Affinity maturation of the RLIP76 Ral binding domain to inform the design of stapled peptides targeting the Ral GTPases
| Autor: | Sarah Ross, Jefferson D. Revell, Darerca Owen, Catherine A. Hurd, Paul Brear, Helen R. Mott |
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| Přispěvatelé: | Brear, Paul [0000-0002-4045-0474], Mott, Helen [0000-0002-7890-7097], Owen, Darerca [0000-0003-0978-5425], Apollo - University of Cambridge Repository |
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
GMPPNP guanosine 5'-[β γ-imido] triphosphate Magnetic Resonance Spectroscopy GTPase stapled peptides DMF N N-dimethylformamide Biochemistry protein-protein interaction SPA scintillation proximity assay RBD Ral binding domain CD circular dichroism RLIP76 RALB ITC isothermal titration calorimetry Chemistry Circular Dichroism GTPase-Activating Proteins Cell biology FP fluorescence polarization Guanine nucleotide exchange factor K-Ras Fmoc 9-fluorenylmethoxycarbonyl Protein Binding Signal Transduction Research Article Binding domain animal structures PPI protein–protein interaction Chemical biology chemical biology Calorimetry Fluorescence drug discovery Protein–protein interaction GEF guanine nucleotide exchange factor TFA trifluoroacetic acid Affinity maturation 03 medical and health sciences FAM carboxyfluorescein Humans cancer cell signaling Molecular Biology GGTase geranylgeranyltransferase 030102 biochemistry & molecular biology CPP cell-penetrating peptide Cell Biology Ral 030104 developmental biology GAP GTPase activating protein Cell-penetrating peptide ATP-Binding Cassette Transporters ral GTP-Binding Proteins |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 0021-9258 |
| Popis: | Ral GTPases have been implicated as critical drivers of cell growth and metastasis in numerous Ras-driven cancers. We have previously reported stapled peptides, based on the Ral effector RLIP76, that can disrupt Ral signaling. Stapled peptides are short peptides that are locked into their bioactive form using a synthetic brace. Here, using an affinity maturation of the RLIP76 Ral-binding domain, we identified several sequence substitutions that together improve binding to Ral proteins by more than 20-fold. Hits from the selection were rigorously analyzed to determine the contributions of individual residues and two 1.5 Å cocrystal structures of the tightest-binding mutants in complex with RalB revealed key interactions. Insights gained from this maturation were used to design second-generation stapled peptides based on RLIP76 that exhibited vastly improved selectivity for Ral GTPases when compared with the first-generation lead peptide. The binding of second-generation peptides to Ral proteins was quantified and the binding site of the lead peptide on RalB was determined by NMR. Stapled peptides successfully competed with multiple Ral-effector interactions in cellular lysates. Our findings demonstrate how manipulation of a native binding partner can assist in the rational design of stapled peptide inhibitors targeting a protein-protein interaction. |
| Databáze: | OpenAIRE |
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