A residue outside the binding site determines the Gα binding

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A Residue outside the Binding Site Determines the Gα Binding Specificity of GoLoco Motifs Chunhua Liu,† Jingwei Weng,† Dan Wang, Maohua Yang, Min Jia, and Wenning Wang* Multiscale Research Institute of Complex Systems, Department of Chemistry, and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, P. R. China

Biochemistry Downloaded from pubs.acs.org by UNIV OF VIRGINIA on 11/19/18. For personal use only.

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ABSTRACT: GoLoco motif-containing proteins regulate the nucleotide-binding state of Gα proteins in various signaling pathways. As guanine nucleotide dissociation inhibitors (GDIs), they bind Gα·GDP and inhibit GDP to GTP exchange. GoLoco proteins show binding selectivity toward different members of the Gα family. Although the Gαi1·GDP/ RGS14 crystal structure explains the specific binding selectivity of the RGS14 GoLoco domain well, the mechanism of selective binding has not been understood for the more general features of short GoLoco domains found in tandem arrays in proteins like GPSM2/LGN/dPins and GPSM1/AGS3. We explored the mechanism of differential interactions of GoLoco protein LGN with hGαi3 and hGαo. By combining mutagenesis experiments and molecular dynamics simulations, we identified a residue (Asp229 in hGαi3) away from the binding interface that remarkably affects the interaction between LGN and hGαi/o. A negatively charged residue at this position is required for high binding affinity. This affinity regulation mechanism was further verified by the cases of hGαi2 and dGαo, suggesting that this pathway is conserved among members of the Gα family.

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the functional role of Gαi is conserved in Drosophila via formation of the Gαi·GDP/Pins/Insc complex during the ACD, Gαo was also reported to be a target of Pins (Figure 1a) and to play a role in GPCR Tre-1-regulated cell polarity in the ACD of the Drosophila neuraoblast (NB).8 Therefore, selectivity of LGN and Pins within Gαi/o family members may implicate functional versatility in different signaling pathways. In other studies of GoLoco proteins, RGS12 and RGS14 were shown to preferentially bind the GDP-loaded Gαi1 and Gαi3 but not Gαo or Gαi2.9,10 In another report, the four GoLoco motifs of LGN exhibited binding specificity for GDPloaded Gαi1/2/3 but could not bind to Gαo·GDP or other members of the Gα family.11 The Gα selectivity of RGS12 and RGS14 was previously explained on the basis of the crystal structure of the Gαi1·GDP/RGS14 complex,9 in which the variable C-terminal tail beyond the conserved GoLoco motif of RGS14 forms an extensive interaction with the helical domain of Gαi1.9,12 It has been shown that the helical domain is responsible for the binding specificity.9,12 However, the biochemical and structural study of interactions between Gαi1/3 and LGN GoLoco motifs revealed that the general mode of binding of GoLoco-containing proteins to Gα·GDP requires only the N-terminal 25 amino acids of each GoLoco domain, and the helical domain is hardly involved in the

he heterotrimeric guanine nucleotide-binding proteins (G proteins) are molecular switches that connect G proteincoupled receptors (GPCRs) to downstream effectors in various signaling pathways.1 The α subunit of the heterotrimeric G proteins (Gα) cycles between the inactive GDP-bound state and the active GTP-bound state.1 Gα·GDP forms a stable complex with Gβγ and is activated by the ligand-bound GPCRs. Activated Gα·GTP interacts with various downstream effectors and finally returns to Gα·GDP because of its innate GTPase activity.1 Besides GPCRs, many other proteins are involved in regulating the nucleotide-binding states of Gα.2,3 Among Gα regulators, the guanine nucleotide dissociation inhibitors (GDIs) make up a group of proteins containing the GoLoco motifs, which specifically bind to and stabilize the GDP-loaded Gαi/o family proteins to inhibit the GDP to GTP exchange.4 One of the central roles for the GoLoco motif-containing proteins is orienting the cortical polarity and mitotic spindle in the asymmetric cell divisions (ACDs).2,4 The multiple-GoLoco motif-containing protein LGN (Drosophila ortholog Pins) is a key protein in regulating of the mitotic spindle orientation. Its GoLoco motif-containing domain (Figure 1a) could simultaneously bind four molecules of Gαi·GDP (for Pins, three molecules of Gαi·GDP are bound), which target LGN to the cell cortex.5,6 On the other hand, LGN interacts with another scaffold protein Inscuteable (Insc) through its TPR domain (Figure 1a), linking the LGN/Gαi·GDP complex to the cell polarity complex Par3/Par6/aPKC.7 The Gαi·GDP/LGN/Insc complex has been shown to determine the apical−basal alignment of the mitotic spindle during the ACDs.6 Though © XXXX American Chemical Society

