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David P Siderovski - One of the best experts on this subject based on the ideXlab platform.
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A role for Regulator of G protein Signaling-12 (RGS12) in the balance between myoblast proliferation and differentiation.
PloS one, 2019Co-Authors: Adam B. Schroer, Melinda D. Willard, Emily A. Oestreich, Junaith S. Mohamed, Vincent Setola, David P SiderovskiAbstract:Regulators of G Protein Signaling (RGS proteins) inhibit G protein-coupled receptor (GPCR) signaling by accelerating the GTP hydrolysis rate of activated Gα subunits. Some RGS proteins exert additional signal modulatory functions, and RGS12 is one such protein, with five additional, functional domains: a PDZ domain, a phosphotyrosine-binding domain, two Ras-binding domains, and a Gα·GDP-binding GoLoco Motif. RGS12 expression is temporospatially regulated in developing mouse embryos, with notable expression in somites and developing skeletal muscle. We therefore examined whether RGS12 is involved in the skeletal muscle myogenic program. In the adult mouse, RGS12 is expressed in the tibialis anterior (TA) muscle, and its expression is increased early after cardiotoxin-induced injury, suggesting a role in muscle regeneration. Consistent with a potential role in coordinating myogenic signals, RGS12 is also expressed in primary myoblasts; as these cells undergo differentiation and fusion into myotubes, RGS12 protein abundance is reduced. Myoblasts isolated from mice lacking Rgs12 expression have an impaired ability to differentiate into myotubes ex vivo, suggesting that RGS12 may play a role as a modulator/switch for differentiation. We also assessed the muscle regenerative capacity of mice conditionally deficient in skeletal muscle Rgs12 expression (via Pax7-driven Cre recombinase expression), following cardiotoxin-induced damage to the TA muscle. Eight days post-damage, mice lacking RGS12 in skeletal muscle had attenuated repair of muscle fibers. However, when mice lacking skeletal muscle expression of Rgs12 were cross-bred with mdx mice (a model of human Duchenne muscular dystrophy), no increase in muscle degeneration was observed over time. These data support the hypothesis that RGS12 plays a role in coordinating signals during the myogenic program in select circumstances, but loss of the protein may be compensated for within model syndromes of prolonged bouts of muscle damage and repair.
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The G42R point mutation prevents Gαi1 from assuming the activated conformation.
2012Co-Authors: Dustin E Bosch, Francis S. Willard, Adam J. Kimple, Melinda D. Willard, Ravikrishna Ramanujam, Naweed I. Naqvi, David P SiderovskiAbstract:Upon binding GDP·AlF4−, the switch regions of Gαi1 undergo a conformational change, burying the switch 2 Trp-211 in a hydrophobic cleft [5]. As a result, the intrinsic tryptophan fluorescence of Gαi1 increases, and the activated switch conformation is protected from trypsin proteolysis, relative to the GDP-bound state. (A) The intrinsic tryptophan fluorescence of wild type Gαi1 increased upon injection of AlF4−, while the response of Gαi1(G42R) was blunted. (B) Gαi1 was relatively resistant to trypsin proteolysis upon loading with either GDP·AlF4− or GTPγS. In contrast, Gαi1(G42R) was efficiently proteolyzed in any nucleotide state. (C) The Gαi1(G42R)·GDP/RGS14 GoLoco crystal structure model of this study (PDB 3QI2) is shown in cyan with the Arg-42 side chain in magenta sticks. GDP and magnesium are represented as green sticks and an orange sphere, respectively. The GoLoco Motif peptide is excluded for clarity. For a complete model, see Figure S2. (D) The activated, GTPγS-bound form of wild type Gαi1 (PDB 1GIA) is shown in gray. Upon binding to the GTP analog, the switch regions (SI-III) of wild type Gαi1 converge on the phosphoryl groups of the nucleotide, resulting in a conformation recognized by effector molecules. However, the mutant Arg-42 side chain extending from the P-loop (superposed in magenta) is not sterically accommodated in a wild type-like activation state; switch 3 residues Leu-234 and Glu-236 would clash with the mutant residue. Thus, Arg-42 does not allow Gαi1(G42R) to assume a typical active conformation, although the critical residues Glu-204 and Arg-178 apparently can be positioned for efficient GTP hydrolysis (see Fig. 2).
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Gαi1(G42R) engages inactive conformation-selective binding partners in two nucleotide states.
2012Co-Authors: Dustin E Bosch, Francis S. Willard, Adam J. Kimple, Melinda D. Willard, Ravikrishna Ramanujam, Naweed I. Naqvi, David P SiderovskiAbstract:(A) Wild type Gαi1 binds Gβ1γ1 only in the GDP-bound state, as determined by SPR, while Gαi1(G42R) displayed no nucleotide state-selectivity of Gβ1γ1 binding when liganded with either GDP or GDP·AlF4−. (B) Similarly, fluorescence polarization experiments showed highly nucleotide state-selective binding of the RGS14 GoLoco Motif to wild-type Gαi1·GDP (KD = 9.0±1.1 nM (s.e.m.)) compared to the AlF4−-bound form (KD = 8.7±1.0 µM (s.e.m.)), but both nucleotide states of Gαi1(G42R) interacted with the GoLoco Motif peptide, with affinity constants of 45±7 nM (s.e.m.) and 168±27 nM (s.e.m.) for GDP and AlF4−, respectively. (C) The activated state-selective peptide KB-1753 preferentially bound the AlF4−-bound form of wild-type Gαi1 (KD = 470±40 nM (s.e.m.)) compared to the GDP-bound form (KD = 6.7±0.4 µM (s.e.m.)), but had low affinity for Gαi1(G42R) in both nucleotide states.
