Mapping the isoprenoid binding pocket of PDEdelta by a semisynthetic, photoactivatable N-Ras lipoprotein.

Biologically functional Ras isoforms undergo post-translational modifications starting with farnesylation of the most C-terminal cysteine. Combined with further processing steps, this isoprenylation allows for the anchoring of these proteins in endomembranes, where signal transduction events take place. The specific localization is subject to dynamic regulation and assumed to modulate the activity of Ras proteins by governing their spatio-temporal distribution. The delta subunit of phosphodiesterase (PDE delta) has attracted attention as a solubilization factor of isoprenylated Ras. In this study, we demonstrate that critical residues in the putative isoprenoid pocket of PDE delta can be mopped by coupling with a semisynthetic N-Ras lipoprotein in which the native farnesyl group of the processed protein was replaced by a photoactivatable geranyl benzophenone moiety. The crosslinked product included ports of beta-sheet 9 of PDE delta, which contains the highly conserved amino acids V145 and L147. Modeling of the PDE delta-geranyl benzophenone (GerBP) complex supports the conclusion that the photolabeled sequence is embedded in the putative isoprenoid pocket of PDE delta.