International Graduate School of Arts and Sciences, Yokohama City Univercity^* Department of Biological Science, Graduate School of Science, Osaka University, Japan^**
○Youichi Naoe^* Kyouhei Arita^* Hiroshi Hashimoto^* Hiroshi Kanazawa^** Mamoru Sato^* Toshiyuki Shimizu^*

CHP1 (Calcineurin B homologous protein 1), is a calcium binding EF-hand protein and shows substantial sequence similarity (~39% identity) with the CNB (Calcineurin B subunit). CHP1 was the essential cofactor of NHEs (Na⁺/H⁺ exchangers), which catalyze the electroneutral influx of extracellular Na⁺ and efflux of intracellular H⁺. CHP1 also binds to DRAK2 (DAP kinase related apoptosis inducing protein kinase 2) and significantly reduced its kinase activity calcium dependently. Interestingly CHP1 biding region of NHE1 and DRAK2 shows little sequence similarity. We have determined crystal structure of calcium-bound CHP1 from rat at 2.2 Å resolution. The overall folding topology of the protein is similar to that of the regulatory B subunit of calcineurin. CHP1 contains a hydrophobic pocket on the opposite side of the protein to the EF-hands. The hydrophobic pocket has been implicated in multiple ligand binding.
DRAK2 is a Ser/Thr kinase which belongs to DAP (Death associated protein) kinase family. Today, DAP kinase family has five members which can induce apoptosis depend on its kinase activity. Recently, DRAK2 is thought to regulate the TCR (T Cell responses) activation threshold during thymocyte selection. But its target molecules and physiological role remains unknown. To clarify the function and the regulation mechanism of DRAK2 by CHP1, we tried to determinate three dimensional structure of DRAK2. We express, purify and crystallize the kinase domain of DRAK2.
We also performed CHP1 and DRAK2 binding experiment by taking size exclusion chromatography and ultracentrifugation. We confirmed that CHP1 strongly bind to DRAK2.