PDBsum entry 3c4x

Transferase

PDB id: 3c4x

Contents

Protein chains

496 a.a. *

Ligands

ATP ×2

Metals

_MG ×2

_CL ×2

Waters ×60

* Residue conservation analysis

PDB id:

3c4x

Name:

Transferase

Title:

Crystal structure of g protein coupled receptor kinase 1 bound to atp and magnesium chloride at 2.9a

Structure:

Rhodopsin kinase. Chain: a, b. Fragment: unp residues 1-535. Synonym: rk, g protein-coupled receptor kinase 1. Engineered: yes

Source:

Bos taurus. Cattle. Organism_taxid: 9913. Cell_line: high 5. Gene: grk1, rhok. Expressed in: trichoplusia ni. Expression_system_taxid: 7111.

Resolution:

2.90Å R-factor: 0.193

Authors:

P.Singh,J.J.G.Tesmer

Key ref:

P.Singh et al. (2008). Structures of rhodopsin kinase in different ligand states reveal key elements involved in G protein-coupled receptor kinase activation. J Biol Chem, 283, 14053-14062. PubMed id: 18339619 DOI: 10.1074/jbc.M708974200

Date:

30-Jan-08 Release date: 11-Mar-08

Protein chains

P28327  (RK_BOVIN) -  Rhodopsin kinase GRK1 from Bos taurus

Seq: 561 a.a.

Struc: 496 a.a.

Enzyme reactions

• Enzyme class: E.C.2.7.11.14 - rhodopsin kinase.
• Reaction:
  1. L-seryl-[rhodopsin] + ATP = O-phospho-L-seryl-[rhodopsin] + ADP + H⁺
  2. L-threonyl-[rhodopsin] + ATP = O-phospho-L-threonyl-[rhodopsin] + ADP + H⁺

L-seryl-[rhodopsin] (Bound ligand (Het Group name = ATP) corresponds exactly) + ATP = O-phospho-L-seryl-[rhodopsin] + ADP + H(+)

L-threonyl-[rhodopsin] (Bound ligand (Het Group name = ATP) corresponds exactly) + ATP = O-phospho-L-threonyl-[rhodopsin] + ADP + H(+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1074/jbc.M708974200

J Biol Chem 283:14053-14062 (2008)

PubMed id: 18339619

Structures of rhodopsin kinase in different ligand states reveal key elements involved in G protein-coupled receptor kinase activation.

P.Singh, B.Wang, T.Maeda, K.Palczewski, J.J.Tesmer.

ABSTRACT

G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate activated heptahelical receptors, leading to their uncoupling from G proteins. Here we report six crystal structures of rhodopsin kinase (GRK1), revealing not only three distinct nucleotide-binding states of a GRK but also two key structural elements believed to be involved in the recognition of activated GPCRs. The first is the C-terminal extension of the kinase domain, which was observed in all nucleotide-bound GRK1 structures. The second is residues 5-30 of the N terminus, observed in one of the GRK1.(Mg2+)2.ATP structures. The N terminus was also clearly phosphorylated, leading to the identification of two novel phosphorylation sites by mass spectral analysis. Co-localization of the N terminus and the C-terminal extension near the hinge of the kinase domain suggests that activated GPCRs stimulate kinase activity by binding to this region to facilitate full closure of the kinase domain.

Selected figure(s)

Figure 1.
FIGURE 1. Overview of GRK1 and its active site. a, GRK1[535]-His[6] crystallized as a homodimer using a conserved interface of the RH domain in all the crystal forms. Shown is the most complete structure, that of crystal form I. The RH terminal subdomain is colored magenta (helices 0-3 and 8-11), and the bundle subdomain (helices 4- 7) is slate blue. The small lobe of the kinase domain (yellow) is composed of six β-strands (orange) and two -helices ( B and C), whereas the large lobe is primarily -helical. The ligand (Mg^2+)[2]·ATP is drawn as spheres. Magnesium atoms are colored black, carbons are white, nitrogens are blue, oxygens are red, phosphates are orange, and chloride ions are cyan. The extreme N-terminal region and the C-terminal extension of the kinase domain are green. b, substrate complex of GRK1. Shown is a [A]-weighted |F[o]| - |F[c]| omit map contoured at 4 , wherein ATP, Mg^2+, and associated waters (green) were excluded from refinement (crystal form I and chain B). Lys^216 (β1 sheet, orange carbons) coordinates the - and β-phosphates. Glu^332 (yellow carbons) coordinates both Mg^2+ atoms. c, product complex of GRK1. Shown is a [A]-weighted |F[o]| - |F[c]| omit map contoured at 5 , wherein ADP, Mg^2+, and associated waters were excluded from refinement (crystal form IV). d, the peptide-binding channel of GRK1. The molecular surface of GRK1 is colored by its electrostatic potential from -7 (red, acidic) to +7 (blue, basic) kT/e^-. The channel has a strikingly basic character, explaining why GRK1 prefers acidic substrates (48, 49) and how it can phosphorylate multiple closely spaced Ser and Thr residues at the C terminus of Rho^*. The channel is also wider in GRK1 than in nucleotide-bound PKB (e), reflecting the more open conformation of the GRK1 kinase domain of GRK1. As a result, the phosphoacceptor oxygen of the modeled peptide is >4 Å from the -phosphate of ATP, which is too far for covalent chemistry to occur. A model of residues 332-345 from the C terminus of Rho^*, is shown as a stick model docked to the large lobe with Ser^338 in position to be phosphorylated (position "+0"). Residues in the F- G loop of the large lobe appear to obstruct the N-terminal end of the peptide-binding site. e, the GSK3β peptide bound to PKB. The PKB kinase domain (Protein Data Bank code 1O6L [PDB] ) is in its closed conformation. The channel is markedly acidic, in line with a preference for basic substrates.

Figure 3.
FIGURE 3. The phosphorylation sites of GRK1. a, the RH-kinase core of GRK1. The structure corresponds to that of crystal form I (with composite C-terminal extension; see Fig. 2). The Ser^5, Thr^8, Ser^21, Ser^488, and Thr^489 phosphorylation sites are drawn as stick models. The expected position of the membrane plane is indicated. Top inset, the Ser^488 and Thr^489 phosphorylation sites correspond to the AGC kinase turn motif. Bottom inset, interaction of Thr(P)^8 with the RH domain. Gln^73 and Glu^93 form direct hydrogen bonds, whereas Lys^69 and Lys^90 complement the charge of the phosphate moiety. These crystals grew at pH 4.35, and so either Glu^93 or the phosphate group could be protonated. b, tandem mass spectrometry spectra of phosphopeptides from GRK1[535]-His[6] (Pool A, pretreated with 4 mM ATP and 2 mM MgCl[2]). Both Ser^5 and Thr^8 sites were identified in a single peptide. The Ser^5 site was also readily observed in endogenous GRK1, as were the previously observed phosphorylation sites at Ser^21, Ser^488, and Thr^489 (supplemental Fig. S7).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2008, 283, 14053-14062) copyright 2008.

Figures were selected by the author.