Preparation and characterization of a single-chain calcineurin-calmodulin complex.
Calcineurin (CN), a Ca(2+)/calmodulin (CaM)-dependent serine/threonine protein phosphatase, is a heterodimer composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). The activity of CNA is under the control of two functionally distinct, but structurally similar Ca(2+)-regulated proteins, CaM and CNB. The crystal structure of the holoenzyme reveals that the N-terminus and C-terminus of CNB and the N-terminus of CNA each have a long arm not involved in the active site. We constructed a fusion of the genes of CaM, CNB and CNA in that order using linker primers containing six and ten codons of glycine. A single-chain CaM-CNB-CNA (CBA) complex was expressed and purified to near homogeneity. The single-chain complex was fully soluble, and had biochemical properties and kinetic parameters similar to single-chain CNB-CNA (BA) activated by CaM. It was not regulated by CaM and CNB, but was strongly stimulated by Mn2+, Ni2+ and Mg2+. Intrinsic fluorescence spectroscopy of the complex showed a change in the environment of tryptophan in the presence of Ca2+ and circular dichroism (CD) spectropolarimetry revealed an increase in alpha-helical content. Our findings suggest that fusion of CaM, CNB and CNA does not prevent the structural changes required for their functioning; in particular, CaM within the complex could still interact correctly with CN in the presence of Ca2+.
Qin
Yunlong
Y
Department of Biochemistry and Molecular Biology, Beijing Normal University, Beijing Key Laboratory, Beijing 100875, PR China.
Liu
Jing
J
Li
Xin
X
Wei
Qun
Q
D001665:Binding Sites; D019703:Calcineurin / Q000737:chemistry / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain / Q000502:physiology; D002413:Cations, Divalent; D002942:Circular Dichroism; D003001:Cloning, Molecular; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D009532:Nickel / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D002147:Calmodulin; D002413:Cations, Divalent; D010455:Peptides; D008345:Manganese; D009532:Nickel; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D008274:Magnesium; D002118:Calcium