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A high throughput fluorescence polarization assay for inhibitors of the GoLoco Motif/G-alpha interaction
Combinatorial chemistry & high throughput screening, 2008Co-Authors: Adam J. Kimple, Francis S. Willard, Adam Yasgar, Mark A. Hughes, Ajit Jadhav, Robin E. Muller, Christopher P. Austin, James Inglese, Gordon C. Ibeanu, David P SiderovskiAbstract:The GoLoco Motif is a short Galpha-binding polypeptide sequence. It is often found in proteins that regulate cell-surface receptor signaling, such as RGS12, as well as in proteins that regulate mitotic spindle orientation and force generation during cell division, such as GPSM2/LGN. Here, we describe a high throughput fluorescence polarization (FP) assay using fluorophore-labeled GoLoco Motif peptides for identifying inhibitors of the GoLoco Motif interaction with the G-protein alpha subunit Galpha (i1). The assay exhibits considerable stability over time and is tolerant to DMSO up to 5%. The Z'-factors for robustness of the GPSM2 and RGS12 GoLoco Motif assays in a 96-well plate format were determined to be 0.81 and 0.84, respectively; the latter assay was run in a 384-well plate format and produced a Z'-factor of 0.80. To determine the screening factor window (Z-factor) of the RGS12 GoLoco Motif screen using a small molecule library, the NCI Diversity Set was screened. The Z-factor was determined to be 0.66, suggesting that this FP assay would perform well when developed for 1,536-well format and scaled up to larger libraries. We then miniaturized to a 4 microL final volume a pair of FP assays utilizing fluorescein- (green) and rhodamine- (red) labeled RGS12 GoLoco Motif peptides. In a fully-automated run, the Sigma-Aldrich LOPAC(1280) collection was screened three times with every library compound being tested over a range of concentrations following the quantitative high throughput screening (qHTS) paradigm; excellent assay performance was noted with average Z-factors of 0.84 and 0.66 for the green- and red-label assays, respectively.
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structure based protocol for identifying mutations that enhance protein protein binding affinities
Journal of Molecular Biology, 2007Co-Authors: Deanne W. Sammond, David P Siderovski, Randall J Kimple, Ziad M. Eletr, Carrie Purbeck, Brian KuhlmanAbstract:The ability to manipulate protein binding affinities is important for the development of proteins as biosensors, industrial reagents, and therapeutics. We have developed a structure-based method to rationally predict single mutations at protein-protein interfaces that enhance binding affinities. The protocol is based on the premise that increasing buried hydrophobic surface area and/or reducing buried hydrophilic surface area will generally lead to enhanced affinity if large steric clashes are not introduced and buried polar groups are not left without a hydrogen bond partner. The procedure selects affinity enhancing point mutations at the protein-protein interface using three criteria: 1) the mutation must be from a polar amino acid to a non-polar amino acid or from a non-polar amino acid to a larger non-polar amino acid, 2) the free energy of binding as calculated with the Rosetta protein modeling program should be more favorable than the free energy of binding calculated for the wild type complex and 3) the mutation should not be predicted to significantly destabilize the monomers. The Rosetta energy function emphasizes short-range interactions: steric repulsion, Van der Waals forces, hydrogen bonding, and an implicit solvation model that penalizes placing atoms adjacent to polar groups. The performance of the computational protocol was experimentally tested on two separate protein complexes; Gαi1 from the heterotrimeric G-protein system bound to the RGS14 GoLoco Motif, and the E2, UbcH7, bound to the E3, E6AP from the ubiquitin pathway. 12 single-site mutations that were predicted to be stabilizing were synthesized and characterized in the laboratory. 9 of the 12 mutations successfully increased binding affinity with 5 of these increasing binding by over 1.0 kcal/mol. To further assess our approach we searched the literature for point mutations that pass our criteria and have experimentally determined binding affinities. Of the 8 mutations identified, 5 were accurately predicted to increase binding affinity, further validating the method as a useful tool to increase protein-protein binding affinities.
Francis S. Willard - One of the best experts on this subject based on the ideXlab platform.
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Letter to the editor Does WAVE1 contain a GoLoco/GPR Motif?
2013Co-Authors: Francis S. WillardAbstract:Heterotrimeric G-protein alpha subunits (Gα) are molecular switches regulated by the binding and hydrolysis of guanosine 5-triphosphate [1]. Nonreceptor proteins that modulate the nucleotide binding and hydrolysis activities of Gα proteins have recently become of considerable interest [2, 3]. One class of Gα modulating proteins is defined by the presence of a GoLoco Motif(s) in their primary sequence [4]. The GoLoco Motif was discovered as a region of sequence homology within novel Gαbinding proteins [5-7], and is also referred to as the G-protein regulatory (GPR) Motif [5]. The GoLoco Motif is a peptide of 20-40 amino acids that binds Gαi/o⋅GDP and prevents the spontaneous release of GDP by Gα, thus acting as a guanine nucleotide dissociatio
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Gαi1(G42R) engages inactive conformation-selective binding partners in two nucleotide states.
