guanosine-triphosphate and 1-6-bismaleimidohexane

Receptors activate the G alpha subunits of heterotrimeric G proteins by binding to the C-terminus and reducing their affinity for bound GDP, therefore promoting exchange of GDP for GTP. Although this general mechanism is the same for all G alpha subunits, different G alpha subunits vary in nucleotide binding and hydrolysis even though the residues that make up the guanine nucleotide binding site are virtually identical. We have shown previously that truncation of 14 amino acids from the C-terminus of G alpha o decreased the apparent affinity for GDP and permitted us to see an activated conformation with GTP [Denker, B. M., et al. (1992) J. Biol. Chem. 267, 9998-10002]. To test whether mutations in the receptor binding region lead to different phenotypes in closely related G alpha subunits, we made the equivalent deletions in G alpha i2, synthesized the proteins in vitro in a rabbit reticulocyte lysate and used the pattern of native tryptic proteolysis as an index of conformation. The phenotype of truncated G alpha i2 was different from that of truncated G alpha o: GDP affinity was reduced, but we could not detect an activated conformation with GTP (although GTP gamma S activated normally). Analysis of shorter deletions showed that loss of three hydrophobic residues (between 11 and 13 residues from the C-terminus) was responsible for the phenotypes. To define the regions of G alpha o and G alpha i2 that were responsible for their different phenotypes, we used a conserved BamHI site (codon 212) to make chimeras. Each chimera truncated at the C-terminus had the phenotype of the donor of the amino-terminal portion. Both truncated chimeras were activated by GTP gamma S-like wild-type proteins, and both had decreased apparent affinity for GDP. Full-length chimeric subunits behaved like wild-type proteins. The crystal structure of G alpha t and G alpha i1 shows that the three hydrophobic amino acids we have identified make contact with residues in the N- and C-terminal portions of the protein. Our studies point to the importance of the contacts in the N-terminal region (start of beta strands 1 and 3) that may stabilize the C-terminal alpha helix, affect nucleotide binding, and determine the characteristic features of different G alpha subunits.

Topics: Amino Acid Sequence; Animals; Centrifugation, Density Gradient; Cross-Linking Reagents; Electrophoresis, Polyacrylamide Gel; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Macromolecular Substances; Maleimides; Models, Molecular; Molecular Sequence Data; Mutagenesis; Peptide Fragments; Polymerase Chain Reaction; Protein Biosynthesis; Protein Structure, Secondary; Rabbits; Rats; Recombinant Fusion Proteins; Reticulocytes; Sequence Deletion; Trypsin

The reversible association of alpha and beta gamma subunits of GTP-binding proteins is important for signal transmission from a variety of cell-surface receptors to intracellular effectors. Previous work showed that 1,6-bis(maleimido)hexane, which crosslinks cysteine residues, crosslinks alpha o and alpha i-1 to beta gamma. These crosslinks are likely to form through a conserved cysteine because 1,6-bis(maleimido)hexane can also crosslink alpha i-2, alpha 1, alpha s and Drosophila alpha 1 to give products of the same apparent molecular weight as crosslinked alpha o beta gamma and alpha i-1 beta gamma. These proteins have only two cysteines in common. Therefore, we mutated each of the two conserved cysteines of alpha o to alanines. Mutation of Cys215 prevents crosslinking to beta gamma, but does not affect binding of guanosine 5'-[gamma-thio]triphosphate or the ability of the mutated alpha subunit to bind beta gamma. In models of the alpha subunit based on the crystal structure of p21ras, Cys215 is located on the face opposite to the GTP-binding site and near an area that changes conformation depending on the nucleotide bound. This surface on the alpha subunit overlaps a putative effector binding region, raising important questions about the spatial organization of the proteins as they form ternary complexes. Mutation of Cys325 has no effect on crosslinking but, surprisingly, decreases by a factor of 10 the affinity of the mutated protein for GDP, relative to wild type, without changing the affinity for guanosine 5'-[gamma-thio]triphosphate. This mutation falls within a region thought to contact receptors and may represent a site through which receptors enhance the release of GDP.

Topics: Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Conserved Sequence; Cross-Linking Reagents; Cysteine; DNA Primers; Drosophila; GTP-Binding Proteins; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Macromolecular Substances; Maleimides; Models, Structural; Molecular Sequence Data; Mutagenesis, Site-Directed; Polymerase Chain Reaction; Protein Structure, Secondary