Structural Determinants of Affinity Enhancement between GoLoco Motifs and G-Protein α Subunit Mutants*

GoLoco motif proteins bind to the inhibitory Gi 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/Gαi 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 Gαi1 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 Gαi1Q147L, affinity enhancement was seen to be driven by favorable changes in binding enthalpy, despite reduced contributions from binding entropy. The crystal structure of Gαi1Q147L 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.

tion of affinity-enhancing point mutations (6 -9). The development of reliable in silico procedures promises to provide a more efficient means of protein design than labor-intensive combinatorial screening techniques such as phage display or ribosome display (10 -12). Previously, a structure-based protocol for predicting affinity-enhancing point mutations was developed (7), in part using as a template the high-resolution crystal structure of the heterotrimeric G-protein subunit G␣ i1 in complex with the RGS14 (regulator of G-protein signaling 14) GoLoco motif (13). Residues at the protein/peptide binding surface were sequentially mutated to nonpolar amino acids in silico, producing 33 predicted affinity-enhancing mutations. Four of six experimentally tested mutations to either G␣ i1 or the GoLoco motif enhanced binding affinity, as predicted by Rosetta (7).
As a critical component of seven-transmembrane domain receptor signaling, G␣ subunits normally cycle between an inactive GDP-bound state and an active GTP-bound state (reviewed in Refs. 14 and 15). GoLoco motif proteins such as LGN, AGS3, RGS12, and RGS14 act as guanine nucleotide dissociation inhibitors, specifically for the adenylyl cyclase inhibitory subclass of G␣ subunits (reviewed in Refs. 16 and 17). GoLoco motifs prevent GDP dissociation by binding across the Ras-like and all-helical domains of G␣, as well as by contributing an arginine side chain from the highly conserved (E/D)QR GoLoco triad (16) to contacts made to the bound guanosine diphosphate (13). Not only are GoLoco motif-containing proteins thought to modulate seven-transmembrane domain receptor signaling in various physiological responses (18 -20), but these proteins are also critical to cell fate determinant sorting and cell division processes in multiple contexts across multiple species (21)(22)(23)(24). Highlighting the value of mutation-derived affinity modulation to cell biological applications, a G␣ i1 N149I mutant with selective loss of GoLoco motif affinity has recently provided direct evidence for the G␣ i /GoLoco protein interaction in these cell division processes (25,26).
We previously predicted that mutation to leucine of one of three polar residues, Glu-116, Gln-147, or Glu-245, on the surface of G␣ i1 enhances binding of G␣ i1 -GDP to the RGS14 GoLoco motif (7,27). Although the algorithms implemented in the Rosetta program predict a favorable change in the Gibbs free energy of binding, our computational tools do not provide a clear mechanism of affinity enhancement. Knowledge of changes in the thermodynamic and/or kinetic parameters of binding, such as the rate of dissociation, will be im-* This work was supported, in whole or in part, by National Institutes of Health Grants R01 CA127152 (to D. P. S.), F30 MH074266 (to A. J. K.), and T32 GM008719 (to D. E. B. and A. J. K.). □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3 and portant in the future application of these computational design strategies to biological and therapeutic questions.
Here, we assessed the effects of these predicted G␣ i1 /GoLoco affinity-enhancing mutations on the kinetics and thermodynamics of binding using fluorescence polarization, isothermal titration calorimetry, and surface plasmon resonance. Although mutations that promote binding to a target protein are desirable, protein design applications often require the preservation of other protein functions. Accordingly, we also determined the effects of these three affinity-enhancing mutations on the nucleotide cycle of G␣ i1 , including GTP binding and hydrolysis. To further elucidate the molecular mechanism of affinity enhancement for one mutant, we determined the x-ray crystal structure of G␣ i1 Q147L -GDP bound to the RGS14 GoLoco motif. In addition, we investigated the conformational flexibility of the GoLoco peptide backbone and side chains surrounding the Leu-147 residue of the G␣ i1 mutant using Monte Carlo simulations.
