The crystal structure of the photoprotein aequorin at 2.3 Å resolution

Abstract

Aequorin is a calcium-sensitive photoprotein originally obtained from the jellyfish Aequorea aequorea¹. Because it has a high sensitivity to calcium ions and is biologically harmless, aequorin is widely used as a probe to monitor intracellular levels of free calcium. The aequorin molecule contains four helix–loop–helix ‘EF-hand’ domains, of which three can bind calcium². The molecule also contains coelenterazine as its chromophoric ligand³. When calcium is added, the protein complex decomposes into apoaequorin, coelenteramide and CO₂, accompanied by the emission of light⁴. Apoaequorin can be regenerated into active aequorin in the absence of calcium by incubation with coelenterazine, oxygen and a thiol agent⁵. Cloning and expression of the complementary DNA for aequorin were first reported in 1985 (refs 2, 6), and growth of crystals of the recombinant protein has been described⁷; however, techniques have only recently been developed to prepare recombinant aequorin of the highest purity⁸, permitting a full crystallographic study. Here we report the structure of recombinant aequorin determined by X-ray crystallography. Aequorin is found to be a globular molecule containing a hydrophobic core cavity that accommodates the ligand coelenterazine-2-hydroperoxide. The structure shows protein components stabilizing the peroxide and suggests a mechanism by which calcium activation may occur.