Research ArticleG Protein Signaling

“Disruptor” residues in the regulator of G protein signaling (RGS) R12 subfamily attenuate the inactivation of Gα subunits

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Science Signaling  12 Jun 2018:
Vol. 11, Issue 534, eaan3677
DOI: 10.1126/scisignal.aan3677
  • Fig. 1 RGS10 and RGS14 have lower GAP activities toward Gαo compared to those of RGS16 and RGS4.

    (A) kGAP constants for RGS16, RGS4, RGS10, and RGS14 for GTP hydrolyzed by Gαo (400 nM) in the presence of RGS protein (20 nM). Data are means ± SEM of at least three independent biological replicates. (B) Dose-response analysis of RGS16, RGS10, and RGS14 activity toward Gαo. The EC50 values (RGS16 = 7 ± 1 nM; RGS10 = 42 ± 2 nM; RGS14 = 63 ± 5 nM) were calculated using three-parameter sigmoidal curves. Data are means ± SEM of experiments performed in triplicate and are representative of three or more independent biological replicates for each RGS tested.

  • Fig. 2 RGS S&C residues interact with the Gα GTPase domain and RGS modulatory residues interact with the GTPase and helical domains of Gα.

    (A) Superimposition of four crystal structures of Gα-RGS complexes (with PDB codes): RGS1-Gαi1 (PDB ID: 2GTP), RGS4-Gαi1 (PDB ID: 1AGR), human RGS16–Gαi1 (PDB ID: 2IK8), and mouse RGS16–Gαo (PDB ID: 3C7K). RGS domains (cyan) and Gα subunits (green and orange) are shown as backbone traces. Gαi in the RGS4-Gαi1 structure is also visualized as a light gray transparent molecular surface. RGS S&C residues are shown as red spheres, and modulatory residues are shown as purple spheres. (B) Four RGS16 modulatory residues (Glu135, Glu164, Lys165, and Lys173, shown as sticks) interact with the helical domain of the Gα subunit. RGS and Gα subunits are shown as ribbon diagrams colored as in (A), rotated 20° about the y axis relative to (A). (C) Four RGS4 modulatory residues (Glu161, Lys162, Arg166, and Lys170; shown as sticks) interact with the helical domain of the Gα subunit, shown in the same orientation as in (B).

  • Fig. 3 R12 RGS residues in critical modulatory positions are predicted to disrupt intermolecular interactions with the Gα helical domain.

    (A) Residue-level sequence map summarizing our structure-based energy calculations of the R4 representative complexes with Gα subunits. Colored boxes mark RGS residues that contribute substantially to interactions with Gα subunits, according to the type of energy contribution: nonpolar (np), electrostatic (elec) contributions from the residue side chain (sc elec) or the main chain (mc elec). S&C residues for all four R4 structures are marked with red asterisks above the alignment, and modulatory positions that contribute in any of the four R4 structures are marked with purple triangles. An open star symbol marks modulatory RGS residues that contribute to interactions with the Gα helical domain across all of the analyzed high-activity R4 subfamily members; a black circle marks a contribution found only in some R4 high-activity representatives. The numbering above the alignment is according to the human RGS16 sequence. (B) Sequence-based prediction of R12 subfamily residue-level contributions, classified into three groups: Conserved S&C residues and conserved modulatory residues are residues that are identical across the relevant R4 and R12 subfamilies members; putative disruptor residues are R12-specific residues in modulatory positions that are different than their counterparts in the high-activity R4 subfamily and are predicted to impair the interaction with the Gα helical domain.

  • Fig. 4 Putative disruptor residues of RGS10, RGS14, and RGS18 are predicted to perturb favorable interactions with the helical domain of Gαi/o.