Fluorescence Polarization Measurements-All polarization experiments were conducted using a PHERAstar microplate reader (BMG Labtech, Offenburg, Germany). Excitation wavelength was 485 Ϯ 6 nm, and emission was recorded at 520 Ϯ 15 nm. Gains of the parallel and perpendicular channels were calibrated so that 350 pM FITC probe alone had a polarization of ϳ35 milli-Polarization units (mP) for each experiment. All experiments were conducted at 25°C in triplicate using 96-well black-bottom plates (Costar, Corning, NY). The final volume of each well was brought to 180 l with PheraBuffer (10 mM Tris (pH 7.5), 150 mM NaCl, 100 M GDP, and 0.05% (v/v) Nonidet P-40). For experiments conducted in the presence of the transition-state mimetic, alumi-num magnesium tetrafluoride (AMF), 4 an additional 10 mM MgCl 2 , 10 mM NaF, and 30 M AlCl 3 were added to the standard PheraBuffer. Polarization was determined from raw intensity values of the parallel and perpendicular channels using PHERAstar Version 1.60 software (BMG Labtech). Binding isotherms were fit and equilibrium dissociation constants were determined using GraphPad Prism Version 5.0 as described previously (28).
Surface Plasmon Resonance Assays-Optical detection of protein/protein interactions by surface plasmon resonance was performed using a Biacore 3000 (GE Healthcare). Carboxymethylated dextran (CM5) sensor chips (GE Healthcare) with covalently bound anti-GST antibody surfaces were created as described previously (30). The GST-GoLoco fusion proteins GST-RGS12 (amino acids 1184 -1228) (29), GST-RGS14 (amino acids 497-532) (29), and GST-GPSM2(GL2) (31), as well as GST alone (serving as a negative control), were loaded onto separate surfaces to levels of 1372, 963, 1290, and 1061 resonance units (RU), respectively. All experiments were performed with the sensor surface, fluidics, and pump equilibrated with Biacore running buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 0.05% (v/v) Nonidet P-40, 100 M GDP, and 50 M EDTA). Injections of 200 l of WT or mutant G␣ i1 at 50 M for kinetic analyses were performed using the KINJECT command with a dissociation phase of 2000 s at a flow rate of 40 l/min. The surface was regenerated between runs by removal of bound G␣ i1 using 325-l injections of AMF buffer (Biacore running buffer with an additional 10 mM MgCl 2 , 10 mM NaF, and 30 M AlCl 3 ). Nonspecific binding to the GST only-loaded surface was subtracted from each curve (BIAevaluation Version 3.0 software, GE Healthcare). To establish off-rates, dissociation-phase sensorgrams were normalized to percent bound, and the averages of triplicate experiments were fit to a single exponential function for dissociation without constraints using Prism Version 5.0.
Nucleotide Binding and Hydrolysis Assays-The [ 35 S]GTP␥S filter-binding assay used to measure rates of spontaneous GDP release was conducted as described previously (32). Briefly, WT or mutant G␣ i1 (100 nM) was incubated in assay buffer (20 mM Tris (pH 7.5), 1 mM C 12 E 10 , 1 mM DTT, 1 mM EDTA, 100 mM NaCl, and 25 mM MgCl 2 ) or nonspecific buffer (assay buffer plus 100 M GTP␥S) at 20°C. Radioligand binding was initiated by the addition of 6.25 nM [ 35 S]GTP␥S. At timed intervals, 100-l aliquots of the reaction were filtered through nitrocellulose membranes and washed four times with 7.5 ml of ice-cold wash buffer (20 mM Tris (pH 7.5), 120 mM NaCl, and 25 mM MgCl 2 ). Filters were dried under a vacuum before measuring ␤-radiation via liquid scintillation counting.
