Poster for the 13th international ATPase conference, Pacific Grove, CA, USA
Darier disease is a skin disorder resulting from mutation of SERCA2b. One of the Darier disease mutations is E917K in the cytoplasmic loop between M8 and M9. Here we have analyzed the functional impact of SERCA2b E917K as well as a series of mutations of the corresponding residue E918 of SERCA1a. SERCA2b E917K as well as SERCA1a E918K reduces the apparent Ca2+ affinity 2- to 3-fold, compared with the respective wild type. The resulting disturbance of Ca2+ homeostasis is probably the reason for the abnormalities of epidermal cells in the Darier disease patients. The reduction of apparent Ca2+ affinity seems to result from a role of the glutamate in the E2 to E1 transition. Hence, the lysine substitution of E917/E918 leads to accumulation of the E2 form during ATP hydrolysis as evaluated by determination of apparent vanadate affinity, and rapid kinetic measurements demonstrated a reduced rate of the E2 to E1 conformational transition. A comparison of the crystal structures of SERCA1a in E1 and E2 conformations indicate that E918 bonds to R819 of the L6-7 loop in E1 (2-3 Å distance), but not in E2 (9-10 Å distance), suggesting that the E918K mutation destabilizes the E1 conformation by mutual repulsion between the two positive side chains. The structural analysis therefore predicts a shift of the conformational equilibrium toward E2, in line with our observations. To further test the role of R819, this residue was also studied by substitution, and R819E gave the same effect on vanadate sensitivity as E918K, thus supporting the proposal that mutual repulsion between the side chains (now negative) in E1 causes accumulation of E2. Moreover the swop mutation E918R/R819E led to gain of function with respect to vanadate affinity. The Na+,K+-ATPase residue at the position corresponding to E917/E918 of SERCA is an arginine, which interacts with the C-terminus. The effects of adding the Na+,K+-ATPase C-terminus to wild-type SERCA1a and E918R will also be presented.