Structural
St
t l and
d functional
f
ti
l studies
t di off
GPCRs
Bernadette Byrne
Department of Life Sciences
Outline of the talk
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Issues associated with membrane protein structural studies
Issues associated with GPCR structural studies
Expression
GPCR modifications
Antibody fragments
Combinations of approaches
Detergents
LCP
NMR
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Structure-function studies
Membrane Protein structural studies
Construct Design
Expression
Crystallisation
Solubilisation
Isolation
Structure determination
Issues associated with GPCR production for structural
studies
•Low/no
Low/no expression
•Aggregation/degradation upon
solubilisation/purification
•Loss of function
•High conformational flexibility
•Low thermal stability
•Aggregation over time
Rhodopsin (1F88, Palczewski
et al, 2000)
Conformational flexibility
βA J
AR
AR*
αAM
AR*G
KB
R
J’
R’
M’
R’G
KA
BR
βB J
BR”
αBM
BR”G
Progress
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First GPCR structure, Rhodopsin, published in 2000 (Palcewski
et al, 2000)
First non-rhodopsin structure, β2 adrenergic receptor, published
in 2007
00 (Rasmussen
( as usse et a
al,, 2007;
00 ; Rosenbaum
ose bau 2007)
00 )
By May (ish) 2013, 17 independent GPCR structures
By August 2014, 26 independent GPCR structures
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Significant methodological developments
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Expression of GPCRs
Expression system
GPCRs
Native tissue (eye)
Rhodopsin, 1F88
Mammalian
Rhodopsin, 2JFY
Insect: Spodptera frugiperda
mGlu1, 4OR2; P2Y12R,
R 4PX2
Insect: Trichoplusia ni
β1AR, 2VT4; A2AR, 2YDO
pastoris
Pichia p
H1R,, 3VG9;; A2AR,, 3RZE
Escherichia coli
CXCR1, 2LNL; NTSR, 4BUO
No structures from S. cerevisiae but reports of high level functional expression
Screening tool-Shiroishi et al, 2012
Receptor modifications
• Removal of extended C-terminus (Parker and Ross,1997)
• Removal of N
N-terminal
terminal residues (Tucker and Grisshammer,
Grisshammer 1996)
• Removal of N-linked glycosylation sites (Yurugi-Kobayashi et al,
2009)
• Addition of tags for purification and stability (Tucker and
Grisshammer 1996; Andre et al, 2006)
Cholesterol
• Cholesterol important for receptor stability in solution
Singh et al, 2010
Cholesterol
consensus motif
[4.39- 4.43(R,K)]—[4.50(W,Y)]—
[4.46(I,V,L)]—[2.41(F,Y)]
Hanson et al, 2008
Wu et al, 2014
Fusion protein approach
• Third ICL tends to be highly
flexible-key for interaction with Gprotein
• Remove ICL and replace with a
fusion partner
• Compact and highly crystallisable
• N and C-termini equidistant with
the ends of helices V and VI
• T4 Lysozyme
y
• Thermostabilised cytochrome
b562 (b562RIL)
Fusion protein approach β2-adrenergic receptor
Residues 231-262 replaced
Rosenbaum et al, 2007
Cherezov et al, 2007; 2RH1
Cytochrome b562RIL
• Fusions with b562RIL an alternative to
T4L
• Selected based on thermostability of
the fusion proteins
• Ability to diffuse in LCP
• Improvements
p
p
possible in resolution
compared to T4L
Liu et al, 2012; 4EIY
Different attachment points
T4L-β2AR
Zou et al, 2012; 4GBR
b562RILRIL
nociceptin/orphanin FQ
receptor
Thompson et al, 2012; 4EA3
Mutagenesis approaches
• 314 of 390 residues of β1
adrenergic receptor
• Thermostability compared to
wild-type
• Final construct 6 point
mutations and Tm 21°C
g
than WT
higher
Serrano-Vega et al, 2008
Altered receptor stability and function
WT
DDM
DM
NM
LDAO
OG
Mutant
Serrano-Vega et al, 2008
Agonist
Antagonist
Possible to select for specific receptor conformations
Structures by mutagenesis
β1AR, Warne et al, 2008;2VT4
A2AR, Lebon et al, 2011; 2YDO
Antibody fragments
• β2AR + Fab; partial structure; 2R4R
T4L
• A2AR + Fab; Hino et al; 3VG9
β2AR
• Nanobodies-single domain proteins
• Engineered from heavy chain only camelid
antibodies
Gαs
Gβ
• Smaller and more stable
• Contributed to the β2AR-G protein complex