Received: August 11, 2018 Revised: November 3, 2018 Published: November 8, 2018 A

DOI: 10.1021/acs.biochem.8b00848 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry

Glu43, which in turn affects the interactions between Gαi/o· GDP and GoLoco. This affinity regulation mechanism was further verified to be conserved among Gαi/o family proteins.

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MATERIALS AND EXPERIMENTAL DETAILS Protein Expression and Purification. The human Gαi3, Gαo, Drosophila Gαo, and mouse LGN GoLoco fragments were individually cloned into a modified version of the pET32a vector. All the mutations were created using the standard polymerase chain reaction-based method and confirmed by DNA sequencing. Recombinant proteins were expressed in Escherichia coli BL21 (DE3) host cells at 16 or 37 °C and purified by using Ni2+-nitrilotriacetic acid-agarose affinity chromatography followed by size exclusion chromatography. Fluorescence Polarization Assay. Fluorescence polarization assays were performed on a PerkinElmer LS-55 fluorimeter equipped with an automated polarizer at 25 °C. Commercial synthesized peptides were labeled with fluorescein 5-isothiocyanate (Invitrogen) at the N-termini. In a typical assay, the FITC-labeled peptide (∼1 μM) was titrated with binding partners in a 50 mM Tris buffer (pH 8.0) containing 100 mM NaCl, 1 mM dithiothreitol, and 1 mM EDTA. The KD values were obtained by fitting the titration data using the classical one-site binding model. Isothermal Titration Calorimetry Measurements. ITC measurements were performed on an ITC200 microcalorimeter (MicroCal) at 25 °C. All protein samples were dissolved in a buffer containing 50 mM Tris (pH 8.0), 100 mM NaCl, and 1 mM EDTA. The titrations were performed by injecting 40 μL of Gαi3·GDP aliquots (0.2 mM) into LGN GL fragments fused to the C-terminal end of thioredoxin (0.02 mM) at 2 min time intervals to ensure that the titration peak returned to the baseline. The titration data were analyzed using Origin7.0 from MicroCal. GDI Activity Assay. Measurements of BODIPY fluorescence were performed with a PerkinElmer Life Sciences LS50B spectrometer with excitation at 485 nm and emission at 530 nm (slit widths of 2.5 nm each). BODIPY FL-GTPγS was diluted to 1 μM in in a buffer that consisted of 50 mM Tris (pH 8.0), 100 mM NaCl, 1 mM EDTA, and 10 mM MgCl2 and equilibrated to 30 °C for 10 min in 1 mL cuvettes. Side by side, 100 nM hGαo, hGαi3, or dGαo protein was preincubated separately or with 10 μM GL fragments at 30 °C for 10 min before being added to the cuvettes. Relative fluorescence levels were recorded. Molecular Dynamics (MD) Simulation. The initial structure of the hGαi3·GDP/LGN-GL4 complex for simulation was obtained from the Protein Data Bank (PDB entry 4G5R). The initial structure of the hGαo·GDP/LGN-GL4 complex was homologously built using Modeler 9v814,15 with the structure mentioned above and the crystal structure of mouse Gαo (PDB entry 3C7K) as templates. Fifty model structures were generated, and the one with the lowest DOPE score was selected. Each initial structure was immersed in a dodecahedral water box with a length of ∼9.6 nm and further ionized with 0.15 mol/L NaCl. The amber99sb force field was used to model the proteins and the ligand,16 and the TIP3P model was used for water.17 The simulation system was then energy optimized by 100000 steps to release potential bad contacts and equilibrated at 300 K for 100 ps, with harmonic restraints on all heavy atoms of the proteins and the ligand. The force constant was 1000 kJ mol−1 nm−2. The restraints on side chains were

