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Crystal structure of the RLIP76 Ral binding domain mutant (E427H/Q433L/K440R) in complex with RalB-GMPPNP
Structural highlights
FunctionRALB_HUMAN Multifunctional GTPase involved in a variety of cellular processes including gene expression, cell migration, cell proliferation, oncogenic transformation and membrane trafficking. Accomplishes its multiple functions by interacting with distinct downstream effectors. Acts as a GTP sensor for GTP-dependent exocytosis of dense core vesicles. Required both to stabilize the assembly of the exocyst complex and to localize functional exocyst complexes to the leading edge of migrating cells. Plays a role in the late stages of cytokinesis and is required for the abscission of the bridge joining the sister cells emerging from mitosis. Required for suppression of apoptosis.[1] Publication Abstract from PubMedRal 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 A 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. Affinity maturation of the RLIP76 Ral binding domain to inform the design of stapled peptides targeting the Ral GTPases.,Hurd CA, Brear P, Revell J, Ross S, Mott HR, Owen D J Biol Chem. 2021 Jan-Jun;296:100101. doi: 10.1074/jbc.RA120.015735. Epub 2020 , Nov 23. PMID:33214225[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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