2012Co-Authors: Dustin E Bosch, Francis S. Willard, Adam J. Kimple, Melinda D. Willard, Ravikrishna Ramanujam, Naweed I. Naqvi, David P SiderovskiAbstract:(A) Wild type Gαi1 binds Gβ1γ1 only in the GDP-bound state, as determined by SPR, while Gαi1(G42R) displayed no nucleotide state-selectivity of Gβ1γ1 binding when liganded with either GDP or GDP·AlF4−. (B) Similarly, fluorescence polarization experiments showed highly nucleotide state-selective binding of the RGS14 GoLoco Motif to wild-type Gαi1·GDP (KD = 9.0±1.1 nM (s.e.m.)) compared to the AlF4−-bound form (KD = 8.7±1.0 µM (s.e.m.)), but both nucleotide states of Gαi1(G42R) interacted with the GoLoco Motif peptide, with affinity constants of 45±7 nM (s.e.m.) and 168±27 nM (s.e.m.) for GDP and AlF4−, respectively. (C) The activated state-selective peptide KB-1753 preferentially bound the AlF4−-bound form of wild-type Gαi1 (KD = 470±40 nM (s.e.m.)) compared to the GDP-bound form (KD = 6.7±0.4 µM (s.e.m.)), but had low affinity for Gαi1(G42R) in both nucleotide states.
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The G42R point mutation prevents Gαi1 from assuming the activated conformation.
2012Co-Authors: Dustin E Bosch, Francis S. Willard, Adam J. Kimple, Melinda D. Willard, Ravikrishna Ramanujam, Naweed I. Naqvi, David P SiderovskiAbstract:Upon binding GDP·AlF4−, the switch regions of Gαi1 undergo a conformational change, burying the switch 2 Trp-211 in a hydrophobic cleft [5]. As a result, the intrinsic tryptophan fluorescence of Gαi1 increases, and the activated switch conformation is protected from trypsin proteolysis, relative to the GDP-bound state. (A) The intrinsic tryptophan fluorescence of wild type Gαi1 increased upon injection of AlF4−, while the response of Gαi1(G42R) was blunted. (B) Gαi1 was relatively resistant to trypsin proteolysis upon loading with either GDP·AlF4− or GTPγS. In contrast, Gαi1(G42R) was efficiently proteolyzed in any nucleotide state. (C) The Gαi1(G42R)·GDP/RGS14 GoLoco crystal structure model of this study (PDB 3QI2) is shown in cyan with the Arg-42 side chain in magenta sticks. GDP and magnesium are represented as green sticks and an orange sphere, respectively. The GoLoco Motif peptide is excluded for clarity. For a complete model, see Figure S2. (D) The activated, GTPγS-bound form of wild type Gαi1 (PDB 1GIA) is shown in gray. Upon binding to the GTP analog, the switch regions (SI-III) of wild type Gαi1 converge on the phosphoryl groups of the nucleotide, resulting in a conformation recognized by effector molecules. However, the mutant Arg-42 side chain extending from the P-loop (superposed in magenta) is not sterically accommodated in a wild type-like activation state; switch 3 residues Leu-234 and Glu-236 would clash with the mutant residue. Thus, Arg-42 does not allow Gαi1(G42R) to assume a typical active conformation, although the critical residues Glu-204 and Arg-178 apparently can be positioned for efficient GTP hydrolysis (see Fig. 2).
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a point mutation to gαi selectively blocks GoLoco Motif binding direct evidence for gα GoLoco complexes in mitotic spindle dynamics
Journal of Biological Chemistry, 2008Co-Authors: Francis S. Willard, Adam J. Kimple, Melinda D. Willard, Zhen Zheng, Juan Guo, Gregory J Digby, Jason M Conley, Christopher A Johnston, Dustin E Bosch, Val J WattsAbstract:Heterotrimeric G-protein Gα subunits and GoLoco Motif proteins are key members of a conserved set of regulatory proteins that influence invertebrate asymmetric cell division and vertebrate neuroepithelium and epithelial progenitor differentiation. GoLoco Motif proteins bind selectively to the inhibitory subclass (Gαi) of Gα subunits, and thus it is assumed that a Gαi·GoLoco Motif protein complex plays a direct functional role in microtubule dynamics underlying spindle orientation and metaphase chromosomal segregation during cell division. To address this hypothesis directly, we rationally identified a point mutation to Gαi subunits that renders a selective loss-of-function for GoLoco Motif binding, namely an asparagine-to-isoleucine substitution in the αD-αE loop of the Gα helical domain. This GoLoco-insensitivity (“GLi”) mutation prevented Gαi1 association with all human GoLoco Motif proteins and abrogated interaction between the Caenorhabditis elegans Gα subunit GOA-1 and the GPR-1 GoLoco Motif. In contrast, the GLi mutation did not perturb any other biochemical or signaling properties of Gαi subunits, including nucleotide binding, intrinsic and RGS protein-accelerated GTP hydrolysis, and interactions with Gβγ dimers, adenylyl cyclase, and seven transmembrane-domain receptors. GoLoco insensitivity rendered Gαi subunits unable to recruit GoLoco Motif proteins such as GPSM2/LGN and GPSM3 to the plasma membrane, and abrogated the exaggerated mitotic spindle rocking normally seen upon ectopic expression of wild type Gαi subunits in kidney epithelial cells. This GLi mutation should prove valuable in establishing the physiological roles of Gαi·GoLoco Motif protein complexes in microtubule dynamics and spindle function during cell division as well as to delineate potential roles for GoLoco Motifs in receptor-mediated signal transduction.