Figure 1. hGαo and hGαi show differential GoLoco bindings and GDI activities. (a) Domain organizations of LGN and its Drosophila ortholog Pins. (b) Interactions between hGαo·GDP/hGαi3·GDP and LGN GL4 measured by a fluorescence polarization assay. (c) LGNGL4 does not have GDI activity for hGαo measured by a BODIPY FL-GTPγS binding assay.

interaction.13 Sequence alignment demonstrates that the crystal structure of the Gαi1/3·GDP/LGN complex represents the general mode of Gα/GoLoco interaction.13 Therefore, what determines the Gα selectivity of LGN/Pins and most of the other GoLoco-containing proteins remains unclear. In this study, we combined biochemical characterization and molecular dynamics (MD) simulations to decipher the mechanism of binding specificities of LGN/Pins-GoLoco for members of the Gαi/o subfamily. We have identified a nonconserved residue (Asp229 in hGαi3 and Gly230 in hGαo) away from GoLoco-binding interface on the Ras-like domain that determines the binding affinity and GDI activity of the GoLoco motif toward Gαi/o·GDP. The residue at this position determines the side chain orientation of the conserved B

DOI: 10.1021/acs.biochem.8b00848 Biochemistry XXXX, XXX, XXX−XXX

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Figure 2. Identification of the determinant of differential bindings of LGN-GoLoco to hGαi3 and hGαo. (a) Surface representation of the crystal structure of the Gαi3·GDP/LGN-GL4 complex. All the residues at the binding interface are conserved between hGαi3 and hGαo (yellow). (b) Surface representation similar to panel a, with the mutated residues out of the binding region colored magenta. Note that Gln171 located on the back side is not surface-exposed. (c) Binding affinities of LGN-GL4 for the mutants of hGαi3. Note that the binding of D229G is significantly weakened. (d) Interactions of WT hGαi3 and the D229G mutant with LGN-GL4 measured by a fluorescence polarization assay.

of other studies.11 In line with the weak binding, the GDI activity of LGN-GL4 toward hGαo could hardly be detected (Figure 1c). For comparison, LGN-GL4 exhibits GDI activity for hGαi3 (Figure S1). Collectively, LGN-GoLoco proteins show obvious binding selectivity between hGαi3 and hGαo. The D229G Mutation Significantly Attenuates the Interaction between hGαi3·GDP and LGN-GL4. The large difference between the binding affinities of LGN-GoLoco for hGαi3 and hGαo is puzzling because the sequences of two Gα proteins are 71% identical. More intriguingly, on the basis of the crystal structure of the hGαi3·GDP/GL4 complex,13 residues at the binding interface are totally conserved in hGαo (Figure 2a). We mapped all the non-identical residues between hGαi3 and hGαo onto the crystal structure of the hGαi3/GL4 complex, showing that these residues are all out of the binding region (Figure S2). To determine the critical residues that affect the binding affinity, we mutated some of the non-identical residues on hGαi3 (soluble proteins could not be obtained for other mutations) to the corresponding amino acids in hGαo (Figure 2b) and measured the binding affinities of these mutants for LGN-GL4 (Figure 2c and Figure S3). Most of the mutations hardly affect the binding affinity, but it was found that the D229G mutation on hGαi3 significantly decreased the GL4 binding affinity (KD) to 2.15 μM (Figure 2d and Figure S3). The Asp229 residue is located on the Raslike domain, between β4 and switch III, and does not have any direct interaction with GL4 or GDP according to the crystal structure (Figure 2b).13 This single mutation unlikely perturbs the structure of hGαi3 because the previously reported crystal structure of Gαo20 is very similar to that of Gαi1/3.13 To further confirm the structural integrity of the D229G mutant, we

removed in a subsequent 20 ns equilibration, and the restraints on all non-Cα atoms were removed in another 20 ns equilibration. The production run started without any restraints and lasted for 200 ns, with the temperature kept at 300 K and the pressure at 1 bar using the Berendsen coupling method.18 The time constants for temperature and pressure coupling were 0.1 and 5.0 ps, respectively. All covalent bonds were constrained, and a time step of 2 fs was used. A 14 Å cutoff was used for van der Waals interactions, and a 12 Å cutoff was used for electrostatic interactions. Longer-range electrostatic interactions were evaluated using the particle mesh Ewald (PME) method.19 The coordinates of the system were saved every 5 ps.

