3onw
From Proteopedia
Structure of a G-alpha-i1 mutant with enhanced affinity for the RGS14 GoLoco motif.
Structural highlights
Function[GNAI1_HUMAN] Guanine nucleotide-binding proteins (G proteins) are involved as modulators or transducers in various transmembrane signaling systems. The G(i) proteins are involved in hormonal regulation of adenylate cyclase: they inhibit the cyclase in response to beta-adrenergic stimuli. The inactive GDP-bound form prevents the association of RGS14 with centrosomes and is required for the translocation of RGS14 from the cytoplasm to the plasma membrane. May play a role in cell division.[1] [2] [RGS14_HUMAN] Acts as a regulator of G protein signaling (RGS). Modulates G protein alpha subunits nucleotide exchange and hydrolysis activities by functioning either as a GTPase-activating protein (GAP), thereby driving G protein alpha subunits into their inactive GDP-bound form, or as a GDP-dissociation inhibitor (GDI). Confers GDI activity on G(i) alpha subunits GNAI1 and GNAI3, but not G(o) alpha subunit GNAO1 and G(i) alpha subunit GNAI2. Confers GAP activity on G(o) alpha subunit GNAI0 and G(i) alpha subunits GNAI2 and GNAI3. May act as a scaffold integrating G protein and Ras/Raf MAPkinase signaling pathways. Inhibits platelet-derived growth factor (PDGF)-stimulated ERK1/ERK2 phosphorylation; a process depending on its interaction with HRAS1 and that is reversed by G(i) alpha subunit GNAI1. Acts as a positive modulator of microtubule polymerisation and spindle organization through a G(i)-alpha-dependent mechanism. Plays a role in cell division. Probably required for the nerve growth factor (NGF)-mediated neurite outgrowth. May be involved in visual memory processing capacity and hippocampal-based learning and memory.[3] [4] Publication Abstract from PubMedGoLoco 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[5] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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Categories: Heterotrimeric G-protein GTPase | Homo sapiens | Bosch, D | Kimple, A J | Kuhlman, B | Machius, M | Miley, M J | Sammond, D W | Siderovski, D P | Willard, F S | Adp-ribosylation | Affinity enhancement | All-helical domain | Arginine finger | Goloco motif | Gtp binding | Guanine nucleotide dissociation inhibitor | Lipoprotein | Nucleotide binding | Protein design | Ras-like domain | Rgs14 goloco | Rosetta | Signaling protein | Transducer
