Cloning, crystallization and preliminary X-ray studies of XC2981 from Xanthomonas campestris, a putative CutA1 protein involved in copper-ion homeostasis

Abstract

Divalent metal ions play key roles in all living organisms, serving as cofactors for many proteins involved in a variety of electron-transfer activities. However, copper ions are highly toxic when an excessive amount is accumulated in a cell. CutA1 is a protein found in all kingdoms of life that is believed to participate in copper-ion tolerance in Escherichia coli, although its specific function remains unknown. Several crystal structures of multimeric CutA1 with different rotation angles and degrees of interaction between trimer interfaces have been reported. Here, the cloning, expression, crystallization and preliminary X-ray analysis of XC2981, a possible CutA1 protein present in the plant pathogen Xanthomonas campestris, are reported. The XC2981 crystals diffracted to a resolution of 2.6 A. They are cubic and belong to space group I23, with unit-cell parameters a = b = c = 130.73 A.

Figure 1

SDS–PAGE monitoring of the overexpression and purification of XC2981. Lane 1, molecular-weight markers in kDa; lane 2, whole cell lysate before IPTG induction; lane 3, whole cell lysate after IPTG induction; lane 4, whole cell lysate after IPTG induction with optional extra SDS; lane 5, purified XC2981 after TEV cleavage without optional extra SDS. It is clear that bands corresponding to monomers (12 kDa), trimers (36 kDa), tetramers (48 kDa) and even hexamers (72 kDa) are present in this gel in the absence of optional extra SDS (lane 5), indicating that XC2981 forms a stable trimer and possibly a hexamer in solution.

Figure 2

Cube-shaped crystals of XC2981 from Xcc grown by the hanging-drop vapour-diffusion method. The crystallization condition used was 0.1 M CAPS buffer pH 10, 2.2 M (NH₄)₂SO₄, 0.2 M Li₂SO₄. The average dimensions of these crystals were all approximately 0.15 × 0.15 × 0.1 mm.

Figure 3

Diffraction pattern of XC2981 collected at NSRRC beamline 13B1 from a flash-frozen crystal in reservoir cryoprotectant. The exposure time was 15 s, with an oscillation range of 1° and a crystal-to-detector distance of 200 mm.