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RESULTS LGN-GoLoco Proteins Show Binding Selectivity between hGαi and hGαo. A previous biochemical study demonstrated that LGN-GoLoco proteins (36−43-amino acid fragments) have Gα selectivity, being functional as GDI for Gαi1/2/3, but bind weakly to Gαo.11 In our previous structural and biochemical study of the interaction between LGNGoLoco proteins and Gαi1/3, it was revealed that the 25-amino acid LGN-GoLoco fragments are sufficient for Gαi1/3 binding and GDI activity.13 Here, we characterized the interaction between LGN-GoLoco proteins and hGαo·GDP by using the 25-amino acid GoLoco fragment. The fluorescence polarization assay showed that the binding between hGαo·GDP and LGN-GL4 (25 amino acids) has a KD of 5.76 μM (Figure 1b). For comparison, the interaction between hGαi3·GDP and LGN-GL4 (25 amino acids) has a KD of 0.20 μM (Figure 1b), which is close to our previous measurement13 and the results C

DOI: 10.1021/acs.biochem.8b00848 Biochemistry XXXX, XXX, XXX−XXX

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Figure 3. MD simulations reveal the structural rearrangements at the Gα·GDP/GL4 binding interface in the WT hGαi3, hGαi3 D229G, hGαo, and hGαo G230D systems. (a) Side chain reorientation in the D229G mutant and hGαo after 200 ns simulations. In WT hGαi3, Glu43 (green) forms interactions with Arg178 and Arg640GL4 simultaneously, while in D229G (purple) and hGαo (yellow), the side chain reorientation of Glu43 disrupts those interactions. (b) Variations of Glu43 side chain dihedral angle χ along the simulation trajectories. (c) Variations of the side chain distances between Glu43 and Arg640GL4 along the simulation trajectories. (d) Variations of the side chain distances between Glu43 and Arg178 (Arg179 in hGαo and the G230D mutant) along the simulation trajectories.

(Figure 3c). At the same time, the interaction between Glu43 and Arg178 was also compromised. The side chain distance between Glu43 and Arg178 is longer than that of WT hGαi3 (Figure 3d). It was previously pointed out that the pairing of Glu43 and Arg178 displaces the side chain of Arg178 from GDP and allows the coordination of the arginine finger on GoLoco to GDP.9 In LGN-GL4, the “double-arginine fingers”13 Arg640GL4 and Agr635GL4 are critical for Gαi·GDP binding and GDI activity.9,13 Crystal structures demonstrate that the arginine fingers insert into the GDP-binding pocket of Gαi1/3 , with their guanidinium groups contacting the phosphate of GDP.9,13 Mutation of the two arginine fingers resulted in an obvious decrease in the binding affinity for Gαi1/3·GDP and the diminished GDI activity.13 We compared the interactions between the two arginine fingers and GDP in different simulation systems. It turns out that in the D229G mutant the hydrogen bond and salt bridge interactions of Arg635GL4 and Arg640GL4 with GDP are totally disrupted or severely impaired, while those in WT hGαi3 are well maintained (Tables S3 and S4). Like that of the hGαi3 D229G mutant, the simulation trajectory of the hGαo·GDP/LGN-GL4 system also demonstrates the side chain rearrangements and the compromised interactions between GL4 and hGαo·GDP. The side chain of Glu43 on hGαo underwent a reorientation similar to that in Gαi3 D229G (Figure 3a,b). The distance between Glu43 and Arg640GL4 increased by >4 Å with respect to that of the WT Gαi3·GDP system (Figure 3c). However, unlike the case of the hGαi3 D229G mutant, the interaction between Glu43 and Arg179 (corresponding to Arg178 in hGαi3) was not

