The macromolecular complex of ICP and falcipain-2 from Plasmodium: preparation, crystallization and preliminary X-ray diffraction analysis

Abstract

The malaria parasite Plasmodium depends on the tight control of cysteine-protease activity throughout its life cycle. Recently, the characterization of a new class of potent inhibitors of cysteine proteases (ICPs) secreted by Plasmodium has been reported. Here, the recombinant production, purification and crystallization of the inhibitory C-terminal domain of ICP from P. berghei in complex with the P. falciparum haemoglobinase falcipain-2 is described. The 1:1 complex was crystallized in space group P4(3), with unit-cell parameters a = b = 71.15, c = 120.09 Å. A complete diffraction data set was collected to a resolution of 2.6 Å.

Figure 1

Isolation of the PbICP-C–FP-2 complex by SEC. (a) SEC elution profiles of PbICP-C (red), FP-2 (blue) and a sample after incubation of both proteins (green). The samples of purified PbICP-C or FP-2 eluted at the volumes expected for monomeric species (PbICP-C, 11.75 ml, 23.6 kDa; FP-2, 10.77 ml, 36.9 kDa). The elution volume of the pre-incubated sample containing both proteins corresponds to the formation of a 1:1 complex between FP-2 and PbICP-C (10.13 ml, 49.3 kDa). (b) SDS–PAGE analysis of purified PbICP-C (lane 1), FP-2 (lane 2) and fractions after SEC confirming the formation of the PbICP-C–FP-2 complex (indicated by green bars above the gel).

Figure 2

Crystals of PbICP-C–FP-2. (a)–(c) Typical crystals with poor diffraction properties grown without CdCl₂. (d) Well diffracting rod-like crystals grown in the presence of CdCl₂ [200 mM sodium acetate, 27.5 mM CdCl₂ and 100 mM MES (pH 5.0)]. Crystals grew within 2–4 weeks to final dimensions of 0.5 × 0.05 × 0.05 mm.

Figure 3

X-ray diffraction pattern at a resolution of 2.6 Å collected with 0.5° oscillation per image on beamline BL14.1 at BESSY (Berlin, Germany); circles indicate resolution shells.