Intrinsic GTP hydrolysis rates of G␣ i1 subunits were assessed by monitoring the production of 32 P-labeled inorganic phosphate during a single round of GTP hydrolysis as described previously (33). In brief, WT or mutant G␣ i1 (100 nM) was incubated for 10 min at 20°C with 1 ϫ 10 6 cpm of [␥-32 P]GTP (specific activity of 6500 dpm/Ci; PerkinElmer Life Sciences) in reaction buffer (50 mM Tris (pH 7.5), 0.05% (v/v) C 12 E 10 , 1 mM DTT, 10 mM EDTA, 100 mM NaCl, and 5 g/ml BSA). The nucleotide-loaded protein was then chilled on ice for 5 min prior to initiation of hydrolysis by the addition of 10 mM MgCl 2 and 100 M GTP␥S (final concentrations). At timed intervals, 100-l aliquots were quenched in 900 l of charcoal slurry (5% (w/v) activated charcoal in 50 mM H 3 PO 4 (pH 3.0)) and centrifuged at 4°C for 10 min at 4000 ϫ g. Subsequently, the ␤-radiation of 600-l aliquots of the supernatant was counted via liquid scintillation.
Intrinsic Tryptophan Fluorescence Measurements of G␣ Activation-Changes in tryptophan fluorescence of G␣ i1 subunits were measured to assess activation by the transitionstate mimetic GDP⅐AlF 4 Ϫ . Activation of G␣ i1 results in translocation of Trp-211, a residue in the conformationally flexible switch II region, to a hydrophobic pocket; the change in environment results in an increased quantum yield of Trp-211 fluorescence (34,35). Tryptophan fluorescence of WT or mutant G␣ i1 (1 M) was measured using a POLARstar Omega plate reader (BMG Labtech) in 187 l of assay buffer (100 mM NaCl, 100 M EDTA, 2 mM MgCl 2 , 2 M GDP, and 20 mM Tris (pH 8.0)). Intrinsic fluorescence was measured using a 280-nm excitation filter and 350-nm emission filter (each with 10-nm cutoffs). After a 20-s equilibration at ambient temperature, activation was induced by sequential injections of 8 l of 0.5 M NaF and 5 l of 1.2 mM AlCl 3 to each well. Tryptophan fluorescence was recorded for 2 min following activation. Fluorescence intensity traces shown represent the average of experiments performed in triplicate.
Isothermal Titration Calorimetry-Isothermal titration calorimetry (ITC) was performed using a MicroCal TM Auto-ITC200 system (GE Healthcare). WT and mutant G␣ i1 subunits were subjected to size-exclusion chromatography in ITC buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 10 M GDP, 5% (v/v) glycerol, and 0.005% (v/v) ␤-mercaptoethanol) and subsequently concentrated to 30 M using a VivaSpin 20 ultrafiltration spin column (Sartorius Stedim Biotech, Göttingen, Germany) at 4°C and 3000 ϫ g. Lyophilized RGS14 GoLoco peptide was dissolved in ITC buffer to a concentration of 350 M. Titrations were performed at 25°C and consisted of 20 2-l injections of peptide into WT G␣ i1 or G␣ i1 Q147L solutions at 250-s intervals. Binding isotherms and curve fittings were obtained using Origin for ITC analysis software (Micro-Cal). Enthalpy of binding was determined for three titrations each of RGS14 GoLoco peptide into solutions of wild-type G␣ i1 or G␣ i1 Q147L , and average values were compared with a Student's t test using Prism Version 5.0. Because ITC sensitivity did not allow us to approach a G␣ i1 or peptide concentration near the K D , Gibbs free energy of binding was calculated from the more accurate fluorescence polarization binding affinity measurements, using ⌬G ϭ ϪR(T)ln(1/K D ), where R is the universal gas constant and T is temperature in Kelvin. Entropy of binding was then estimated with ⌬S ϭ (⌬H Ϫ ⌬G)/T, where ⌬H is the average enthalpy of binding derived from isothermal titrations.
