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Publication Abstract from PubMed

GoLoco motif proteins bind to the inhibitory Gi subclass of G-protein alpha subunits and slow the release of bound GDP; this interaction is considered critical to asymmetric cell division and neuro-epithelium and epithelial progenitor differentiation. To provide protein tools for interrogating the precise cellular role(s) of GoLoco motif/Galphai complexes, we have employed structure-based protein design strategies to predict gain-of-function mutations that increase GoLoco-motif binding affinity. Here, we describe fluorescence polarization and isothermal titration calorimetry measurements showing three predicted Galphai1 point mutations, E116L, Q147L, and E245L, each increase affinity for multiple GoLoco motifs. A component of this affinity enhancement results from a decreased rate of dissociation between the Galpha mutants and GoLoco motifs. For Galphai1Q147L, affinity enhancement was seen to be driven by favorable changes in binding enthalpy, despite reduced contributions from binding entropy. The crystal structure of Galphai1Q147L bound to the RGS14 GoLoco motif revealed disorder among three peptide residues surrounding a well-defined Leu-147 side-chain. Monte Carlo simulations of the peptide in this region showed a sampling of multiple backbone conformations in contrast to the wildtype complex. We conclude that mutation of Glu-147 to leucine creates a hydrophobic surface favorably buried upon GoLoco peptide binding, yet the hydrophobic Leu-147 also promotes flexibility among residues 511-513 of the RGS14 GoLoco peptide.

Structural determinants of affinity enhancement between GoLoco motifs and G-protein alpha subunit mutants.,Bosch DE, Kimple AJ, Sammond DW, Muller RE, Miley MJ, Machius M, Kuhlman B, Willard FS, Siderovski DP J Biol Chem. 2010 Nov 29. PMID:21115486^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.