Syntaxin (STX) is a N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein that binds to the plasma membrane and regulates ion channels and neurotransmitter transporters. Experiments have established the involvement of the N-terminal segment of STX in direct protein-protein interactions and have suggested a critical role for the phosphorylation of serine 14 (S14) by casein kinase-2 (CK2). Because the organization of STX in the plasma membrane was shown to be regulated by phosphatidylinositol 4,5-biphosphate (PIP2) lipids, we investigated the mechanistic involvement of PIP2 lipids in modulating both the membrane interaction and the phosphorylation of STX, using a computational strategy that integrates mesoscale continuum modeling of protein-membrane interactions, with all-atom molecular dynamics (MD) simulations. Iterative applications of this protocol produced quantitative evaluations of lipid-type demixing due to the protein and identified conformational differences between STX immersed in PIP2-containing and PIP2-depleted membranes. Specific sites in STX were identified to be important for the electrostatic interactions with the PIP2 lipids attracted to the protein, and the segregation of PIP2 lipids near the protein is shown to have a dramatic effect on the positioning of the STX N-terminal segment with respect to the membrane/water interface. This PIP2-dependent repositioning is shown to modulate the extent of exposure of S14 to large reagents representing the CK2 enzyme and hence the propensity for phosphorylation. The prediction of STX sites involved in such PIP2-dependent regulation of STX phosphorylation at S14 offers experimentally testable probes of the mechanisms and models presented in this study, through structural modifications that can modulate the effects. © 2012 American Chemical Society.