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A high throughput fluorescence polarization assay for inhibitors of the GoLoco Motif/G-alpha interaction
Combinatorial chemistry & high throughput screening, 2008Co-Authors: Adam J. Kimple, Francis S. Willard, Adam Yasgar, Mark A. Hughes, Ajit Jadhav, Robin E. Muller, Christopher P. Austin, James Inglese, Gordon C. Ibeanu, David P SiderovskiAbstract:The GoLoco Motif is a short Galpha-binding polypeptide sequence. It is often found in proteins that regulate cell-surface receptor signaling, such as RGS12, as well as in proteins that regulate mitotic spindle orientation and force generation during cell division, such as GPSM2/LGN. Here, we describe a high throughput fluorescence polarization (FP) assay using fluorophore-labeled GoLoco Motif peptides for identifying inhibitors of the GoLoco Motif interaction with the G-protein alpha subunit Galpha (i1). The assay exhibits considerable stability over time and is tolerant to DMSO up to 5%. The Z'-factors for robustness of the GPSM2 and RGS12 GoLoco Motif assays in a 96-well plate format were determined to be 0.81 and 0.84, respectively; the latter assay was run in a 384-well plate format and produced a Z'-factor of 0.80. To determine the screening factor window (Z-factor) of the RGS12 GoLoco Motif screen using a small molecule library, the NCI Diversity Set was screened. The Z-factor was determined to be 0.66, suggesting that this FP assay would perform well when developed for 1,536-well format and scaled up to larger libraries. We then miniaturized to a 4 microL final volume a pair of FP assays utilizing fluorescein- (green) and rhodamine- (red) labeled RGS12 GoLoco Motif peptides. In a fully-automated run, the Sigma-Aldrich LOPAC(1280) collection was screened three times with every library compound being tested over a range of concentrations following the quantitative high throughput screening (qHTS) paradigm; excellent assay performance was noted with average Z-factors of 0.84 and 0.66 for the green- and red-label assays, respectively.
Randall J Kimple - One of the best experts on this subject based on the ideXlab platform.
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structure based protocol for identifying mutations that enhance protein protein binding affinities
Journal of Molecular Biology, 2007Co-Authors: Deanne W. Sammond, David P Siderovski, Randall J Kimple, Ziad M. Eletr, Carrie Purbeck, Brian KuhlmanAbstract:The ability to manipulate protein binding affinities is important for the development of proteins as biosensors, industrial reagents, and therapeutics. We have developed a structure-based method to rationally predict single mutations at protein-protein interfaces that enhance binding affinities. The protocol is based on the premise that increasing buried hydrophobic surface area and/or reducing buried hydrophilic surface area will generally lead to enhanced affinity if large steric clashes are not introduced and buried polar groups are not left without a hydrogen bond partner. The procedure selects affinity enhancing point mutations at the protein-protein interface using three criteria: 1) the mutation must be from a polar amino acid to a non-polar amino acid or from a non-polar amino acid to a larger non-polar amino acid, 2) the free energy of binding as calculated with the Rosetta protein modeling program should be more favorable than the free energy of binding calculated for the wild type complex and 3) the mutation should not be predicted to significantly destabilize the monomers. The Rosetta energy function emphasizes short-range interactions: steric repulsion, Van der Waals forces, hydrogen bonding, and an implicit solvation model that penalizes placing atoms adjacent to polar groups. The performance of the computational protocol was experimentally tested on two separate protein complexes; Gαi1 from the heterotrimeric G-protein system bound to the RGS14 GoLoco Motif, and the E2, UbcH7, bound to the E3, E6AP from the ubiquitin pathway. 12 single-site mutations that were predicted to be stabilizing were synthesized and characterized in the laboratory. 9 of the 12 mutations successfully increased binding affinity with 5 of these increasing binding by over 1.0 kcal/mol. To further assess our approach we searched the literature for point mutations that pass our criteria and have experimentally determined binding affinities. Of the 8 mutations identified, 5 were accurately predicted to increase binding affinity, further validating the method as a useful tool to increase protein-protein binding affinities.
