The crystal structure of a novel calcium binding protein AtCBL2 from Arabidopsis thaliana

Masamichi Nagae,^a Akira Nozawa,^b Nozomu Koizumi,^b Hiroshi Sano,^b Mamoru Sato,^a and Toshiyuki Shimizu^b

^aGraduate School of Integrated Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokoham, Kanagawa, Japan; ^bLaboratory of Plant Molecular Breeding, Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Ikoma, Nara, Japan (nagae@tsurumi.yokohama-cu.ac.jp).

AtCBL2 is a member of a new family of calcineurin B-like calcium binding proteins that has recently been identified in Arabidopsis thaliana. These proteins have been shown to interact with novel family of serine-threonine protein kinases (AtCIPKs) in a calcium dependent manner. The signal pathway of AtCBL/AtCIPK plays critical roles in stress response such as salt, drought, light. Despite the great importance of intracellular signaling pathways in plants, no structural information has been obtained for AtCBL/AtCIPK system. Here, we report the crystal structure of AtCBL2 and propose a unique target recognition mecanizm of AtCBL/AtCIPK. Crystal of calcium bound AtCBL2 was obtained in space group C222₁ with unit-cell parameters a=83.9 Å, b=118.1 Å, c=49.1 Å. Using a synchrotron-radiation source, the crystal diffracts to 2.1 Å, with an overall R_merge of 6.0 % and a completeness of 98.8 %. One independent molecule was estimated to be present in the asymmetric unit, with a solvent content of 48 %. Phase to 2.5 Å was determined by conventional multiple isomorphous replacement with anomalous scattering method (MIRAS). The final model refined to an R value of 20.4 % and free R value of 24.8 % at 2.1 Å resolution. The three-dimensional structure of AtCBL2 reveals a compact α-helical structure with two pairs of EF-hands and the overall fold resembles that of calcineurin B and neuronal calcium sensor 1, but significant structural differences are observed. The first and fourth EF-hands differ from the canonical sequence, but calcium ions are coordinated in the EF-hand. The rest of the EF-hands are maintained in the open form by internal hydrogen bonds despite lacking calcium ions. A possible site for the target binding based on the three-dimensional structure is discussed.