Abstract
Peptide inhibitors of Methuselah (Mth), a G protein-coupled receptor (GPCR), were reported that can extend the life span of Drosophila melanogaster. Mth is a class B GPCR, which is characterized by a large, N-terminal ectodomain that is often involved with ligand recognition. The crystal structure of the Mth ectodomain, which binds to the peptide inhibitors with high affinity, was previously determined. Here we report the predicted structures for RWR motif peptides in complex with the Mth ectodomain. We studied representatives of both Pro-class and Arg-class RWR motif peptides and identified ectodomain residues Asp139, Phe130, Asp127, and Asp78 as critical in ligand binding. To validate these structures, we predicted the effects of various ligand mutations on the structure and binding to Mth. The binding of five mutant peptides to Mth was characterized experimentally by surface plasmon resonance, revealing measured affinities that are consistent with predictions. The electron density map calculated from our MD structure compares well with the experimental map of a previously determined peptide/Mth crystal structure and could be useful in refining the current low-resolution data. The elucidation of the ligand binding site may be useful in analyzing likely binding sites in other class B GPCRs.