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Structure-based Protocol for Identifying Mutations that Enhance Protein–Protein Binding Affinities
Journal of molecular biology, 2007Co-Authors: Deanne W. Sammond, David P Siderovski, Randall J Kimple, Ziad M. Eletr, Carrie Purbeck, Brian KuhlmanAbstract:The ability to manipulate protein binding affinities is important for the development of proteins as biosensors, industrial reagents, and therapeutics. We have developed a structure-based method to rationally predict single mutations at protein-protein interfaces that enhance binding affinities. The protocol is based on the premise that increasing buried hydrophobic surface area and/or reducing buried hydrophilic surface area will generally lead to enhanced affinity if large steric clashes are not introduced and buried polar groups are not left without a hydrogen bond partner. The procedure selects affinity enhancing point mutations at the protein-protein interface using three criteria: 1) the mutation must be from a polar amino acid to a non-polar amino acid or from a non-polar amino acid to a larger non-polar amino acid, 2) the free energy of binding as calculated with the Rosetta protein modeling program should be more favorable than the free energy of binding calculated for the wild type complex and 3) the mutation should not be predicted to significantly destabilize the monomers. The Rosetta energy function emphasizes short-range interactions: steric repulsion, Van der Waals forces, hydrogen bonding, and an implicit solvation model that penalizes placing atoms adjacent to polar groups. The performance of the computational protocol was experimentally tested on two separate protein complexes; Gαi1 from the heterotrimeric G-protein system bound to the RGS14 GoLoco Motif, and the E2, UbcH7, bound to the E3, E6AP from the ubiquitin pathway. 12 single-site mutations that were predicted to be stabilizing were synthesized and characterized in the laboratory. 9 of the 12 mutations successfully increased binding affinity with 5 of these increasing binding by over 1.0 kcal/mol. To further assess our approach we searched the literature for point mutations that pass our criteria and have experimentally determined binding affinities. Of the 8 mutations identified, 5 were accurately predicted to increase binding affinity, further validating the method as a useful tool to increase protein-protein binding affinities.
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gα selectivity and inhibitor function of the multiple GoLoco Motif protein gpsm2 lgn
Biochimica et Biophysica Acta, 2005Co-Authors: Christopher R Mccudden, Randall J Kimple, Melinda D. Hains, Francis S. Willard, Christopher A Johnston, Miller B Jones, David P SiderovskiAbstract:Abstract GPSM2 (G-protein signalling modulator 2; also known as LGN or mammalian Pins) is a protein that regulates mitotic spindle organization and cell division. GPSM2 contains seven tetratricopeptide repeats (TPR) and four Gαi/o–Loco (GoLoco) Motifs. GPSM2 has guanine nucleotide dissociation inhibitor (GDI) activity towards both Gαo- and Gαi-subunits; however, a systematic analysis of its individual GoLoco Motifs has not been described. We analyzed each of the four individual GoLoco Motifs from GPSM2, assessing their relative binding affinities and GDI potencies for Gαi1, Gαi2, and Gαi3 and Gαo. Each of the four GPSM2 GoLoco Motifs (36–43 amino acids in length) was expressed in bacteria as a GST-fusion protein and purified to homogeneity. The binding of each of the four GST–GoLoco Motifs to Gαi1-, Gαo-, and Gαs-subunits was assessed by surface plasmon resonance; all of the Motifs bound Gαi1, but exhibited low affinity towards Gαo. GDI activity was assessed by a fluorescence-based nucleotide-binding assay, revealing that all four GoLoco Motifs are functional as GDIs for Gαi1, Gαi2, and Gαi3. Consistent with our binding studies, the GDI activity of GPSM2 GoLoco Motifs on Gαo was significantly lower than that toward Gαi1, suggesting that the in vivo targets of GPSM2 are most likely to be Gαi-subunits.
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d2 dopamine receptor activation of potassium channels is selectively decoupled by gαi specific GoLoco Motif peptides
Journal of Neurochemistry, 2005Co-Authors: David P Siderovski, Randall J Kimple, Francis S. Willard, Christina K Webb, Christopher R Mccudden, Gerry Stephen OxfordAbstract:The GoLoco Motif is a short polypeptide sequence found in G-protein signaling regulators such as regulator of G-protein signaling proteins type 12 and 14 and activator of G-protein signaling protein type 3. A unique property of the GoLoco Motifs from these three proteins is their preferential interaction with guanosine diphosphate (GDP)-bound Gαi1, Gαi3 and, sometimes, Gαi2 subunits over Gαo subunits. This interaction prevents both spontaneous guanine nucleotide release and reassociation of Gαi-GDP with Gβγ. We utilized this property of the GoLoco Motif to examine dopamine (D2 and D3) and somatostatin receptor coupling to G-protein-regulated inwardly rectifying potassium (GIRK) channels in mouse AtT20 cells. GoLoco Motif peptides had no effect on either basal channel activity or the initial responses to agonists, suggesting that the GoLoco Motif cannot disrupt pre-formed G-protein heterotrimers. GoLoco Motif peptides did, however, interfere with human D2(short) receptor coupling to GIRK channels as demonstrated by the progressively diminished responses after repeated agonist application. This behavior is consistent with some form of compartmentalization of D2 receptors and GIRK channels such that Gβγ subunits, freed by local receptor activation and prevented from reforming a heterotrimeric complex, are not functionally constrained within the receptor–channel complex and thus are unable to exert a persistent activating effect. In contrast, GoLoco Motif peptides had no effect on either D3 or somatostatin coupling to GIRK channels. Our results suggest that GoLoco Motif-based peptides will be useful tools in examining the specificity of G-protein-coupled receptor–effector coupling.
