Crystallization and preliminary crystallographic study of a trypsin-resistant catalytic domain of human calcineurin

Authors: Jin, Lei; Roehrl, Michael H. A.; Xiao, Li; He, Xiuyun; Li, Haibin; Ge, Linhu; Shi, Bingyi

Source: Acta Crystallographica Section F, Volume 68, Number 5, 1 May 2012 , pp. 574-579(6)

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Abstract:

Calcineurin, a Ca²⁺/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 P2₁2₁2, 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.

Document Type: Research article

DOI: http://dx.doi.org/10.1107/S1744309112007890

Publication date: 2012-05-01