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 alpha-subunit of transducin (G alpha(t)-GTP) activates the cGMP phosphodiesterase (PDE) by binding the inhibitory gamma-subunit (PDE gamma). Significant progress in understanding the interaction between G alpha(t) and PDE gamma was achieved by solving the crystal structure of the PDE gamma C-terminal peptide bound to G alpha(t) 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 PDE gamma C terminus and activated G alpha(t) bound to guanosine 5'-O-(3-thio)-triphosphate (GTP gamma S) using a series of full-length PDE gamma photoprobes generated by intein-mediated expressed protein ligation. For each of seven PDE gamma 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 PDE gamma C-terminal positions to the G alpha(t)-GTP gamma S N terminus, particularly from PDE gamma residue 73. The overall percentage of cross-linking to the G alpha(t)-GTP gamma S N terminus was analyzed using a far Western method for examining G alpha(t)-GTP gamma S proteolytic digestion patterns. Furthermore, mass spectrometric analysis of cross-links to G alpha(t) from a benzoyl-phenylalanine replacement at PDE gamma position 86 localized the region of photoinsertion to G alpha(t) N-terminal residues G alpha(t)-(22 - 26). This novel G alpha(t)/PDE gamma interaction suggests that the transducin N terminus plays an active role in signal transduction.