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guanine nucleotide dissociation inhibitor activity of the triple GoLoco Motif protein g18 alanine to aspartate mutation restores function to an inactive second GoLoco Motif
Biochemical Journal, 2004Co-Authors: Randall J Kimple, Melinda D. Hains, Francis S. Willard, Miller B Jones, Gift K Nweke, David P SiderovskiAbstract:GoLoco ('Galpha(i/o)-Loco' interaction) Motif proteins have recently been identified as novel GDIs (guanine nucleotide dissociation inhibitors) for heterotrimeric G-protein alpha subunits. G18 is a member of the mammalian GoLoco-Motif gene family and was uncovered by analyses of human and mouse genomes for anonymous open-reading frames. The encoded G18 polypeptide is predicted to contain three 19-amino-acid GoLoco Motifs, which have been shown in other proteins to bind Galpha subunits and inhibit spontaneous nucleotide release. However, the G18 protein has thus far not been characterized biochemically. Here, we have cloned and expressed the G18 protein and assessed its ability to act as a GDI. G18 is capable of simultaneously binding more than one Galpha(i1) subunit. In binding assays with the non-hydrolysable GTP analogue guanosine 5'-[gamma-thio]triphosphate, G18 exhibits GDI activity, slowing the exchange of GDP for GTP by Galpha(i1). Only the first and third GoLoco Motifs within G18 are capable of interacting with Galpha subunits, and these bind with low micromolar affinity only to Galpha(i1) in the GDP-bound form, and not to Galpha(o), Galpha(q), Galpha(s) or Galpha12. Mutation of Ala-121 to aspartate in the inactive second GoLoco Motif of G18, to restore the signature acidic-glutamine-arginine tripeptide that forms critical contacts with Galpha and its bound nucleotide [Kimple, Kimple, Betts, Sondek and Siderovski (2002) Nature (London) 416, 878-881], results in gain-of-function with respect to Galpha binding and GDI activity.
Brian Kuhlman - One of the best experts on this subject based on the ideXlab platform.
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structure based protocol for identifying mutations that enhance protein protein binding affinities
Journal of Molecular Biology, 2007Co-Authors: Deanne W. Sammond, David P Siderovski, Randall J Kimple, Ziad M. Eletr, Carrie Purbeck, Brian KuhlmanAbstract:The ability to manipulate protein binding affinities is important for the development of proteins as biosensors, industrial reagents, and therapeutics. We have developed a structure-based method to rationally predict single mutations at protein-protein interfaces that enhance binding affinities. The protocol is based on the premise that increasing buried hydrophobic surface area and/or reducing buried hydrophilic surface area will generally lead to enhanced affinity if large steric clashes are not introduced and buried polar groups are not left without a hydrogen bond partner. The procedure selects affinity enhancing point mutations at the protein-protein interface using three criteria: 1) the mutation must be from a polar amino acid to a non-polar amino acid or from a non-polar amino acid to a larger non-polar amino acid, 2) the free energy of binding as calculated with the Rosetta protein modeling program should be more favorable than the free energy of binding calculated for the wild type complex and 3) the mutation should not be predicted to significantly destabilize the monomers. The Rosetta energy function emphasizes short-range interactions: steric repulsion, Van der Waals forces, hydrogen bonding, and an implicit solvation model that penalizes placing atoms adjacent to polar groups. The performance of the computational protocol was experimentally tested on two separate protein complexes; Gαi1 from the heterotrimeric G-protein system bound to the RGS14 GoLoco Motif, and the E2, UbcH7, bound to the E3, E6AP from the ubiquitin pathway. 12 single-site mutations that were predicted to be stabilizing were synthesized and characterized in the laboratory. 9 of the 12 mutations successfully increased binding affinity with 5 of these increasing binding by over 1.0 kcal/mol. To further assess our approach we searched the literature for point mutations that pass our criteria and have experimentally determined binding affinities. Of the 8 mutations identified, 5 were accurately predicted to increase binding affinity, further validating the method as a useful tool to increase protein-protein binding affinities.
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Structure-based Protocol for Identifying Mutations that Enhance Protein–Protein Binding Affinities
Journal of molecular biology, 2007Co-Authors: Deanne W. Sammond, David P Siderovski, Randall J Kimple, Ziad M. Eletr, Carrie Purbeck, Brian KuhlmanAbstract:The ability to manipulate protein binding affinities is important for the development of proteins as biosensors, industrial reagents, and therapeutics. We have developed a structure-based method to rationally predict single mutations at protein-protein interfaces that enhance binding affinities. The protocol is based on the premise that increasing buried hydrophobic surface area and/or reducing buried hydrophilic surface area will generally lead to enhanced affinity if large steric clashes are not introduced and buried polar groups are not left without a hydrogen bond partner. The procedure selects affinity enhancing point mutations at the protein-protein interface using three criteria: 1) the mutation must be from a polar amino acid to a non-polar amino acid or from a non-polar amino acid to a larger non-polar amino acid, 2) the free energy of binding as calculated with the Rosetta protein modeling program should be more favorable than the free energy of binding calculated for the wild type complex and 3) the mutation should not be predicted to significantly destabilize the monomers. The Rosetta energy function emphasizes short-range interactions: steric repulsion, Van der Waals forces, hydrogen bonding, and an implicit solvation model that penalizes placing atoms adjacent to polar groups. The performance of the computational protocol was experimentally tested on two separate protein complexes; Gαi1 from the heterotrimeric G-protein system bound to the RGS14 GoLoco Motif, and the E2, UbcH7, bound to the E3, E6AP from the ubiquitin pathway. 12 single-site mutations that were predicted to be stabilizing were synthesized and characterized in the laboratory. 9 of the 12 mutations successfully increased binding affinity with 5 of these increasing binding by over 1.0 kcal/mol. To further assess our approach we searched the literature for point mutations that pass our criteria and have experimentally determined binding affinities. Of the 8 mutations identified, 5 were accurately predicted to increase binding affinity, further validating the method as a useful tool to increase protein-protein binding affinities.
