei
HW
Yang
Wei
W
Shealy
Sarah
S
Yang
Jenny J
JJ
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D018801:CD2 Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D007811:Lanthanum / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D013725:Terbium / Q000737:chemistry / Q000378:metabolism*
D018801:CD2 Antigens; D002147:Calmodulin; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D013725:Terbium; D007811:Lanthanum; D002118:Calcium