PDBsum entry: 1bh2

Signal transduction protein

PDB id: 1bh2

Contents

Protein chain

315 a.a. *

Ligands

GSP

Metals

_MG

Waters ×116

* Residue conservation analysis

PDB id:

1bh2

Name:

Signal transduction protein

Title:

A326s mutant of an inhibitory alpha subunit

Structure:

Guanine nucleotide-binding protein. Chain: a. Synonym: gi-alpha-1. Engineered: yes. Mutation: yes

Source:

Rattus norvegicus. Norway rat. Organism_taxid: 10116. Cell_line: bl21. Organ: brain. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

2.10Å R-factor: 0.190 R-free: 0.250

Authors:

M.B.Mixon,B.A.Posner,M.A.Wall,A.G.Gilman,S.R.Sprang

Key ref:

B.A.Posner et al. (1998). The A326S mutant of Gialpha1 as an approximation of the receptor-bound state. J Biol Chem, 273, 21752-21758. PubMed id: 9705312 DOI: 10.1074/jbc.273.34.21752

Date:

12-Jun-98 Release date: 04-Nov-98

Protein chain

P10824  (GNAI1_RAT) -  Guanine nucleotide-binding protein G(i) subunit alpha-1

Seq: 354 a.a.

Struc: 315 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

 Gene Ontology (GO) functional annotation 

• Biological process: signal transduction (2 terms)
• Biochemical function: signal transducer activity (3 terms)

DOI no: 10.1074/jbc.273.34.21752

J Biol Chem 273:21752-21758 (1998)

PubMed id: 9705312

The A326S mutant of Gialpha1 as an approximation of the receptor-bound state.

B.A.Posner, M.B.Mixon, M.A.Wall, S.R.Sprang, A.G.Gilman.

ABSTRACT

Agonist-bound heptahelical receptors activate heterotrimeric G proteins by catalyzing exchange of GDP for GTP on their alpha subunits. In search of an approximation of the receptor-alpha subunit complex, we have considered the properties of A326S Gialpha1, a mutation discovered originally in Gsalpha (Iiri, T., Herzmark, P., Nakamoto, J. M., Van Dop, C., and Bourne, H. R. (1994) Nature 371, 164-168) that mimics the effect of receptor on nucleotide exchange. The mutation accelerates dissociation of GDP from the alphai1beta1gamma2 heterotrimer by 250-fold. Nevertheless, affinity of mutant Gialpha1 for GTPgammaS is high in the presence of Mg2+, and the mutation has no effect on the intrinsic GTPase activity of the alpha subunit. The mutation also uncouples two activities of betagamma: stabilization of the GDP-bound alpha subunit (which is retained) and retardation of GDP dissociation from the heterotrimer (which is lost). For wild-type and mutant Gialpha1, beta gamma prevents irreversible inactivation of the alpha subunit at 30 degreesC. However, the mutation accelerates irreversible inactivation of alpha at 37 degreesC despite the presence of beta gamma. Structurally, the mutation weakens affinity for GTPgammaS by steric crowding: a 2-fold increase in the number of close contacts between the protein and the purine ring of the nucleotide. By contrast, we observe no differences in structure at the GDP binding site between wild-type heterotrimers and those containing A326S Gialpha1. However, the GDP binding site is only partially occupied in crystals of G protein heterotrimers containing A326S Gialpha1. In contrast to original speculations about the structural correlates of receptor-catalyzed nucleotide exchange, rapid dissociation of GDP can be observed in the absence of substantial structural alteration of a Galpha subunit in the GDP-bound state.

Selected figure(s)

Figure 3.
Fig. 3. Intrinsic GTPase activity of wild-type and A326S G[i 1]. Single turnover GTPase assays were conducted at 30 °C for wild-type G[i 1] ( ) and A326S G[i 1] ( circle ) as described under "Materials and Methods."

Figure 5.
Fig. 5. Effect of Mg2+ on steady-state hydrolysis of GTP by A326S G[i 1]. The final concentrations of protein and nucleotide were 50 nM and 40 µM, respectively.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (1998, 273, 21752-21758) copyright 1998.

Figures were selected by an automated process.