Structural determinants of affinity enhancement between GoLoco motifs and G-protein alpha subunit mutants.
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.
Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Dustin E
DE
Bosch
Adam J
AJ
Kimple
Deanne W
DW
Sammond
Robin E
RE
Muller
Michael J
MJ
Miley
Mischa
M
Machius
Brian
B
Kuhlman
Francis S
FS
Willard
David P
DP
Siderovski
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D044385:GTP-Binding Protein alpha Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D056004:Molecular Dynamics Simulation; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D013816:Thermodynamics
D044385:GTP-Binding Protein alpha Subunits; D010455:Peptides