PDBsum entry 3onw

Signaling protein

PDB id: 3onw

Contents

Protein chains

316 a.a. *

35 a.a. *

34 a.a. *

Ligands

GDP ×2

SO4 ×3

Waters ×97

* Residue conservation analysis

PDB id:

3onw

Name:

Signaling protein

Title:

Structure of a g-alpha-i1 mutant with enhanced affinity for the rgs14 goloco motif.

Structure:

Guanine nucleotide-binding protein g(i) subunit alpha-1. Chain: a, b. Synonym: adenylate cyclase-inhibiting g alpha protein. Engineered: yes. Mutation: yes. Regulator of g-protein signaling 14. Chain: c, d. Synonym: rgs14. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: gnai1. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes. Other_details: the rgs14 goloco motif peptide was synthesized according to the human rgs14 sequence, identical to that used in PDB

Resolution:

2.38Å R-factor: 0.230 R-free: 0.265

Authors:

D.Bosch,A.J.Kimple,D.W.Sammond,M.J.Miley,M.Machius,B.Kuhlman, F.S.Willard,D.P.Siderovski

Key ref:

D.E.Bosch et al. (2011). Structural determinants of affinity enhancement between GoLoco motifs and G-protein alpha subunit mutants. J Biol Chem, 286, 3351-3358. PubMed id: 21115486 DOI: 10.1074/jbc.M110.190496

Date:

30-Aug-10 Release date: 24-Nov-10

Protein chains

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

Seq: 354 a.a.

Struc: 316 a.a.*

Protein chain

O43566  (RGS14_HUMAN) -  Regulator of G-protein signaling 14 from Homo sapiens

Seq: 566 a.a.

Struc: 35 a.a.

Protein chain

O43566  (RGS14_HUMAN) -  Regulator of G-protein signaling 14 from Homo sapiens

Seq: 566 a.a.

Struc: 34 a.a.

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

Enzyme reactions

• Enzyme class: Chains A, B: E.C.3.6.5.- - ?????

DOI no: 10.1074/jbc.M110.190496

J Biol Chem 286:3351-3358 (2011)

PubMed id: 21115486

Structural determinants of affinity enhancement between GoLoco motifs and G-protein alpha subunit mutants.

D.E.Bosch, A.J.Kimple, D.W.Sammond, R.E.Muller, M.J.Miley, M.Machius, B.Kuhlman, F.S.Willard, D.P.Siderovski.

ABSTRACT

GoLoco motif proteins bind to the inhibitory G(i) subclass of G-protein α subunits and slow the release of bound GDP; this interaction is considered critical to asymmetric cell division and neuro-epithelium and epithelial progenitor differentiation. To provide protein tools for interrogating the precise cellular role(s) of GoLoco motif/Gα(i) complexes, we have employed structure-based protein design strategies to predict gain-of-function mutations that increase GoLoco motif binding affinity. Here, we describe fluorescence polarization and isothermal titration calorimetry measurements showing three predicted Gα(i1) point mutations, E116L, Q147L, and E245L; each increases affinity for multiple GoLoco motifs. A component of this affinity enhancement results from a decreased rate of dissociation between the Gα mutants and GoLoco motifs. For Gα(i1)(Q147L), affinity enhancement was seen to be driven by favorable changes in binding enthalpy, despite reduced contributions from binding entropy. The crystal structure of Gα(i1)(Q147L) bound to the RGS14 GoLoco motif revealed disorder among three peptide residues surrounding a well defined Leu-147 side chain. Monte Carlo simulations of the peptide in this region showed a sampling of multiple backbone conformations in contrast to the wild-type complex. We conclude that mutation of Glu-147 to leucine creates a hydrophobic surface favorably buried upon GoLoco peptide binding, yet the hydrophobic Leu-147 also promotes flexibility among residues 511-513 of the RGS14 GoLoco peptide.