recorded the circular dichroism (CD) spectra of wild-type (WT) hGαi3 and D229G. It was found that D229G has a CD spectrum similar to that of the WT protein (Figure S4). MD Simulations Reveal the Role of Asp229 in the Interaction with LGN-GL4. To understand why Asp229 is important for the interaction between hGαi3·GDP and the GoLoco motif, we performed parallel MD simulations of hGαi3, hGαi3 D229G, and hGαo in complex with the LGNGL4 peptide to compare the binding interactions. The initial structures of the hGαi3 D229G/LGN-GL4 and hGαo·GDP/ LGN-GL4 complexes were built on the basis of the crystal structures of the hGαi3·GDP/LGN-GL4 complex13 and mouse Gαo.20 After 200 ns simulations, the overall structures of all three systems are stable (Figure S5). However, different rearrangements at the binding interface between various forms of Gα·GDP and GL4 were observed. The most prominent difference is the side chain reorientation of Glu43 on the Ploop (Figure 3a). In the crystal structure of the hGαi3/LGNGL4 complex, Glu43 forms electrostatic and hydrogen bonding interactions with both Arg640 on GL4 (Arg654GL4) and Arg178 on switch I simultaneously (Figure 3a). In the corresponding MD simulation system of WT hGαi3, these interactions were maintained (Figure 3a) and the χ angle of the side chain of Glu43 fluctuated around the initial value of −60° (Figure 3b). However, in the hGαi3 D229G mutant, the side chain of Glu43 was reoriented dramatically to approximately −175° (Figure 3a,b), and the interaction between Glu43 and Arg640GL4 was disrupted (Tables S1 and S2). The distance between the side chains of Glu43 and Arg640GL4 is longer than that in the WT hGαi3 system by >3 Å D

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Figure 4. Allosteric regulation mechanism that determines the GoLoco binding selectivity. (a) The residue at position 229 determines the side chain orientation of Glu43, which in turn affects the binding between Gα·GDP and GoLoco. Residues of WT hGαi3 are colored green, and residues of the D229G mutant are colored purple. Residues of LGN-GL4 are colored cyan. (b) Comparison of the distributions of the side chain distances between Glu43 and Arg242 (Arg243 in hGαo and the G230D mutant) based on the 200 ns MD simulations of the three systems. (c) Sequence alignment of Gαi/Gαo family proteins. Asp229 in hGαi3 and its counterparts in other members are highlighted with a red frame. (d) Binding of hGαi2·GDP to LGN-GL4 is much weaker than that to hGαi3·GDP. (e) Interactions between dGαo·GDP and Pins GoLoco proteins measured by a fluorescence polarization assay. (f) GDI activity of Pins-GL1 with dGαo·GDP measured by a BODIPY FL-GTPγS binding assay.

between Gαi/o and GDP, and between LGN-GL4 and GDP in the three systems. It is demonstrated that the total numbers of both hydrogen bonds and salt bridges decrease in the hGαi3 D229G mutant and hGαo with respect to the number in WT hGαi3 (Tables S1−S4), in agreement with the weak binding affinities for LGN-GL4 in the mutant and hGαo system (Figures 1b and 2d).

perturbed. The inter-residue Glu43−Arg179 distance is similar to its counterpart in WT hGαi3 (Figure 3d). Accordingly, in the hGαo·GDP/LGN-GL4 system, the interactions between the two arginine figures and GDP were hardly disturbed (Tables S3 and S4). We next systematically examined the hydrogen bonding and electrostatic interactions between LGN-GL4 and Gαi/o, E

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Figure 5. Enhanced binding affinities of two gain-of-function mutants, (a) hGαi2 A230D and (b) hGαo G230D, for the LGN-GL4 peptide measured by a polarized fluorescence assay.

