Mapping of phosphatidylserine recognition region on CD36 ectodomain

CD36-PS interaction is an important affair to identify and remove dead/aged cells to control inflammation. CD36 ectodomain was cloned, over-expressed in bacterial expression system and purified to homogeneity. The dot-blot analysis shows that the CD36_ecto selectively binds PS vesicles blotted on the nitrocellulose membrane. PS binds strongly to CD36_ecto with a dissociation constant K-D of 53.7 +/- 0.48 jiM. The stoichiometry of interaction between CD36 and PS is 1:2. The hCD36_ecto-PS thermogram revealed that the hydrophobic and salt bridge interactions play crucial role in their interactions. PS docked nicely into the predicted pharmacophoric site with a binding energy of 5.1 kcal/mol. Analysis of CD36-PS molecular model showed that the residues R63, R96, N118, D270 and E418 were forming hydrogen bonds with PS. Molecular dynamics simulations indicate that R63 mutation has disrupted the integrity of biophoric constituents, directly affecting the hydrogen bonding from R96, N118 and D270. ITC thermogram analysis of mutant protein with PS vesicles indicate complete loss of binding with R63A and very low affinity of PS vesicles with D270A. Dot blot analysis further confirmed the ITC results. These finding may help to design suitable agents mimicking PS biophore with potentials in diagnostics of apoptotic cells and cardiovascular intervention.