Crystallization and preliminary crystallographic study of a trypsin-resistant catalytic domain of human calcineurin.
Calcineurin, a Ca(2+)/calmodulin-dependent serine/threonine protein phosphatase, plays a key role in a number of cellular pathways, including T-cell activation, and is an important molecular target of the immunosuppressive drugs cyclosporin A and FK506. To understand the structural basis underlying the activation of calcineurin by calmodulin, X-ray crystallography was employed to solve the three-dimensional structure of the free calcineurin catalytic domain (residues 20-347 of the A subunit). To accomplish this, a bacterially expressed glutathione S-transferase (GST) fusion protein of the human calcineurin catalytic domain was first purified by GST-affinity chromatography. After limited digestion by trypsin, the catalytic domain (Cncat) was purified using anion-exchange and size-exclusion chromatography. Crystallization of Cncat was achieved by the hanging-drop vapour-diffusion method at pH 6.5 using PEG 6000 as precipitant. The diffraction results showed that the Cncat crystal belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 161.6, b = 87.4, c = 112.0 Å. There are four Cncat molecules in the asymmetric unit, with 49.5% solvent content. An X-ray diffraction data set was collected to 2.87 Å resolution and a clear molecular-replacement solution was obtained. The active site of Cncat is open to the solvent channels in the crystal packing.
Organ Transplant Center, Beijing 309 Hospital, 17A Hei-shan-hu Road, Haidian District, Beijing 100091, People's Republic of China.
Lei
L
Jin
Michael H A
MH
Roehrl
Li
L
Xiao
Xiuyun
X
He
Haibin
H
Li
Linhu
L
Ge
Bingyi
B
Shi
D019703:Calcineurin / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D003460:Crystallization; D018360:Crystallography, X-Ray; D006801:Humans; D008958:Models, Molecular; D020836:Protein Structure, Quaternary; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism
D019703:Calcineurin; D014357:Trypsin