Purification and crystallization of the human EF-hand tumour suppressor protein S100A2

Abstract

S100A2 is a Ca(2+)-binding EF-hand protein that is mainly localized in the nucleus. There, it acts as a tumour suppressor by binding and activating p53. Wild-type S100A2 and a S100A2 variant lacking cysteines have been purified. CD spectroscopy showed that there are no changes in secondary-structure composition. The S100A2 mutant was crystallized in a calcium-free form. The crystals, with dimensions 30 x 30 x 70 microm, diffract to 1.7 A and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 43.5, b = 57.8, c = 59.8 A, alpha = beta = gamma = 90 degrees. Preliminary analysis of the X-ray data indicates that there are two subunits per asymmetric unit.

Figure 1

Secondary-structure analysis of S100A2wt and S100A2ΔCys by CD spectroscopy. (a) CD spectrum of S100A2wt. (b) CD spectrum of S100A2ΔCys. The spectra were recorded in 20 mM Tris–HCl, 5 mM MgCl₂ pH 7.6. Analysis of the spectra using CDNN revealed 51% α-helix, 9% β-sheet, 16% β-turn and 25% random coil for both proteins.

Figure 2

Oxidation kinetics of cysteines in S100A2wt.

Figure 3

Crystals of recombinant human S100A2ΔCys. Crystal dimensions are 0.3 × 0.075 × 0.05 mm.

Figure 4

Typical diffraction pattern of a crystal of S100A2ΔCys.