Extended Data Fig. 8: Interactions of GRK1 with intracellular loops of Rho* and development of autophosphorylation mimetic variants.
From: Structures of rhodopsin in complex with G-protein-coupled receptor kinase 1
a, Interactions of GRK1 αN with the cytoplasmic cleft and H8 of Rho*. GRK1-Ser5 was modelled in a phosphorylated state to demonstrate proximity to Rho*-Lys311 and Arg314. b, GRK1 AST interaction with ICL1. The key participating residues are shown with stick side chains. Ser488 and Thr489 are autophosphorylation sites in GRK1. c–g, Kinetic and crosslinking analysis of GRK1 and phosphomimetic mutants of Rho* autophosphorylation (S5E; S488E and T489E (EE); S488D and T489D (DD); S5E, S488E, and T489E (S5E/EE)). One-way ANOVA followed by Dunnett’s multiple comparison test was carried out to compare each mutant with GRK1. Reactions were performed in 50 mM HEPES (pH 8.0), 10 mM MgCl2 for 2 min at room temperature. Data were normalized to the Vmax,Rho or Vmax,ATP of GRK1. For Rho kinetics: S5E, n = 4; EE, n = 5; DD, n = 3; S5E/EE, n = 3. For ATP kinetics: S5E, n = 3; EE, n = 3; DD, n = 4; S5E/EE, n = 4 (all technical replicates). All data shown as mean ± s.d. f, Time courses of tubulin phosphorylation by GRK1 and variants are similar. Data were normalized to the phosphorylation level of tubulin by GRK1 at 30 min (n = 3 technical replicates). g, Crosslinking yield of GRK1 variants relative to GRK1 (n = 3 technical replicates). For gel source data, see Supplementary Fig. 5. h, Interaction of the GRK1 small lobe with ICL2 of Rho*. The GRK1 α0 helix from a basal ATP-bound structure (PDB entry 3C4Z49) is modelled by aligning its small lobe with that in the Rho*−GRK1(S5E/EE)−Fab1 complex, demonstrating a potential clash between ICL2 and α0. i, Interaction of GRK1 AST with ICL3.
