Structural modeling of perforin missense mutations in the C2 domain. (A) Homology model of the perforin C2 domain based on the C2 domain of 1DSY (protein kinase Cα) bound to Ca and phosphatidylserine. Res spheres represent Ca ions; phosphatidylserine are sticks colored according to atom type, and side chains of T435 and Y438 are orange sticks. The box highlights the zoomed view shown in panel B. (B) Magnified view of Ca-binding region. An overlay is shown of perforin C2 models based on 1DSY (green, as in panel A) and 2CHD, a Ca-free C2 domain from rabphilin-3A (cyan). The Ca-binding BC loop is highlighted to illustrate the structural differences. Note that T435 adopts opposite orientations in the Ca-bound versus Ca-free models. (C) Side view overlay of 4 representative Ca-bound type I perforin C2 domain models built by the PS2 server, based on 1A25, 1DQV, 1DSY, and 2CM6. Note the similar positions of T435 and Y438 side chains in all models. (D) Side view of the perforin C2 model based on the Ca-free 2CHD structure, with the same orientation as panel C. Note that the BC loop in the Ca-free form would clash with the Ca ions (transparent red spheres). (E,F) Top views of perforin C2 models based on Ca-bound 1DSY (E) or Ca-free 2CHD (F). In both panels, the C2 domain has been sliced identically by an approximately horizontal plane just above the Ca ions in the 1DSY model. This view reveals that, in the Ca-bound model, the T435 side chain is pointed inward and surrounded by atoms from other residues. In contrast, T435 is directed outward and is solvent-accessible in the Ca-free form. Thus, the T435M mutation would be tolerated only in the Ca-free conformation of the C2 domain.