    (A) RGS16 residues that contribute to favorable electrostatic interactions with the Gα helical domain, shown as sticks. Electrostatic interactions or hydrogen bonds are marked with dashed lines. The complexes of human RGS16–Gαi1 and mouse RGS16–Gαo are superimposed and shown as ribbon diagrams: RGS16 (cyan) and Gα GTPase domain (green; bottom) and helical domain (orange; top and bottom). (B) Corresponding RGS10 and RGS14 putative disruptor residues, shown in stick form as in (A). RGS10 (yellow) and RGS14 (tan) were superimposed onto the complexes of human RGS16–Gαi1 and mouse RGS16–Gαo, with Gα colored as described in (A). (C) Electrostatic interaction between Arg166, a unique modulatory residue in RGS4 (blue), and the Gαi1 helical domain. (D) RGS10 (yellow) and RGS14 (tan) residues corresponding to RGS4-Arg166, shown with the Gαi1 helical domain residue that interacts with RGS4 Arg166. (E) RGS18 (pink) putative disruptor residues (sticks), shown with the helical domain of Gαi/o with Gα colored as in (A). RGS18 was superimposed onto RGS16-Gαi1 and RGS16-Gαo, as described in (B).

  • Fig. 5 Replacement of RGS16 and RGS4 modulatory residues that interact with the Gα helical domain with the corresponding putative disruptor residues impairs GAP activity toward Gαo.

    (A) kGAP constants for WT RGS16 and the following RGS16 mutants: RGS16-to-RGS10 mutants E164K-K165Y (EK>KY) and E135Q-E164K-K165Y (EEK>QKY); RGS16-to-RGS14 mutants E164K-K165F (EK>KF) and E135Q-E164K-K165F (EEK>QKF); the RGS16-to-RGS18 mutant E135H-K165Q (E-K>H-Q); and the RGS16 mutants in noncontributing residues adjacent to Glu164 and Lys165, Y168A and P169A. The kGAP values are means ± SEM of at least three independent biological replicates. (B) Dose-response analysis of WT RGS16, the EEK>QKY mutant, and the EEK>QKF mutant GAP activity toward Gαo. EC50 values (RGS16 = 7 ± 1 nM; EEK>QKY = 26 ± 2 nM; EEK>QKF = 74 ± 3 nM) are means ± SEM of experiments performed in triplicate and are representative of at least three independent biological replicates each. (C) kGAP constants for WT RGS4 and the following RGS4 mutants: RGS4-to-RGS14 mutants E161K-K162F and E161K-K162F-R166A and the RGS4 S&C residue mutant N128S. kGAP values are means ± SEM of at least three independent biological replicates.

  • Fig. 6 Mutating all four RGS16 modulatory positions that interact with the Gα helical domain into alanines does not reduce the GAP activity of RGS16 toward Gαo.

    Representative single-turnover GTPase assays of Gαo (400 nM) with RGS16 (20 nM; black circles and solid line) and the RGS16-Ala4 mutant E135A-E164A-K165A-K173A (20 nM; black triangles and dashed line). The reaction rate constant (k) for RGS16 was 1.5 ± 0.3 min−1, and that for the RGS16-Ala4 mutant was 1.6 ± 0.2 min−1. Data are means ± SEM of at least three independent biological replicates.

  • Fig. 7 Replacement of RGS18 disruptor residues with the corresponding RGS16 modulatory residues led to a gain of function.

    The kGAP constants for WT RGS18, the RGS18-to-RGS16 mutants Q186K and H156E-Q186K, and WT RGS16 were calculated as in described in Fig. 1. Data are means ± SEM of at least three independent biological replicates.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/534/eaan3677/DC1

    Fig. S1. The RGS interface with Gα subunits is predominantly electrostatic and polar.

    Fig. S2. A pair of RGS1 and RGS4 residues contributes favorably to electrostatic interactions with the Gα helical domain.

    Fig. S3. A chimera of RGS14 and RGS16 exhibits increased GAP activity.

  • Supplementary Materials for:

    "Disruptor" residues in the regulator of G protein signaling (RGS) R12 subfamily attenuate the inactivation of Gα subunits

    Ali Asli, Isra Sadiya, Meirav Avital-Shacham, Mickey Kosloff*

    *Corresponding author. Email: kosloff{at}sci.haifa.ac.il

    This PDF file includes:

    • Fig. S1. The RGS interface with Gα subunits is predominantly electrostatic and polar.
    • Fig. S2. A pair of RGS1 and RGS4 residues contributes favorably to electrostatic interactions with the Gα helical domain.
    • Fig. S3. A chimera of RGS14 and RGS16 exhibits increased GAP activity.

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    © 2018 American Association for the Advancement of Science

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