The N Terminus of GTPγS-activated Transducin α-Subunit Interacts with the C Terminus of the cGMP Phosphodiesterase γ-Subunit*

Dynamic regulation of G-protein signaling in the phototransduction cascade ensures the high temporal resolution of vision. In a key step, the activated α-subunit of transducin (Gαt-GTP) activates the cGMP phosphodiesterase (PDE) by binding the inhibitory γ-subunit (PDEγ). Significant progress in understanding the interaction between Gαt and PDEγ was achieved by solving the crystal structure of the PDEγ C-terminal peptide bound to Gαt in the transition state for GTP hydrolysis (Slep, K. C., Kercher, M. A., He, W., Cowan, C. W., Wensel, T. G., and Sigler, P. B. (2001) Nature 409, 1071–1077). However, some of the structural elements of each molecule were absent in the crystal structure. We have probed the binding surface between the PDEγ C terminus and activated Gαt bound to guanosine 5′-O-(3-thio)-triphosphate (GTPγS) using a series of full-length PDEγ photoprobes generated by intein-mediated expressed protein ligation. For each of seven PDEγ photoprobe species, expressed protein ligation allowed one benzoyl-l-phenylalaine substitution at selected hydrophobic C-terminal positions, and the addition of a biotin affinity tag at the extreme C terminus. We have detected photocross-linking from several PDEγ C-terminal positions to the Gαt-GTPγS N terminus, particularly from PDEγ residue 73. The overall percentage of cross-linking to the Gαt-GTPγSN terminus was analyzed using a far Western method for examining Gαt-GTPγS proteolytic digestion patterns. Furthermore, mass spectrometric analysis of cross-links to Gαt from a benzoyl-phenylalanine replacement at PDEγ position 86 localized the region of photoinsertion to Gαt N-terminal residues Gαt-(22–26). This novel Gαt/PDEγ interaction suggests that the transducin N terminus plays an active role in signal transduction.