Crystallization and Structure Determination-The complex of G␣ i1 Q147L and RGS14 GoLoco peptide was obtained by mixing G␣ i1 Q147L and GoLoco peptide to concentrations of 400 and 600 M, respectively, in 10 mM Tris (pH 7.5), 1 mM MgCl 2 , 10 M GDP, 5% (v/v) glycerol, and 5 mM DTT. Crystals of the G␣ i1 Q147L ⅐GDP/GoLoco peptide heterodimer were obtained by vapor diffusion from hanging drops containing a 1:1 (v/v) ratio of protein/peptide solution to well solution (1.5-1.6 M ammonium sulfate, 100 mM MgCl 2 , 100 mM sodium acetate (pH 4.8), and 10% (v/v) glycerol). Crystals (ϳ200 ϫ 200 ϫ 50 m) formed in 1-4 days at 18°C exhibited the symmetry of space group P222 1 (a ϭ 70.3, b ϭ 83.7, and c ϭ 190.1 Å, ␣ ϭ ␤ ϭ ␥ ϭ 90°) and contained two G␣ i1 Q147L / GoLoco heterodimers in the asymmetric unit. For data collection at 100 K, crystals were transferred to well solution supplemented with 25% saturated sucrose for ϳ30 s, followed by plunging into liquid nitrogen. A native data set was collected at the SER-CAT 22-BM beamline at the Advanced Photon Source (Argonne National Laboratory). Data were processed using the HKL-2000 program (36). The crystal structure of the WT G␣ i1 /RGS14 GoLoco heterodimer (Protein Data Bank code 2OM2 (7)), excluding the GoLoco motif peptide, nucleotide, waters, and magnesium, was used as a search model for molecular replacement using the Phaser program (37). Refinement was carried out using the Refmac5 program (38), consisting of conjugate gradient minimization and refinement of individual atomic displacement and translationlibration-screw parameters, interspersed with manual revisions of the model using the Coot program (39). The current model contains two G␣ i1 Q147L ⅐GDP/GoLoco peptide heterodimers in the asymmetric unit. For data collection and refinement statistics and a list of residues that could not be located in the electron density, see supplemental Table S1. All structural images were made with PyMOL (Schrödinger LLC, Portland, OR) unless indicated otherwise.

RESULTS AND DISCUSSION
Comparing Different Point Mutations in G␣ i1 That Enhance Affinity for GoLoco Motifs-Three polar-to-nonpolar point mutations in G␣ i1 (E116L, Q147L, and E245L) have been described individually (7,27) as enhancing GoLoco motif binding affinity; the location of these three positions within G␣ i1 is highlighted in supplemental Figs. S1 and S2. To determine and compare directly the affinities of these three separate G␣ i1 point mutants (G␣ i1 E116L , G␣ i1 Q147L , and G␣ i1 E245L ) for the GoLoco motif of RGS14, we used a synthetic FITC-RGS14 GoLoco motif peptide probe in fluorescence polarization (FP) binding assays similar to those performed with RGS12 and WT G␣ i1 (28). Holding the peptide concentration at 350 pM, we added increasing concentrations of WT or mutant G␣ i1 protein (30 pM to 200 nM) and generated equilibrium binding isotherms (Fig. 1). The dissociation constants (K D ) were determined to be 14, 1.6, 2.3, and 0.69 nM for GDP-bound G␣ i1 WT , G␣ i1 E116L , G␣ i1 Q147L , and G␣ i1 E245L , respectively. These results confirm our earlier reports of affinity enhancement obtained with a tenascin-GoLoco fusion FP probe (7) and, for G␣ i1 E245L , mutation in the context of other affinitymodifying changes (27). Consistent with the selective binding of GoLoco motifs to ground-state G␣ i subunits (i.e. G␣ i ⅐GDP) (reviewed in Ref. 16), the transition-state mimetic form (i.e. FEBRUARY 4, 2011 • VOLUME 286 • NUMBER 5
These affinity-enhancing mutations of G␣ i1 were computationally predicted based on the known structure of the WT G␣ i1 /RGS14 GoLoco motif heterodimer (7,27). To determine whether these mutations differentially affected binding to other known GoLoco motifs, we performed similar FP assays with GoLoco motifs from RGS12 and GPSM2 (the latter protein is also known as LGN or mammalian Pins). Both G␣ i1 E116L ⅐GDP and G␣ i1 E245L ⅐GDP had enhanced affinities toward the RGS12 and GPSM2 GoLoco motif FP probes, whereas the K D values obtained for G␣ i1 Q147L ⅐GDP were statistically indistinguishable from those for G␣ i1 WT ⅐GDP for both probes (Fig. 2).
