Recoverin (Rv) is a small Ca2+-binding protein that prolongs the active state of rhodopsin by binding rhodopsin kinase, a Ser/Thr kinase responsible for rhodopsin phosphorylation in rod photoreceptor cells. The Rv structure is composed of four EF hand motifs, but only two are functional (EF2 and EF3). The active form of Rv is well known to require Ca2+ bound to both EF hands, but in over two decades of studying this protein, none of the published crystal structures have had Ca2+ ions bound to both EF hands. All of the structures have Ca2+ bound to EF3, with EF2 in an open, empty conformation. We suspected that this was due to an intermolecular crystal contact between a surface glutamate (E153) and T80 in EF2. This crystal contact, observed in most of the published structures, prevents the closure of EF2 loop and hence Ca2+ ion coordination in EF2. We constructed a E153A mutant of Rv and determined its structure by X-ray crystallography. This mutation has no effect on the ability of Rv to bind Ca2+ or rhodopsin kinase in solution, but the crystal structure of the E153A Rv mutant is significantly different from that of wild type with Ca2+ ions bound in both EF2 and EF3. In addition, we observe several reorientations of amino acids leading from EF2 to the N-terminal tail, which were not seen before in crystal structures and are not observed in a second structure of the E153A mutant in which a Na+ instead of Ca2+ is bound to EF2.

Crystal contact deletion enables recoverin to crystallize with a calcium ion bound in EF-hands 2 and 3

250th National Meeting (2015)

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