3ONW

Structure of a G-alpha-i1 mutant with enhanced affinity for the RGS14 GoLoco motif.

Summary for 3ONW

• Entry DOI: 10.2210/pdb3onw/pdb
• Related: 1kjy 2om2
• Descriptor: Guanine nucleotide-binding protein G(i) subunit alpha-1, Regulator of G-protein signaling 14, GUANOSINE-5'-DIPHOSPHATE, ... (5 entities in total)
• Functional Keywords: rgs14 goloco, rosetta, protein design, affinity enhancement, ras-like domain, all-helical domain, goloco motif, arginine finger, signaling protein, lipoprotein, transducer, guanine nucleotide dissociation inhibitor, gtp binding, nucleotide binding, adp-ribosylation
• Biological source: Homo sapiens (human); More
• Cellular location: Nucleus (By similarity): P63096 O43566
• Total number of polymer chains: 4
• Total formula weight: 84195.11

Authors

Bosch, D.,Kimple, A.J.,Sammond, D.W.,Miley, M.J.,Machius, M.,Kuhlman, B.,Willard, F.S.,Siderovski, D.P. (deposition date: 2010-08-30, release date: 2010-11-24, Last modification date: 2023-09-06)

Primary citation

Bosch, D.E.,Kimple, A.J.,Sammond, D.W.,Muller, R.E.,Miley, M.J.,Machius, M.,Kuhlman, B.,Willard, F.S.,Siderovski, D.P.
Structural Determinants of Affinity Enhancement between GoLoco Motifs and G-Protein {alpha} Subunit Mutants.
J.Biol.Chem., 286:3351-3358, 2011

PubMed Abstract: GoLoco motif proteins bind to the inhibitory G(i) subclass of G-protein α 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/Gα(i) 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 Gα(i1) point mutations, E116L, Q147L, and E245L; each increases affinity for multiple GoLoco motifs. A component of this affinity enhancement results from a decreased rate of dissociation between the Gα mutants and GoLoco motifs. For Gα(i1)(Q147L), affinity enhancement was seen to be driven by favorable changes in binding enthalpy, despite reduced contributions from binding entropy. The crystal structure of Gα(i1)(Q147L) 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 wild-type 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.

PubMed: 21115486
DOI: 10.1074/jbc.M110.190496

Experimental method

X-RAY DIFFRACTION (2.38 Å)