FIGURE 10.

Molecular modeling and characterization of the interaction between calumenin-B and SERCA2-L4. A, the multiple sequence alignment of SERCA isoforms using the ClustalX program (53). SERCA2-L4 region is shown in blue. Four hydrophobic residues of SERCA2-L4 involved in the interaction with calumenin-B are indicated by arrows. The dashed line indicates the disulfide bond (Cys⁸⁷⁵–Cys⁸⁸⁷). B, Coomassie Blue staining of the purified GST control, GST-SERCA2-L4, GST-SERCA2-L4-F866A, GST-SERCA2-L4-Y867A, GST-SERCA2-L4-L869A, and GST-SERCA2-L4-L873A fusion proteins. C, pulldown assay was performed using equivalent amounts of GST control protein and different GST-SERCA2-L4 fusion peptides bound to Sepharose 4B by incubating with cardiac homogenates. The pulldown samples were separated by SDS-PAGE and immunoblotted with anti-calumenin antibody. D, molecular modeling of SERCA2 structure. The model for SERCA2 structure was built using the structure of SERCA1 in E1 state (pdb id 1SU4). The luminal region of SERCA2 model structure contains five loops (L1–L5). L4 is colored blue. L1, L2, L3, and L5 are colored orange. More open conformational state of SERCA2-L4 may be required upon calumenin binding (arrow). E, molecular surface representation of the SERCA2-L4 binding site in calumenin-B. The residues (Phe⁸⁶⁶, Tyr⁸⁶⁷, Leu⁸⁶⁹, and Leu⁸⁷³) of SERCA2-L4 involved in the hydrophobic interaction with calumenin-B are shown as ball-and-stick models. The surface of calumenin-B binding to SERCA2-L4 is shown in yellow.