PDBsum entry: 1y3a

Signaling protein

PDB id: 1y3a

Contents

Protein chains

299 a.a. *

12 a.a. *

Ligands

GDP ×4

Waters ×136

* Residue conservation analysis

PDB id:

1y3a

Name:

Signaling protein

Title:

Structure of g-alpha-i1 bound to a gdp-selective peptide provides insight into guanine nucleotide exchange

Structure:

Guanine nucleotide-binding protein g(i), alpha-1 subunit. Chain: a, b, c, d. Fragment: sequence database residues 25-353. Synonym: adenylate cyclase-inhibiting g alpha protein, g- alpha-i1. Engineered: yes. Kb752 peptide. Chain: e, f, g, h.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: gnai1. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008. Expressed in: escherichia coli. Expression_system_taxid: 562. Other_details: protein selected by phage display

Biol. unit:

Dimer (from PQS)

Resolution:

2.50Å R-factor: 0.240 R-free: 0.280

Authors:

C.A.Johnston,F.S.Willard,M.R.Jezyk,Z.Fredericks,E.T.Bodor, M.B.Jones,R.Blaesius,T.K.Harden,J.Sondek,V.J.Watts, J.K.Ramer,D.P.Siderovski

Key ref:

C.A.Johnston et al. (2005). Structure of Galpha(i1) bound to a GDP-selective peptide provides insight into guanine nucleotide exchange. Structure, 13, 1069-1080. PubMed id: 16004878 DOI: 10.1016/j.str.2005.04.007

Date:

24-Nov-04 Release date: 12-Jul-05

Protein chains

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

Seq: 354 a.a.

Struc: 299 a.a.

Protein chain

No UniProt id for this chain

 Gene Ontology (GO) functional annotation 

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

DOI no: 10.1016/j.str.2005.04.007

Structure 13:1069-1080 (2005)

PubMed id: 16004878

Structure of Galpha(i1) bound to a GDP-selective peptide provides insight into guanine nucleotide exchange.

C.A.Johnston, F.S.Willard, M.R.Jezyk, Z.Fredericks, E.T.Bodor, M.B.Jones, R.Blaesius, V.J.Watts, T.K.Harden, J.Sondek, J.K.Ramer, D.P.Siderovski.

ABSTRACT

Heterotrimeric G proteins are molecular switches that regulate numerous signaling pathways involved in cellular physiology. This characteristic is achieved by the adoption of two principal states: an inactive, GDP bound state and an active, GTP bound state. Under basal conditions, G proteins exist in the inactive, GDP bound state; thus, nucleotide exchange is crucial to the onset of signaling. Despite our understanding of G protein signaling pathways, the mechanism of nucleotide exchange remains elusive. We employed phage display technology to identify nucleotide state-dependent Galpha binding peptides. Herein, we report a GDP-selective Galpha binding peptide, KB-752, that enhances spontaneous nucleotide exchange of Galpha(i) subunits. Structural determination of the Galpha(i1)/peptide complex reveals unique changes in the Galpha switch regions predicted to enhance nucleotide exchange by creating a GDP dissociation route. Our results cast light onto a potential mechanism by which Galpha subunits adopt a conformation suitable for nucleotide exchange.

Selected figure(s)

