Engineering a minimal G protein to facilitate crystallisation of G protein-coupled receptors in their active conformation.
G protein-coupled receptors (GPCRs) modulate cytoplasmic signalling in response to extracellular stimuli, and are important therapeutic targets in a wide range of diseases. Structure determination of GPCRs in all activation states is important to elucidate the precise mechanism of signal transduction and to facilitate optimal drug design. However, due to their inherent instability, crystallisation of GPCRs in complex with cytoplasmic signalling proteins, such as heterotrimeric G proteins and β-arrestins, has proved challenging. Here, we describe the design of a minimal G protein, mini-Gs, which is composed solely of the GTPase domain from the adenylate cyclase stimulating G protein GsMini-Gsis a small, soluble protein, which efficiently couples GPCRs in the absence of Gβγ subunits. We engineered mini-Gs, using rational design mutagenesis, to form a stable complex with detergent-solubilised β1-adrenergic receptor (β1AR). Mini G proteins induce similar pharmacological and structural changes in GPCRs as heterotrimeric G proteins, but eliminate many of the problems associated with crystallisation of these complexes, specifically their large size, conformational dynamics and instability in detergent. They are therefore novel tools, which will facilitate the biochemical and structural characterisation of GPCRs in their active conformation.
MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus , Francis Crick Avenue, Cambridge CB2 0QH, UK byronc@mrc-lmb.cam.ac.uk cgt@mrc-lmb.cam.ac.uk.
Byron
B
Carpenter
MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus , Francis Crick Avenue, Cambridge CB2 0QH, UK byronc@mrc-lmb.cam.ac.uk cgt@mrc-lmb.cam.ac.uk.
Christopher G
CG
Tate
D001665:Binding Sites; D003460:Crystallization; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D016296:Mutagenesis; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D000072417:Protein Domains; D015202:Protein Engineering; D055550:Protein Stability; D043705:Receptor, Adenosine A2A / Q000737:chemistry* / Q000378:metabolism; D013696:Temperature
D010446:Peptide Fragments; D043705:Receptor, Adenosine A2A; D019204:GTP-Binding Proteins
G protein; G protein-coupled receptor; GPCR; Gs
; complex; mini G protein; mini-Gs

Trends Biochem Sci. 2012 Sep;37(9):343-52
22784935
Nature. 2013 Dec 5;504(7478):101-6
24256733
Nature. 1996 Jan 25;379(6563):311-9
8552184
Nature. 2011 Jul 19;477(7366):549-55
21772288
Nature. 2009 May 21;459(7245):356-63
19458711
Science. 1988 Aug 12;241(4867):832-5
3136547
Nature. 2013 Oct 24;502(7472):575-9
24056936
Proc Natl Acad Sci U S A. 1981 Jan;78(1):152-6
6264430
Trends Biochem Sci. 2015 Jan;40(1):49-57
25544475
Nature. 2015 Aug 13;524(7564):173-9
26147082
Nature. 2011 May 18;474(7352):521-5
21593763
J Biol Chem. 2014 Aug 29;289(35):24475-87
25037222
Proc Natl Acad Sci U S A. 2008 Jan 22;105(3):877-82
18192400
Annu Rev Biochem. 1997;66:639-78
9242920
Nature. 2011 Jan 13;469(7329):241-4
21228877
Protein Expr Purif. 2009 Jun;65(2):204-13
19297694
Nature. 2011 Jan 13;469(7329):236-40
21228876
J Mol Biol. 2011 Oct 28;413(3):628-38
21907721
Science. 2015 Jun 19;348(6241):1361-5
26089515
EMBO J. 2002 May 1;21(9):2095-106
11980706
Nature. 2008 Sep 25;455(7212):497-502
18818650
J Biol Chem. 1992 Aug 25;267(24):17040-6
1512243
Science. 1993 Dec 17;262(5141):1895-901
8266082
Naunyn Schmiedebergs Arch Pharmacol. 2011 Jul;384(1):71-91
21547538
J Mol Biol. 2011 Jun 10;409(3):298-310
21501622
Science. 2011 Apr 15;332(6027):322-7
21393508
J Biol Chem. 1987 Jan 15;262(2):762-6
3100519
Proc Natl Acad Sci U S A. 2011 Jun 7;108(23):9420-4
21606326
Trends Biochem Sci. 1991 Sep;16(9):338-41
1949155
Nature. 2015 Aug 20;524(7565):315-21
26245379
J Biol Chem. 2006 Oct 6;281(40):30234-41
16847064
Nat Struct Mol Biol. 2006 Sep;13(9):772-7
16892066
Nucleic Acids Res. 2010 Jul;38(Web Server issue):W545-9
20457744
Nat Struct Mol Biol. 2015 Sep;22(9):686-94
26258638
Nat Rev Drug Discov. 2008 Apr;7(4):339-57
18382464
Nature. 1984 Oct 18-24;311(5987):659-61
6090950
Proc Natl Acad Sci U S A. 2011 Sep 20;108(38):16086-91
21914848
Biochemistry. 1990 Jun 26;29(25):6058-65
2200519
Nature. 2016 Aug 4;536(7614):104-7
27462812
Nat Protoc. 2007;2(9):2212-21
17853878
Eur J Biochem. 1989 Oct 1;184(3):687-98
2509200
J Biol Chem. 1980 Aug 10;255(15):7108-17
6248546
Nature. 2011 Jan 13;469(7329):175-80
21228869
Biochim Biophys Acta. 2003 Feb 17;1610(1):133-40
12586387
Nat Struct Mol Biol. 2014 Jan;21(1):56-63
24292645
PLoS One. 2014 Mar 24;9(3):e92727
24663151
Science. 1997 Dec 12;278(5345):1943-7
9395396