ABSTRACT
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 x AlF4(-)- and GTPgammaS-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) x GDP x AlF4(-) reveals binding to a conserved hydrophobic groove between switch II and alpha3 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.
Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Aluminum Compounds/chemistry , Amino Acid Sequence , Amino Acid Substitution , Bacterial Proteins/metabolism , Crystallography, X-Ray , Dimerization , Fluorides/chemistry , GTP-Binding Protein alpha Subunits, Gi-Go/chemistry , Guanosine Diphosphate/chemistry , Humans , Luminescent Proteins/metabolism , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Structure, Secondary , RGS Proteins/metabolism , Recombinant Fusion Proteins/metabolism , Structure-Activity RelationshipABSTRACT
Signaling via G-protein coupled receptors is initiated by receptor-catalyzed nucleotide exchange on Galpha subunits normally bound to GDP and Gbetagamma. Activated Galpha . GTP then regulates effectors such as adenylyl cyclase. Except for Gbetagamma, no known regulators bind the adenylyl cyclase-stimulatory subunit Galphas in its GDP-bound state. We recently described a peptide, KB-752, that binds and enhances the nucleotide exchange rate of the adenylyl cyclase-inhibitory subunit Galpha(i). Herein, we report that KB-752 binds Galpha(s) . GDP yet slows its rate of nucleotide exchange. KB-752 inhibits GTPgammaS-stimulated adenylyl cyclase activity in cell membranes, reflecting its opposing effects on nucleotide exchange by Galpha(i) and Galpha(s).
Subject(s)
Adenylyl Cyclase Inhibitors , GTP-Binding Protein alpha Subunits, Gs/antagonists & inhibitors , Peptides/pharmacology , Signal Transduction/drug effects , Cells, Cultured , GTP-Binding Protein alpha Subunits, Gi-Go/drug effects , HumansABSTRACT
Heterotrimeric G proteins are molecular switches that regulate numerous signaling pathways involved in cellular physiology. This characteristic is achieved by the adoption of two principal states: an inactive, GDP bound state and an active, GTP bound state. Under basal conditions, G proteins exist in the inactive, GDP bound state; thus, nucleotide exchange is crucial to the onset of signaling. Despite our understanding of G protein signaling pathways, the mechanism of nucleotide exchange remains elusive. We employed phage display technology to identify nucleotide state-dependent Galpha binding peptides. Herein, we report a GDP-selective Galpha binding peptide, KB-752, that enhances spontaneous nucleotide exchange of Galpha(i) subunits. Structural determination of the Galpha(i1)/peptide complex reveals unique changes in the Galpha switch regions predicted to enhance nucleotide exchange by creating a GDP dissociation route. Our results cast light onto a potential mechanism by which Galpha subunits adopt a conformation suitable for nucleotide exchange.
Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go/chemistry , Amino Acid Motifs , Amino Acid Sequence , Biosensing Techniques , Buffers , Catalytic Domain , Crystallography, X-Ray , Dimerization , Electrons , Enzyme-Linked Immunosorbent Assay , Guanine Nucleotide Exchange Factors/chemistry , Guanine Nucleotides/chemistry , Kinetics , Magnesium/chemistry , Models, Molecular , Molecular Sequence Data , Nucleotides/chemistry , Peptide Library , Peptides/chemistry , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Signal Transduction , Stereoisomerism , Surface Plasmon Resonance , Time FactorsABSTRACT
Interleukin-1 (IL-1) blockade by IL-1 receptor antagonist benefits some arthritis patients by reducing joint damage. This fact inspired us to develop antagonist human therapeutic antibodies against IL-1R(I) using phage libraries that display single-chain variable fragment (scFv) antibody fragments. Panning libraries against human IL-1R(I) generated 39 unique scFv-phage whose binding to IL-1R(I) was competed by IL-1 ligands. Fifteen of these scFv-phage, identified using IL-1R(I)-binding assays and dissociation rate ranking, were reformatted as scFv-Fc and IgG(4) molecules. The ease of producing antibodies in the scFv-Fc format permitted rapid identification of four lead clones (C10, C13, C14, C15) that inhibit NF-kappaB nuclear translocation induced by IL-1. Reformatting these clones as IgG(4) molecules increased their inhibition potency by