Assessment of Nucleotide Binding and Hydrolysis Properties of the Affinity-enhanced G␣ i1 Mutants-Using our 2.2-Å crystal structure of the WT G␣ i1 /RGS14 GoLoco motif interface (Protein Data Bank code 2OM2 (7)), we employed the Rosetta program to predict mutations with the goal of increasing GoLoco motif binding affinity while maintaining other G␣ functionality. To determine whether the G␣ i1 mutants retain their ability to bind GTP, we performed radionucleotidebinding assays with [ 35 S]GTP␥S. Whereas all three G␣ i1 mutants were capable of binding GTP␥S, G␣ i1 E245L had a noticeably slower rate of binding (k obs ), 0.04 min Ϫ1 compared with  , respectively (Fig. 4A). To determine any effects on the intrinsic GTP hydrolysis rate, we also performed single-turnover [␥-32 P]GTP hydrolysis assays (Fig. 4B). G␣ i1 WT , G␣ i1 E116L , and G␣ i1 Q147L were observed to have similar k cat values of 0.48, 0.58, and 0.65 min Ϫ1 , respectively, whereas

Basis of Enhanced Affinity of G␣ i1 Mutants for GoLoco Motifs
E245L had a reduced rate of GTP hydrolysis of 0.15 min Ϫ1 (95% confidence interval (CI), 0.12-0.17). Despite the rate changes observed with G␣ i1 E245L , all four G␣ i1 subunits demonstrated identical rates of activation by aluminum tetrafluoride, as measured by increased Trp-211 intrinsic fluorescence (supplemental Fig. S3). However, the reduced rates of GTP binding and hydrolysis exhibited by G␣ i1 E245L led us to exclude this particular mutant from further pursuit.
Assessment of Thermodynamic Binding Parameters of the Affinity-enhanced G␣ i1 Q147L Mutant-Rosetta makes predictions of changes in Gibbs free energy (⌬G) upon mutation of single residues, but because there is no explicit entropy term provided as output from the program, one cannot accurately predict whether affinity enhancement arises from favorable enthalpic (⌬H) or entropic (⌬S) thermodynamic changes (40).

Structural Determinants of the G␣ i1
Q147L Mutant Bound to the RGS14 GoLoco Motif-To elucidate the structural determinants of GoLoco affinity enhancement for G␣ i1 Q147L , we obtained x-ray diffraction data to 2.38-Å resolution from a single crystal of the G␣ i1 Q147L ⅐GDP/RGS14 GoLoco motif peptide complex. A structural model was obtained by molecular replacement using the model of the WT G␣ i1 ⅐GDP/RGS14 complex  (Protein Data Bank code 2OM2) as a search model and subsequently refined (supplemental Table S1). The overall conformations of the G␣ i1 Q147L subunit and bound GoLoco motif peptide were essentially identical (root mean square deviation of 0.38 Å, 2545 atoms used in comparison) (supplemental Fig. S1) to those observed in the WT crystal structure (7). The N-terminal helix of the RGS14 GoLoco motif contacts the Ras-like domain of G␣ i1 Q147L in a manner similar to the WT structure (Protein Data Bank code 2OM2 (7)), whereas the N-terminal helix of a WT structure reported earlier (code 1KJY (13)) is shifted by one halfturn. The conserved (E/D)QR GoLoco triad (Asp-514, Gln-515, Arg-516) loops across the nucleotide-binding pocket, positioning the Arg-516 side chain to contact the ␣and ␤-phosphate groups of GDP, and thus inhibits nucleotide dissociation (13). However, very weak electron density was observed for the preceding three GoLoco motif residues Gly-511, Ala-512, and His-513 that surround a well defined leucine side chain on G␣ i1 at position 147 (i.e. site of the Q147L substitution) (Fig. 6). Thus, the placement of a hydrophobic leucine on the surface of G␣ i1 appears to lead to disorder in the surrounding GoLoco motif peptide region. These findings suggest an increased conformational flexibility of the RGS14 GoLoco motif peptide in the region of amino acids 511-513 upon binding to G␣ i1 Q147L compared with WT G␣ i1 .

