Heo, Jiyoung and Ja, William W. and Benzer, Seymour and Goddard, William A., III (2008) The Predicted Binding Site and Dynamics of Peptide Inhibitors to the Methuselah GPCR from Drosophila melanogaster. Biochemistry, 47 (48). pp. 12740-12749. ISSN 0006-2960 http://resolver.caltech.edu/CaltechAUTHORS:HEOb08
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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.
|Additional Information:||Copyright © 2008 American Chemical Society. Received July 15, 2008; Revised Manuscript Received August 18, 2008. Publication Date (Web): November 7, 2008. We thank J. Kaiser for help in calculating the density map, A.P. West, Jr., for providing the experimental density map, and J. Vielmetter and the Protein Expression Center of the Beckman Institute for technical assistance on the Biacore T100. This work was supported by funding from the NIH (K99AG030493 to W.W.J.; AG16630 to S.B.). The computational parts of this study were supported in part by the NSF (CTS-0608889) and NIH (1 RO1 CA 112293-01). [S.B.] Deceased Nov 30, 2007. Supporting Information: Kinetics analysis of peptide binding by surface plasmon resonance (Table S1), binding charateristics of the LR2 ligand into the Mth ectodomain (Figure S1), changes in distance during the 2 ns MD simulation of key intermolecular interactions in mutant complexes (Figure S2), and SPR sensorgrams of LP1 wild-type (WT) and mutant peptides with the Mth ectodomain (Figure S3). This material is available free of charge via the Internet at http://pubs.acs.org.|
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