(Figure 4e), comparable with those between human Gαi3·GDP (hGαi3·GDP) and LGN GoLoco motifs.13 The three Pins GoLoco motifs also exhibit GDI activities toward dGαo, but GL3 has a GDI potency slightly lower than those of the other two GoLoco motifs (Figure 4f and Figure S6). Following the same logic, a “reverse mutation”, such as hGαi2 A230D, should exhibit a gain-of-function result, i.e., have a GoLoco binding affinity higher than that of the WT protein. To verify this assumption, we generated two gain-of-function mutants: hGαi2 A230D and hGαo G230D. Both mutants show LGN-GL4 binding affinities that are higher than those of the WT proteins (Figure 5). hGαi2 A230D has a dramatic enhancement of LGN-GL4 binding. The dissociation constant (KD = 0.42 μM) is nearly 2 orders of magnitude lower than that of WT hGαi2 (KD = 13.24 μM). The binding affinity of hGαo G230D, however, is just slightly higher than that of WT hGαo. We also performed MD simulations of the hGαo G230D/LGN-GL4 complex. Consistent with our expectation, the side chain orientation of Glu43 in G230D is very similar to that of WT hGαi3 and different from those of WT hGαo and the hGαi3 D229G mutant (Figure 3b). The inter-residue distances (E43−R640, E43−R178, and E43−R242) exhibit the same trend, indicating that the residue identity at position 230/229 determines the side chain orientation of Glu43. Salt bridge and hydrogen bond analyses also demonstrate that the numbers of polar interactions are larger in the hGαo G230D mutant than in WT hGαo (Tables S1−S4). Overall, all of the above evidence demonstrates that an evolutionary nonconserved residue located between β4 and switch III (Asp229 in Gαi1/3, Gly230 in hGαo, and Glu230 in dGαo) plays a crucial role in regulating the binding affinity of GoLoco motifs for different members of the Gα protein family.

The Allosteric Regulation Pathway Initiated from Asp229. The next question is how the reduced hydrogen bond/electrostatic interactions are related to the D229G mutation. To answer this question, we scrutinized the simulation trajectories and found that the reorientations of Glu43 in hGαi3 D229G and hGαo can be attributed to Asp229 (Gαi3) and Gly230 (Gαo). The negatively charged Asp229 pushes the like-charged Glu43 away, which interacts with Arg178 in hGαi3, while in the D229G mutant and hGαo, Glu43 does not experience this repulsion and forms an ion pair with Arg242Gαi3/Arg243Gαo (Figure 4a). The distances between the side chains of Glu43 and Arg242 (Arg243) in both the hGαi3 D229G mutant and hGαo systems are obviously shorter than that in the WT hGαi3 system (Figure 4b). Therefore, although Asp229 (Gly230 in Gαo) is not directly involved in the interaction with the GoLoco peptide, it allosterically regulates the GoLoco binding affinity of Gα proteins. It should be noted that the term “allosteric regulation” originally refers to regulation of protein (enzyme) activity by reversible ligand binding, and it is used here only in an analogous sense. The nature of the residue at position 229 or 230 determines the side chain orientation and interaction partner of Glu43 on the P-loop, and this in turn affects the interaction between Glu43 and Arg640 on LGN-GL4 as well as other Gαi/o/GL4 interactions and the coordination of GDP. A sequence alignment shows that Asp229 varies among different classes of Gα proteins (Figure 4c). For example, unlike hGαi1 and hGαi3, hGαi2 has an alanine residue at this position (Figure 4c). According to the affinity regulation mechanism described above, we predict that Gαi2·GDP would have a low binding affinity for GoLoco motifs. We purified hGαi2 and characterized the interaction between hGαi2·GDP and LGN-GL4. As expected, the binding affinity (KD = 13.24 μM) is much lower than those of hGαi1 and hGαi3 (Figure 4d). On the other hand, different from hGαo, Drosophila Gαo (dGαo) has a glutamate acid Glu230 at this position (Figure 4c). Therefore, we predict that dGαo·GDP would have high affinities for Pins (LGN homologue protein in Drosophila) GoLoco motifs. We purified dGαo and measured the binding affinities with three fragments of Pins GoLoco motifs (25 amino acids), i.e., GoLoco1 (GL1), GoLoco2 (GL2), and GoLoco3 (GL3), using a fluorescence polarization binding assay (Figure 4e). dGαo·GDP binds the three GoLoco motifs with similar affinities (0.21, 0.61, and 0.44 μM, respectively)

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DISCUSSION

On the basis of the crystal structure of the Gαi1·GDP/RGS14 complex,9 previous studies attributed the Gα selectivity of the GoLoco proteins to the nonconserved helical domain of the Gα subunit and the GoLoco C-terminal residues.9,12 However, the Gαi1·GDP/RGS14 complex was shown to be a special case of the Gα−GoLoco interaction because of the unique sequence of RGS14-GoLoco.13 The more recent crystal structures of the Gαi1/3·GDP/LGN complexes13 do not support this mechanism because the GoLoco peptides do not have direct contact with the helical domain and the F