Adam J. Kimple - One of the best experts on this subject based on the ideXlab platform.
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The G42R point mutation prevents Gαi1 from assuming the activated conformation.
2012Co-Authors: Dustin E Bosch, Francis S. Willard, Adam J. Kimple, Melinda D. Willard, Ravikrishna Ramanujam, Naweed I. Naqvi, David P SiderovskiAbstract:Upon binding GDP·AlF4−, the switch regions of Gαi1 undergo a conformational change, burying the switch 2 Trp-211 in a hydrophobic cleft [5]. As a result, the intrinsic tryptophan fluorescence of Gαi1 increases, and the activated switch conformation is protected from trypsin proteolysis, relative to the GDP-bound state. (A) The intrinsic tryptophan fluorescence of wild type Gαi1 increased upon injection of AlF4−, while the response of Gαi1(G42R) was blunted. (B) Gαi1 was relatively resistant to trypsin proteolysis upon loading with either GDP·AlF4− or GTPγS. In contrast, Gαi1(G42R) was efficiently proteolyzed in any nucleotide state. (C) The Gαi1(G42R)·GDP/RGS14 GoLoco crystal structure model of this study (PDB 3QI2) is shown in cyan with the Arg-42 side chain in magenta sticks. GDP and magnesium are represented as green sticks and an orange sphere, respectively. The GoLoco Motif peptide is excluded for clarity. For a complete model, see Figure S2. (D) The activated, GTPγS-bound form of wild type Gαi1 (PDB 1GIA) is shown in gray. Upon binding to the GTP analog, the switch regions (SI-III) of wild type Gαi1 converge on the phosphoryl groups of the nucleotide, resulting in a conformation recognized by effector molecules. However, the mutant Arg-42 side chain extending from the P-loop (superposed in magenta) is not sterically accommodated in a wild type-like activation state; switch 3 residues Leu-234 and Glu-236 would clash with the mutant residue. Thus, Arg-42 does not allow Gαi1(G42R) to assume a typical active conformation, although the critical residues Glu-204 and Arg-178 apparently can be positioned for efficient GTP hydrolysis (see Fig. 2).
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Gαi1(G42R) engages inactive conformation-selective binding partners in two nucleotide states.
2012Co-Authors: Dustin E Bosch, Francis S. Willard, Adam J. Kimple, Melinda D. Willard, Ravikrishna Ramanujam, Naweed I. Naqvi, David P SiderovskiAbstract:(A) Wild type Gαi1 binds Gβ1γ1 only in the GDP-bound state, as determined by SPR, while Gαi1(G42R) displayed no nucleotide state-selectivity of Gβ1γ1 binding when liganded with either GDP or GDP·AlF4−. (B) Similarly, fluorescence polarization experiments showed highly nucleotide state-selective binding of the RGS14 GoLoco Motif to wild-type Gαi1·GDP (KD = 9.0±1.1 nM (s.e.m.)) compared to the AlF4−-bound form (KD = 8.7±1.0 µM (s.e.m.)), but both nucleotide states of Gαi1(G42R) interacted with the GoLoco Motif peptide, with affinity constants of 45±7 nM (s.e.m.) and 168±27 nM (s.e.m.) for GDP and AlF4−, respectively. (C) The activated state-selective peptide KB-1753 preferentially bound the AlF4−-bound form of wild-type Gαi1 (KD = 470±40 nM (s.e.m.)) compared to the GDP-bound form (KD = 6.7±0.4 µM (s.e.m.)), but had low affinity for Gαi1(G42R) in both nucleotide states.
