Identifying Functionally Important Conformational Changes in Proteins: Activation of the Yeast α-factor Receptor Ste2p

Abstract

We have developed a procedure in which disulfide crosslinks are used to identify regions of proteins that undergo functionally important intramolecular motion. The approach was applied to the identification of disulfide bonds that stabilize the active state of the yeast α-mating pheromone receptor Ste2p, a member of the superfamily of G Protein Coupled Receptors. Cysteine residues were introduced at random positions in targeted regions of a starting allele of Ste2p that completely lacks cysteines. Libraries of mutated receptors were then screened for alleles that exhibit constitutive signaling. Two strongly activated alleles were recovered containing cysteine residues in transmembrane segments 5 and 6. Constitutive activity of these alleles was dependent on the presence of both introduced cysteines and was sensitive to reducing agent. Crosslinked peptides derived from the mutant receptors were detected by immunoblotting. Additional sites of crosslinking between transmembrane segments 5 and 6 that did not lead to constitutive activation were also identified. These results indicate that relative motion of the transmembrane segments 5 and 6 in the extracellular half of the membrane is sufficient to activate the receptor and that transmembrane segment 6, but not transmembrane segment 5, exhibits rotational mobility that is not associated with receptor activation.

Identification of Destabilizing and Stabilizing Mutations of Ste2p, a G Protein-Coupled Receptor in Saccharomyces cerevisiae

“…Full-length alleles of STE2 consisted of fusions to three copies of the influenza HA epitope tag. C-terminally truncated alleles of STE2 were fused to a triple c-myc epitope tag, as described previously 75 . For immunoblotting of cell lysates, strains were cultured in SD-ura to an OD 600 of 1.…”

Functional fusions of T4 lysozyme in the third intracellular loop of a G protein-coupled receptor identified by a random screening approach in yeast

“…Plasmid pMD1887, containing full duplication of the 16-residues IC3 loop, but no inserted T4L, was created by oligonucleotide-directed mutagenesis (oligonucleotide ON 1424) of plasmid pMD1886, recovered from the library screen, that contained 16 residues of IC3, followed by T4L, followed by 15 residues of IC3. Yeast strain A2953 (Taslimi et al, 2012) was used for screening of insertional libararies. Strain A575 (Sommers et al, 2000) was used for retransformation with identified STE2 alleles for further characterization.…”

Distinguishing Critical, Beneficial, Neutral and Harmful Mutations Uncovered in the Directed Evolution of a Yeast Membrane Receptor

“…In another study, the K225C mutation ablated functionality of the native Ste2p receptor [15], so it is noteworthy that a substitution to a similarly nucleophilic residue (K225T) did not ablate receptor functionality here. K225T is on the border of the third intracellular loop and the fifth transmembrane region, the relative motion of which is required for receptor activation [11]. If the K225T mutation is destabilizing, a possible explanation for the beneficial nature of the K225T mutation in the Mut2 receptor is that the mutation destabilizes the receptor enough to accept a nonnative ligand, but the M218T mutation, which was noted as a rescuing mutation earlier in this discussion, rescues this destabilization.…”

“…M218T is located in the fifth transmembrane region, the relative motion of which is required for receptor activation [11]. In the context of the native Ste2p, M218T on its own is a neutral mutation, and residue 218 is believed to interact with residues on the third transmembrane region.…”