PDBsum entry: 2ebc

Signaling protein

PDB id: 2ebc

Contents

Protein chain

342 a.a. *

Ligands

GDP

Waters ×81

* Residue conservation analysis

PDB id:

2ebc

Name:

Signaling protein

Title:

Mechanism underlying the critical contribution of a switch ii residue in a heterotrimeric g-protein alpha subunit during c. Elegans asymmetric cell division

Structure:

Guanine nucleotide-binding protein g(i), alpha-1 subunit. Chain: a. Synonym: adenylate cyclase-inhibiting g alpha protein. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

2.24Å R-factor: 0.229 R-free: 0.236

Authors:

C.A.Johnston,D.P.Siderovski,F.S.Willard

Key ref:

C.A.Johnston et al. (2008). Structural determinants underlying the temperature-sensitive nature of a Galpha mutant in asymmetric cell division of Caenorhabditis elegans. J Biol Chem, 283, 21550-21558. PubMed id: 18519563 DOI: 10.1074/jbc.M803023200

Date:

08-Feb-07 Release date: 19-Feb-08

Protein chain

P63096  (GNAI1_HUMAN) -  Guanine nucleotide-binding protein G(i) subunit alpha-1

Seq: 354 a.a.

Struc: 342 a.a.*

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

 Gene Ontology (GO) functional annotation 

• Cellular component: intracellular (11 terms)
• Biological process: cell cycle (9 terms)
• Biochemical function: nucleotide binding (11 terms)

DOI no: 10.1074/jbc.M803023200

J Biol Chem 283:21550-21558 (2008)

PubMed id: 18519563

Structural determinants underlying the temperature-sensitive nature of a Galpha mutant in asymmetric cell division of Caenorhabditis elegans.

C.A.Johnston, K.Afshar, J.T.Snyder, G.G.Tall, P.Gönczy, D.P.Siderovski, F.S.Willard.

ABSTRACT

Heterotrimeric G-proteins are integral to a conserved regulatory module that influences metazoan asymmetric cell division (ACD). In the Caenorhabditis elegans zygote, GOA-1 (Galpha(o)) and GPA-16 (Galpha(i)) are involved in generating forces that pull on astral microtubules and position the spindle asymmetrically. GPA-16 function has been analyzed in vivo owing notably to a temperature-sensitive allele gpa-16(it143), which, at the restrictive temperature, results in spindle orientation defects in early embryos. Here we identify the structural basis of gpa-16(it143), which encodes a point mutation (G202D) in the switch II region of GPA-16. Using Galpha(i1)(G202D) as a model in biochemical analyses, we demonstrate that high temperature induces instability of the mutant Galpha. At the permissive temperature, the mutant Galpha was stable upon GTP binding, but switch II rearrangement was compromised, as were activation state-selective interactions with regulators involved in ACD, including GoLoco motifs, RGS proteins, and RIC-8. We solved the crystal structure of the mutant Galpha bound to GDP, which indicates a unique switch II conformation as well as steric constraints that suggest activated GPA-16(it143) is destabilized relative to wild type. Spindle severing in gpa-16(it143) embryos revealed that pulling forces are symmetric and markedly diminished at the restrictive temperature. Interestingly, pulling forces are asymmetric and generally similar in magnitude to wild type at the permissive temperature despite defects in the structure of GPA-16(it143). These normal pulling forces in gpa-16(it143) embryos at the permissive temperature were attributable to GOA-1 function, underscoring a complex interplay of Galpha subunit function in ACD.

Selected figure(s)

Figure 2.
Protein-protein interactions of wild type and Gly-to-Asp mutant Gα subunits. Interactions between wild type (WT) or indicated Gly-to-Asp mutated Gα subunits and Gβ[1]γ[1] (A and B), PCP-2 (C and D), Ric-8A (E and F), the RGS domain of RGS14 (G and H), and PDEγ(aa 63–87) (I and J) were measured using surface plasmon resonance. Proteins were immobilized using biotin-streptavidin coupling (A, B, I, and J) or anti-GST antibody capture (C–H). Indicated concentrations of Gα subunits in the GDP (blue), GTPγS(red), or (green) loaded forms were injected over biosensor surfaces at a flow rate of 20 μl/min as denoted by arrows. Binding curves were generated after subtracting nonspecific binding to mNOTCH peptide (A, B, I, and J) or GST (C–H) control surfaces.

Figure 6.
Pulling forces on spindle poles at 16 °C are normal in gpa-16(it143) but not gpa-16(it143)/goa-1(RNAi) embryos. Average peak velocities of the anterior (A) and posterior (P) spindle poles (±S.E.) following spindle severing of C. elegans embryos of the indicated genotypes. Experiments were performed as indicated either at 16 °C (this study) or at 25 °C (as previously described in Refs. 7, 9, 10 or in this study for gpa-16(it143)/goa-1(RNAi) embryos). For values and statistical tests, see supplemental Table 2.

The above figures are reprinted from an Open Access publication published by the ASBMB: J Biol Chem (2008, 283, 21550-21558) copyright 2008.

Figures were selected by the author.