Modeling of the Q147L Substitution by Monte Carlo
Simulations-The G␣ i1 Q147L /GoLoco crystal structure suggest that residues 511-513 may be more dynamic in the mutant than in WT G␣ i1 , and therefore, an increase in conformational entropy may contribute to the enhanced affinity of G␣ i1 Q147L for GoLoco motifs. However, the ITC results show that the net change in the entropy component upon binding (Fig. 5) is more favorable for WT G␣ i1 than for G␣ i1 Q147L . In general, it is very difficult to predict relative changes in binding entropies and enthalpies because enthalpy/entropy compensation is a dominant feature of solvated systems (41). Changes in water networks in either the bound or unbound state could change the number of hydrogen bonds within the system and also change the rotational freedom of each water. Given our observation of disorder within the RGS14 GoLoco motif, we used Rosetta loop modeling simulations to examine the structural preferences of GoLoco motif residues 510 -513 when bound to either G␣ i1 WT or G␣ i1 Q147L . For each case, 100 independent structure prediction simulations were performed. With WT G␣ i1 , all 100 simulations predicted a loop conformation similar to that observed in the crystal structure (Fig. 7A). However, with G␣ i1 Q147L , two alternative conformations were observed for residues 510 -513 (Fig. 7B). In 40% of FIGURE 6. Differences in electron density observed within the GoLoco motif between WT and Q147L mutant G␣ i1 ⅐GDP/RGS14 GoLoco motif complexes. A crystal structure (using diffraction data to 2.38-Å resolution) of the G␣ i1 Q147L ⅐GDP/RGS14 GoLoco motif complex (Protein Data Bank code 3ONW) was obtained. Overall, the G␣ i1 Q147L protein binds the RGS14 GoLoco motif protein similarly (root mean square deviation of 0.38 Å) to G␣ i1 WT (code 2OM2 (7)). However, electron density for the GoLoco motif peptide residues Gly-511, Ala-512, and His-513 is much weaker in the G␣ i1 Q147L structure (B) than in the WT structure (code 2OM2) (A), indicating disorder in the peptide surrounding the mutated G␣ i1 residue Leu-147. Both 2F o Ϫ F c electron density maps shown are contoured at ϭ 1.5. the models, residues 510 -513 adopt a conformation similar to the WT structure; in the other 60%, residues 511 and 512 are displaced by Ͼ4 Å. The alternate conformation places the backbone groups adjacent to both methyl groups on the G␣ i1 residue Leu-147. The solvation energy term in Rosetta disfavors the alternate conformation for G␣ i1 WT because both the nitrogen and oxygen atoms of Gln-147 are desolvated without the formation of a compensating hydrogen bond. These results are consistent with the poor electron density observed for residues 511-513 in the GoLoco/G␣ i1 Q147L crystal structure. Conclusions-Our findings confirm the ability of Rosetta to predict point mutations that enhance protein/protein binding affinities, a technique that should be readily applicable to other systems with high-resolution structural information available (7). Furthermore, our findings have provided insight into the kinetic, thermodynamic, and structural mechanisms underlying the favorable accommodation of affinity-enhancing mutations. Two of the three G␣ i1 mutants studied (G␣ i1 Q147L and G␣ i1 E116L ) exhibit enhanced binding to GoLoco motifs without disrupting guanine nucleotide binding or hydrolysis functions (an important consideration for their utility in examining selective G␣ activities in an integrated cellular context). The selective loss of GoLoco motif binding of the G␣ i1 N149I point mutant was instrumental in establishing direct involvement of a G␣ i /GoLoco motif complex in microtubule dynamics underlying spindle orientation and chromosomal segregation during cell division (25). Other groups have since employed the G␣ i1 N149I mutant to demonstrate that cell polarization-mediated spatial restriction of spindle orientation determinants is critical for epithelial morphogenesis (26) and that the multiple-GoLoco motif protein AGS4 may couple G␣ subunits to receptor-proximal events affected by agonist application (42). The verified gain-of-function mutants G␣ i1 Q147L and G␣ i1 E116L described in this work should complement the loss-of-function G␣ i1 N149I mutant and find equal utility in investigating the role of G␣/GoLoco motif interactions in asymmetric cell division, epithelial progenitor differentiation, and G-protein-coupled receptor signaling regulation.