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a point mutation to gαi selectively blocks GoLoco Motif binding direct evidence for gα GoLoco complexes in mitotic spindle dynamics
Journal of Biological Chemistry, 2008Co-Authors: Francis S. Willard, Adam J. Kimple, Melinda D. Willard, Zhen Zheng, Juan Guo, Gregory J Digby, Jason M Conley, Christopher A Johnston, Dustin E Bosch, Val J WattsAbstract:Heterotrimeric G-protein Gα subunits and GoLoco Motif proteins are key members of a conserved set of regulatory proteins that influence invertebrate asymmetric cell division and vertebrate neuroepithelium and epithelial progenitor differentiation. GoLoco Motif proteins bind selectively to the inhibitory subclass (Gαi) of Gα subunits, and thus it is assumed that a Gαi·GoLoco Motif protein complex plays a direct functional role in microtubule dynamics underlying spindle orientation and metaphase chromosomal segregation during cell division. To address this hypothesis directly, we rationally identified a point mutation to Gαi subunits that renders a selective loss-of-function for GoLoco Motif binding, namely an asparagine-to-isoleucine substitution in the αD-αE loop of the Gα helical domain. This GoLoco-insensitivity (“GLi”) mutation prevented Gαi1 association with all human GoLoco Motif proteins and abrogated interaction between the Caenorhabditis elegans Gα subunit GOA-1 and the GPR-1 GoLoco Motif. In contrast, the GLi mutation did not perturb any other biochemical or signaling properties of Gαi subunits, including nucleotide binding, intrinsic and RGS protein-accelerated GTP hydrolysis, and interactions with Gβγ dimers, adenylyl cyclase, and seven transmembrane-domain receptors. GoLoco insensitivity rendered Gαi subunits unable to recruit GoLoco Motif proteins such as GPSM2/LGN and GPSM3 to the plasma membrane, and abrogated the exaggerated mitotic spindle rocking normally seen upon ectopic expression of wild type Gαi subunits in kidney epithelial cells. This GLi mutation should prove valuable in establishing the physiological roles of Gαi·GoLoco Motif protein complexes in microtubule dynamics and spindle function during cell division as well as to delineate potential roles for GoLoco Motifs in receptor-mediated signal transduction.
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A high throughput fluorescence polarization assay for inhibitors of the GoLoco Motif/G-alpha interaction
Combinatorial chemistry & high throughput screening, 2008Co-Authors: Adam J. Kimple, Francis S. Willard, Adam Yasgar, Mark A. Hughes, Ajit Jadhav, Robin E. Muller, Christopher P. Austin, James Inglese, Gordon C. Ibeanu, David P SiderovskiAbstract:The GoLoco Motif is a short Galpha-binding polypeptide sequence. It is often found in proteins that regulate cell-surface receptor signaling, such as RGS12, as well as in proteins that regulate mitotic spindle orientation and force generation during cell division, such as GPSM2/LGN. Here, we describe a high throughput fluorescence polarization (FP) assay using fluorophore-labeled GoLoco Motif peptides for identifying inhibitors of the GoLoco Motif interaction with the G-protein alpha subunit Galpha (i1). The assay exhibits considerable stability over time and is tolerant to DMSO up to 5%. The Z'-factors for robustness of the GPSM2 and RGS12 GoLoco Motif assays in a 96-well plate format were determined to be 0.81 and 0.84, respectively; the latter assay was run in a 384-well plate format and produced a Z'-factor of 0.80. To determine the screening factor window (Z-factor) of the RGS12 GoLoco Motif screen using a small molecule library, the NCI Diversity Set was screened. The Z-factor was determined to be 0.66, suggesting that this FP assay would perform well when developed for 1,536-well format and scaled up to larger libraries. We then miniaturized to a 4 microL final volume a pair of FP assays utilizing fluorescein- (green) and rhodamine- (red) labeled RGS12 GoLoco Motif peptides. In a fully-automated run, the Sigma-Aldrich LOPAC(1280) collection was screened three times with every library compound being tested over a range of concentrations following the quantitative high throughput screening (qHTS) paradigm; excellent assay performance was noted with average Z-factors of 0.84 and 0.66 for the green- and red-label assays, respectively.
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a high throughput fluorescence polarization assay for inhibitors of the GoLoco Motif g alpha interaction
Combinatorial Chemistry & High Throughput Screening, 2008Co-Authors: Adam J. Kimple, Francis S. Willard, Adam Yasgar, Mark A. Hughes, Ajit Jadhav, Robin E. Muller, Christopher P. Austin, James Inglese, Gordon C. IbeanuAbstract:The GoLoco Motif is a short Galpha-binding polypeptide sequence. It is often found in proteins that regulate cell-surface receptor signaling, such as RGS12, as well as in proteins that regulate mitotic spindle orientation and force generation during cell division, such as GPSM2/LGN. Here, we describe a high throughput fluorescence polarization (FP) assay using fluorophore-labeled GoLoco Motif peptides for identifying inhibitors of the GoLoco Motif interaction with the G-protein alpha subunit Galpha (i1). The assay exhibits considerable stability over time and is tolerant to DMSO up to 5%. The Z'-factors for robustness of the GPSM2 and RGS12 GoLoco Motif assays in a 96-well plate format were determined to be 0.81 and 0.84, respectively; the latter assay was run in a 384-well plate format and produced a Z'-factor of 0.80. To determine the screening factor window (Z-factor) of the RGS12 GoLoco Motif screen using a small molecule library, the NCI Diversity Set was screened. The Z-factor was determined to be 0.66, suggesting that this FP assay would perform well when developed for 1,536-well format and scaled up to larger libraries. We then miniaturized to a 4 microL final volume a pair of FP assays utilizing fluorescein- (green) and rhodamine- (red) labeled RGS12 GoLoco Motif peptides. In a fully-automated run, the Sigma-Aldrich LOPAC(1280) collection was screened three times with every library compound being tested over a range of concentrations following the quantitative high throughput screening (qHTS) paradigm; excellent assay performance was noted with average Z-factors of 0.84 and 0.66 for the green- and red-label assays, respectively.