DOI: 10.1021/acs.biochem.8b00848 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry residues at the binding interface of Gα are totally conserved. Here, we solved this puzzle by identifying a key residue on the Ras-like domain out of the binding interface that determines the binding affinity. The identity of this residue (Asp229 in hGαi3 and Gly230 in hGαo) determines the side chain orientation of Glu43 and thereby affects the interactions between Gαi/o·GDP and GoLoco. An acidic residue at this position pushes Glu43 away from Arg242 due to like-charge repulsion, leading to favorable interactions between GoLoco motifs and Gα. Otherwise, a neutral residue at this position (like Ala or Gly in Gαi2 and hGαo) would compromise the GoLoco binding affinity. Thus, the differential binding affinities of Gα family members for GoLoco are thus regulated by the identity of a residue outside the binding interface. Though the residue at the Asp229 position varies among Gα family members, the amino acids involved in the affinity regulation are highly conserved around the nucleotide-binding site, such as Arg178 on switch I and Glu43 on the P-loop, which have been shown to be crucial for Gα activation and ATP hydrolysis.21 This implies that the regulation mechanism discovered in this work may have more general functional importance. The Gα selectivity of GoLoco proteins could be important in cell division. For the interactions between LGN/Pins and Gαi/o, only strong binding could disrupt the intramolecular interaction between TPR and GoLoco domains and open the autoinhibition conformation of LGN/Pins.22 According to the mechanism proposed in this study, Drosophila Gαo should have a binding affinity for Pins that is higher than that of dGαi. It has long been established that dGαi is involved in the regulation of the spindle orientation in Drosophila ACDs by associating with Pins and targeting the Pins/Insc (Mud) complex to the apical cell cortex.23−25 However, dGαo was also implicated in orienting the spindle during ACD,8,26,27 and its effect is stronger than that of dGαi in Drosophila sensory organ precursor (SOP) cells.26 Therefore, Gαo and Gαi may work cooperatively in regulating Pins localization and spindle orientation. In previous studies of Drosophila Gαo and Gαi, it was found that both GDP- and GTP-loaded dGαo could bind to the GoLoco domain of Pins while Pins-GoLoco binds only GDP-loaded Gαi.27,28 In addition, a trimeric complex of Gαo, Pins, and Gαi could be formed in Drosophila.27 In contrast to the case of Drosophila, mammalian Gαi1/3 has a binding affinity for LGN that is higher than that of hGαo, and Gαi1/3 was also reported to be involved in spindle reorientation in mammalian cell ACD.7 In this case, Gαi1/3 could efficiently bind LGN and determines the cell localization of LGN. On the other hand, the role of Gαo in mammalian cell division remains unclear. In summary, the Gα binding selectivity of GoLoco proteins, such as LGN, could not be explained on the basis of the complex crystal structures. In this study, we identified a key residue outside the binding interface that determines the binding specificity. Moreover, the regulation mechanism modulates the residues at the nucleotide-binding site of the Gα subunit and thereby may have more general functional importance.

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Four tables of hydrogen bond and salt bridge numbers between the GoLoco motif and Gα (or GDP) in the MD simulations, GDI activities of LGN-GL4 toward hGαi3, the nonconserved residues among Gαi/o family members on the surface of the Gαi3/GL4 complex, binding affinities of Gαi3 mutants for the GL4 peptide, CD spectra of Gαi3 and the D229G mutant, backbond root-mean-square deviation variation along the MD simulations, and GDI activity of Pins-GL2 and GL3 toward dGαo (PDF)

AUTHOR INFORMATION

Corresponding Author

*Telephone: +86-21-31243985. E-mail: [email protected]. cn. ORCID

Wenning Wang: 0000-0002-8712-0536 Author Contributions †

C.L. and J.W. contributed equally to this work.

Funding

This work was supported by the National Key Research and Development Program of China (2016YFA0501702) and the National Science Foundation of China (21773038 and 21473034). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research made use of the resources of the computer clusters at the computer center at Fudan University. REFERENCES

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DOI: 10.1021/acs.biochem.8b00848 Biochemistry XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.biochem.8b00848 Biochemistry XXXX, XXX, XXX−XXX