Berbamine specifically binds to the ATP-binding pocket of CaMKII γ and inhibits its kinase activity. (A) BBM physically interacts with CaMKII γ proteins. Two proteins of ∼ 62 kDa and 54 kDa were captured specifically by BBM as shown by silver staining (lane 1). These 2 bands were identified as CaMKIIγ isoform 1 (62 kDa) and isoform 3 (54 kDa), respectively, by mass spectrometry (MS) and confirmed by Western blot with total and phospho-CaMKIIγ antibodies (lanes 3-4). (B) BBM directly binds to CaMKII γ protein. Purified GST-CaMKIIγ protein was incubated with biotinylated BBM and the complex was isolated with streptavidin agarose. Western blot with anti-GST antibody revealed that BBM specifically pulled down CaMKIIγ proteins (lane 1), but not GST protein (lane 2). GST protein was used as negative control. (C) Confocal images of BBM colocalization with CaMKII γ in 293T cells. Green: EGFP-tagged CaMKIIγ protein. Red: biotin-labled BBM visualized by Rhodamine-labeled streptavidin. Yellow: BBM was predominantly colocalized with CaMKIIγ protein in 293T cells. (D) Berbamine selectively binds to ATP binding pocket of CaMKIIγ. (E) BBM molecule is located within the ATP binding pocket of CaMKIIγ protein. The model of the CaMKIIγ in complex with calmodulin was built using Modeller based on the x-ray crystal of CaMKIIδ/calmodulin complex. (F) Best docking poses of ATP (blue), XBA24 (red), and berbamine (green) at the ATP-binding pocket of CaMKIIγ. The ligand is clamped by 4 surface residues in CaMKIIγ docking groove: K43 (purple), V74 (black), F90 (yellow), and D157 (brown).