structure
Gγ
Nanobody
Rasmussen et al, Nature, 2011; PDB 3SN6
Westfield et al, 2011
Comparison of the approaches-A2AR
T4L fusion protein, Jaakola et al, 2008; 3EML
Thermostabilization, Doré et al, 2011; 3PWH
Antibody fragment, Hino et al, 2012; 3VG9
Extracellular
Intracellular
Combinations of approaches
O approach
One
h iis nott always
l
enough
h
NTSR; White et al,
2012; 4GRV
mGlu5R; Dore et al, 2014; 4OO9
Novel detergents
MNG-1
MPA-12
MNG 2
MNG-2
DDM
MNG-3
DM
SDS
DDM 0 min
DDM 120 min
MNG-3 0 min
MNG-3 120 min
Chae et al, Nature
Methods 2010
Lipidic Cubic Phase crystallisation
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Bicontinuous lipid matrix
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Monoolein + Cholesterol
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GPCR-detergent mixture placed
over tiny droplets of LCP
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GPCR slowly exchanges into the
lipid phase
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3D crystals with packing
resembling 2D crystals
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Slower crystallisation
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MNG-3 has proven to be an
excellent stabiliser during LCP
crystallisation
http://cherezov.scripps.edu/images/LCPsm.jpg
Alternative ways of structurally characterising GPCRs
Solution state NMR: Sensory
Rhodopsin II
Solid state NMR: CXCR1 in
phospholipid bilayers
Gautier et al, 2010; 2KSY
Park et al,
al 2012; 2LNL
Shukla et al 2014, β2AR-β-arrestin 1 complex, single particle EM and HDX-MS
Thermostabilised mutants of GPCR
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Rag 23 (Tm: +5°C): F79A, A184L, R199A, L208A, L272A.
Rant 5 (Tm: +11°C): A54L, T88A, V239A
R t 21(Tm:
Rant
21(T +10°C):
10°C) A54L,
A54L T88A
T88A, K122A,
K122A V239A.
V239A
Magnani et al, PNAS, 2008
Aim
• Understand at a molecular level what make these A2AR
mutants thermostable
• Develop a tool we could use for other GPCRs to screen
potential stable mutants.
Method
Dowell and Brown, 2007
Functional analysis
Bertheleme et al, 2013, BJP
Extra mutant
• Generated intermediate mutant Rag23.1: back mutation of the
L208A (located in ICL3)
• WT and Rag23.1 have similar efficacy suggesting similar affinity for
G protein
Bertheleme et al, 2013, BJP
G-protein coupling
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P
Presence
off G-protein
G
t i iincreases affinity
ffi it off receptor
t ffor liligand
d
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If G-protein coupling has not been affected then we should see a
similar shift in Kd of receptors in the presence and absence of Gprotein
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Used two cell strains: MMY24 and MMY11 and looked at change
in binding affinity of NECA
G-protein coupling
WT + G-protein
G protein Kd
= 8.6±3.7 nM
Rag23.1 + G-protein
Kd = 1.6±0.2 nM
WT - G-protein
G protein Kd
= 123.9±7.1 nM
Rag23.1 - G-protein
Kd = 14.4±1.3 nM
Bertheleme et al, 2013, BJP
Summary
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Loss of constitutive activity
y is correlated with increased
stability of A2AR
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Constitutively active conformation is distinct from the agonist
induced activity conformation
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The effect of mutagenesis on proteins is difficult to predict
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C provide insight into protein ffunction
Can
Summary
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Rapid progress in GPCR structural studies
Several novel engineering approaches
Antibodies/Nanobodies
Combinations of approaches
Novel detergents
LCP crystallisation
• Structures
St t
complemented
l
t db
by ffunctional
ti
l analysis
l i
Acknowledgments
Nicolas
Rohini
Shweta
Yil
Yilmaz
Prof Philip Strange,
University of Reading
James
Prof Sam Gellman, Dr Pil-Seok
Chae, University of Wisconsin
Dr Simon Dowell