Minimal determinants for binding activated Gα from the structure of a Gαi1-peptide dimer

G-Proteins cycle between an inactive GDP-bound state and an active GTP-bound state, serving as molecular switches that coordinate cellular signaling. We recently used phage display to identify a series of peptides that bind G alpha subunits in a nucleotide-dependent manner [ Johnston, C. A., Willard, F. S., Jezyk, M. R., Fredericks, Z., Bodor, E. T., Jones, M. B., Blaesius, R., Watts, V. J., Harden, T. K., Sondek, J., Ramer, J. K., and Siderovski, D. P. (2005) Structure 13, 1069 - 1080]. Here we describe the structural features and functions of KB-1753, a peptide that binds selectively to GDP, AlF4- - and GTP gamma S-bound states of G alpha(i) subunits. KB-1753 blocks interaction of G alpha(transducin) with its effector, cGMP phosphodiesterase, and inhibits transducin-mediated activation of cGMP degradation. Additionally, KB-1753 interferes with RGS protein binding and resultant GAP activity. A fluorescent KB-1753 variant was found to act as a sensor for activated G alpha in vitro. The crystal structure of KB-1753 bound to G alpha(i1)center dot GDP center dot AlF4- reveals binding to a conserved hydrophobic groove between switch II and alpha 3 helices and, along with supporting biochemical data and previous structural analyses, supports the notion that this is the site of effector interactions for G alpha(i) subunits.