Crystallographic analysis of the Arabidopsis thaliana BAG5-calmodulin protein complex.

Abstract

Arabidopsis thaliana BAG5 (AtBAG5) belongs to the plant BAG (Bcl-2-associated athanogene) family that performs diverse functions ranging from growth and development to abiotic stress and senescence. BAG family members can act as nucleotide-exchange factors for heat-shock protein 70 (Hsp70) through binding of their evolutionarily conserved BAG domains to the Hsp70 ATPase domain, and thus may be involved in the regulation of chaperone-mediated protein folding in plants. AtBAG5 is distinguished from other family members by the presence of a unique IQ motif adjacent to the BAG domain; this motif is specific for calmodulin (CaM) binding, indicating a potential role in the plant calcium signalling pathway. To provide a better understanding of the IQ motif-mediated interaction between AtBAG5 and CaM, the two proteins were expressed and purified separately and then co-crystallized together. Diffraction-quality crystals of the complex were grown using the sitting-drop vapour-diffusion technique from a condition consisting of 0.1 M Tris-HCl pH 8.5, 2.5 M ammonium sulfate. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 64.56, b = 74.89, c = 117.09 Å. X-ray diffraction data were recorded to a resolution of 2.5 Å from a single crystal using synchrotron radiation. Assuming the presence of two molecules in the asymmetric unit, a Matthews coefficient of 2.44 Å(3) Da(-1) was calculated, corresponding to a solvent content of approximately 50%.