Figure 5.
Figure 5. Biochemical Confirmation of the Overall Structural Features of the Ga[i1]/KB-752 Interaction
(A) Ribbon trace of KB-752 (red) bound between the a2 ("switch II") and a3 helices of the Ga[i1] Ras-like domain (blue). No contacts are made between KB-752 and the all-helical domain (yellow) or bound GDP (magenta). Switch regions are denoted in green.
(B) Structural basis for nucleotide selective binding of KB-752 to Ga[i1]. KB-752 peptide (red, translucent) binds Ga[i1] between switch II and the a3 helix; the conformations of these two helices are shown for Ga[i1] operator GDP/KB-752 (green), Ga[i1] operator GTPgS (yellow), and Ga[i1] operator GDP operator AlF[4]^ - (magenta). Whereas the a3 helix is not significantly altered, switch II is displaced to accommodate KB-752 binding. Switch II in both Ga[i1] operator GTPgS and Ga[i1] operator GDP operator AlF[4]^ - assumes an extended a-helical conformation that is stabilized relative to Ga[i1] operator GDP (Mixon et al., 1995; Sprang, 1997). This conformation of switch II is not permissive to KB-752 binding, as it creates extensive steric hindrance. In particular, W211 of switch II (shown in space filling) is in a restrictive position relative to W5 of KB-752.
(C) The GoLoco motif of RGS14 (orange) is also seen to bind, in an a-helical conformation, between switch II and the a3 helix of Ga[i1] (PDB ID 1KJY); the critical arginine finger which contacts GDP is highlighted within the Ca carbon ribbon trace of the GoLoco peptide. Other features are colored as in (A).
(D) KB-752 GEF activity does not rely on the all-helical domain. 100 nM Ga[i1] or a chimeric Ga containing the Ras-like domain of Ga[i1] and the all-helical domain of Ga[o] ("Gaioi" [Remmers et al., 1999]) was incubated in the absence or presence of 50 µM KB-752, and [35S]GTPgS binding after 10 min at 30°C was measured as described in the Experimental Procedures. Data are expressed as a percentage of GTPgS bound relative to Ga protein in the absence of KB-752 ("Control") and are the average ± SEM of four independent experiments. Data shown are the mean ± SEM for five independent experiments conducted in triplicate.
(E) The KB-752 binding site on Ga[i1] overlaps that of GoLoco motif peptides. Ga[i1] (50 nM) was incubated in the absence or presence of the indicated concentrations of a peptide representing the GoLoco motif of RGS12 (R12GL) (Kimple et al., 2002). GTPgS binding was then measured in the presence of the indicated concentrations of KB-752. Data are expressed as fmol of GTPgS bound above that measured in the absence of KB-752 and are from a representative experiment of three independent experiments.
(F) The binding of KB-752 has no effect on the kinetics of Ga[i1] activation by AlF[4]^ -, unlike the slowed activation rate seen upon GoLoco peptide binding. Ga[i1]-CFP (200 nM) and YFP-RGS4 (280 nM) fusion proteins, previously shown to generate increased fluorescence resonance energy transfer (FRET) upon Ga[i1] activation by AlF[4]^ - and subsequent RGS-box binding (Willard et al., 2004), were mixed together and preincubated with either 10 µM KB-752 peptide or 5 µM GoLoco consensus peptide (AGS3Con [Kimple et al., 2002]), prior to the addition of NaF and AlCl[3] to final concentrations of 20 mM and 30 µM, respectively, at the 150 s mark.

Figure 8.
Figure 8. Comparison of Switch Regions and Core Catalytic Residues of KB-752 Bound Ga[i1] with Other States of Ga[i1]
(A) Movement of switch I in the Ga[i1] operator GDP/KB-752 complex (green), versus its position in the Ga[i1]b[1]g[2] heterotrimer (blue) and the Ga[i1] operator GDP/R14GL complex (orange), results in disruption of a salt bridge (black dotted line) between R178 and E43 that normally stabilizes bound GDP (magenta) within Ga[i1] when complexed to a GDI (Gbg or GoLoco peptide).
(B) Electron density of the R178 side chain in the Ga[i1] operator GDP/KB-752 complex (from a 2F[o] - F[c] simulated annealing composite omit map contoured to a level of 1s) is denoted by white mesh. In the background is the b phosphate of the bound GDP (bP).
(C and D) Switch region comparisons with activated Ga[i1] states. Switch regions of Ga[i1] operator GDP/KB-752 (green), Ga[i1] operator GDP operator AlF[4]^ - (PDB code 1GFI; magenta; [C]), and Ga[i1] operator GTPgS (PDB code 1GIA; yellow; [D]) are shown along with the residues critical for GTP hydrolysis (R178 and T181 within switch I and Q204 within switch II). GDP from the Ga[i1] operator GDP/KB-752 structure is shown for reference in each case. Overall conformation of the switch regions of Ga[i1] operator GDP operator AlF[4]^ - and Ga[i1] operator GTPgS are very similar, save for key changes in the position of catalytic residue side chains (Wall et al., 1998). Whereas switch I of Ga[i1] operator GDP/KB-752 is very similar to that of the activated forms, both switch II and III are dramatically removed from the guanine nucleotide to allow for GDP release. The catalytic Q204 residue within switch II is far removed from the bound nucleotide and active site for GTP hydrolysis in the Ga[i1] operator GDP/KB-752 structure. However, R178 and T181 of switch I are in a strikingly similar position to that of the Ga[i1] operator GDP operator AlF[4]^ - structure.

The above figures are reprinted from an Open Access publication published by Cell Press: Structure (2005, 13, 1069-1080) copyright 2005.

Figures were selected by the author.