PDBsum entry 2v4z

Cell cycle

PDB id: 2v4z

Contents

Protein chains

316 a.a. *

122 a.a. *

Ligands

GDP-ALF

Metals

_MG

Waters ×18

* Residue conservation analysis

PDB id:

2v4z

Name:

Cell cycle

Title:

The crystal structure of the human g-protein subunit alpha (gnai3) in complex with an engineered regulator of g-protein signaling type 2 domain (rgs2)

Structure:

Guanine nucleotide-binding protein g(k) subunit alpha. Chain: a. Fragment: subunit alpha, residues 4-350. Synonym: g(i) alpha-3, guanine nucleotide-binding protein g k subunit alpha g i alpha-3. Engineered: yes. Regulator of g-protein signaling 2. Chain: b. Fragment: rgs domain, residues 71-209.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 469008. Expression_system_variant: r3-prare2.

Resolution:

2.80Å R-factor: 0.210 R-free: 0.254

Authors:

A.K.Roos,M.Soundararajan,A.C.W.Pike,C.H.Arrowsmith,J.Weigelt, A.Edwards,C.Bountra,S.Knapp

Key ref:

A.J.Kimple et al. (2009). Structural Determinants of G-protein {alpha} Subunit Selectivity by Regulator of G-protein Signaling 2 (RGS2). J Biol Chem, 284, 19402-19411. PubMed id: 19478087 DOI: 10.1074/jbc.M109.024711

Date:

30-Sep-08 Release date: 04-Nov-08

Protein chain

P08754  (GNAI3_HUMAN) -  Guanine nucleotide-binding protein G(i) subunit alpha-3 from Homo sapiens

Seq: 354 a.a.

Struc: 316 a.a.

Protein chain

P41220  (RGS2_HUMAN) -  Regulator of G-protein signaling 2 from Homo sapiens

Seq: 211 a.a.

Struc: 122 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

DOI no: 10.1074/jbc.M109.024711

J Biol Chem 284:19402-19411 (2009)

PubMed id: 19478087

Structural Determinants of G-protein {alpha} Subunit Selectivity by Regulator of G-protein Signaling 2 (RGS2).

A.J.Kimple, M.Soundararajan, S.Q.Hutsell, A.K.Roos, D.J.Urban, V.Setola, B.R.Temple, B.L.Roth, S.Knapp, F.S.Willard, D.P.Siderovski.

ABSTRACT

"Regulator of G-protein signaling" (RGS) proteins facilitate the termination of G protein-coupled receptor (GPCR) signaling via their ability to increase the intrinsic GTP hydrolysis rate of Galpha subunits (known as GTPase-accelerating protein or "GAP" activity). RGS2 is unique in its in vitro potency and selectivity as a GAP for Galpha(q) subunits. As many vasoconstrictive hormones signal via G(q) heterotrimer-coupled receptors, it is perhaps not surprising that RGS2-deficient mice exhibit constitutive hypertension. However, to date the particular structural features within RGS2 determining its selectivity for Galpha(q) over Galpha(i/o) substrates have not been completely characterized. Here, we examine a trio of point mutations to RGS2 that elicits Galpha(i)-directed binding and GAP activities without perturbing its association with Galpha(q). Using x-ray crystallography, we determined a model of the triple mutant RGS2 in complex with a transition state mimetic form of Galpha(i) at 2.8-A resolution. Structural comparison with unliganded, wild type RGS2 and of other RGS domain/Galpha complexes highlighted the roles of these residues in wild type RGS2 that weaken Galpha(i) subunit association. Moreover, these three amino acids are seen to be evolutionarily conserved among organisms with modern cardiovascular systems, suggesting that RGS2 arose from the R4-subfamily of RGS proteins to have specialized activity as a potent and selective Galpha(q) GAP that modulates cardiovascular function.

Selected figure(s)

Figure 6.
The triple mutant RGS2(C106S,N184D,E191K), but not wild type RGS2, inhibits dopamine D2-receptor influence on forskolin-stimulated cAMP production. HEK293T cells were transiently co-transfected with expression vectors for the GloSensor cAMP biosensor and the G[i]-coupled dopamine D2-receptor with empty vector, wild type RGS2, or the RGS2(triple) mutant. Inhibition of forskolin-stimulated cAMP production was determined after activation of the D2 receptor with various concentrations of quinpirole as indicated. The IC[50] (95% CI) for quinpirole was determined to be 18 (12–26), 14 (9–22), and 762 (498–1170) nm in the presence of empty vector, wild type RGS2, and the triple mutant, respectively. Inset, post-transfection cell lysates were immunoblotted with anti-HA epitope tag antibody to confirm the equivalent overexpression of HA-RGS2 and HA-RGS2(C106S,N184D,E191K) proteins.

Figure 8.
Particular Gα selectivity determinants inferred from the structural model of the triple mutant RGS2(C106S,N184D,E191K) bound to Gα[i3].A, illustration of the αVII–αVIII region of the RGS domain to highlight the intramolecular interaction between the highly conserved αVIII helix arginine (Arg^188 of RGS2) and position 184 (asparagine in wild type RGS2 and aspartate in the triple mutant). RGS2(C106S,N184D,E191K) triple mutant (yellow-green; PDB code 2V4Z), unliganded wild type RGS2 (gray; PDB code 2AF0), and the Gα[i1]-bound RGS16 (dark green; PDB code 2IK8) were aligned by sequence and then structure (Cα atoms) using the Align command with default align settings of MacPyMOL (DeLano Scientific, Palo Alto, CA), resulting in root mean square deviations of 0.92 and 0.80 Å, respectively. The conserved Arg^188 makes salt bridges with the terminal oxygens of the Asp^184 side chain in the RGS2(C106S,N184D,E191K) mutant and the analogous asparate side chain in RGS16; however, only one contact can be made between Arg^188 and the Asn^184 side chain of wild type RGS2. Loss of the second salt bridge creates a torsion in the wild type RGS2 Asp^184 residue, resulting of the loss of the stabilizing hydrogen bond to Thr^182 in switch I of the Gα subunit. B, critical contacts between the three mutated positions of RGS2(C106S,N184D,E191K) (yellow-green) and its Gα binding partner (Ras-like domain in red; all-helical domain in blue; switch regions in cyan; bound GDP in magenta). The modeled terminal atoms of the Lys^191 side chain (spheres) within RGS2(C106S,N184D,E191K) are in close enough proximity to make a hydrogen bond with Glu^65 of the Gα all-helical domain. Asp^184 makes two hydrogen bonds with Arg^188 and an additional bond with the backbone amine of the peptide bond connecting Thr^181 and Thr^182, both located within switch I of Gα. Ser^106 of the RGS2 triple mutant is tightly packed with the backbone carbonyl and γ-hydroxyl of Gα Thr^182, both being less than 3.9 Å from β-carbon of Ser^106. Additionally, the Gα switch II residue Lys^210 is 3.8 Å from the Ser^106 α-carbon.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2009, 284, 19402-19411) copyright 2009.

Figures were selected by the author.