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Nanobodies for the structural analysis
of GPCR transmembrane signaling
Strasbourg
September 2011
Steyaert lab
Structural Biology Brussels
GPCRs are complex
allosteric switches
Ortosteric ligands
Allosteric ligands
Biased ligands
G proteins
β arrestins
GRKs
Basal activity
100
50
Bio
log
ica
lre
sp
on
s (
%)
Log drug concentration
Basal states
Conformational complexity of
GPCR behaviour
G protein
Agonist
G protein
Partial agonist
Full agonist
Inverse
agonist
Inverse agonistG proteinG protein
Capturing motions?
Basal states
G protein G protein
Agonist
Inverse agonist
Neutral antagonist
100
50
Bio
log
ica
lre
sp
on
s (
%)
Log drug concentration
Inverse
agonist
Ways to reduce the complexity
in conformation rich proteins
Mutagenesis
Mabs
T4L insertions
G protein G protein
Conformation selective Nanobodies?
G protein G protein
Conventional antibodies
CH3
CH2
Fc
Large
Two chains
Hybridoma technology
Two cDNAs
Unstable
Difficult to express in E.coli
Fab scFv
Conventional versus
Heavy-chain antibodies
CH3
CH2
FcCH2
CH3
Fc
CH1 domain Long hinge
hydrophobic VH-VL interface hydrophilic surface
6 diversity regions 3 diversity regions
VHH or Nanobody
CH2
CH3
Fc
Camel Heavy-Chain
antibody
Immunoglobulin fold
12 Kd
VH versus VHH
VHVH
N
C
CDR1 CDR2 CDR3VHH
V37
G44
L45
W47
F37
E44
R45
G47
VHH
N
C
CDR1 CDR2 CDR3
Key Nb features
• Immunoglobulin fold
• Long CDR1
• Longer CDR3, key player
• CDR1-S-S-CDR3
• Cavity binding
• Conformational epitope
Nanobody discovery platform
Immunize llama
with native antigen
Collect a blood sample
after 6 to 12 weeks
Nanobody discovery platform
Immunize llama
with native antigen
Select in vivo
matured nanobodies
by phage display
Collect a blood sample
after 6 to 12 weeks
Generation of β2-AR binders
Recombinant receptor
reconstituted in lipid vesicles
DOPC:Lipid A (10:1)
+-50
0
50
100
-14 -12 -10 -8 -6 -4
log(agonist)
G protein G protein
Generation of β2-AR binders
Receptor+
GERBU
Receptor+
GERBU
Receptor+
GERBUReceptor Receptor
Day 1 Day 14 Day 28 Day 42 Day 56
CDR1 CDR2 CDR3 His-Tag
β2-AR specific Nbs
Selection of conformational binders
Folded β2-AR
+ BI187107
Heat denaturated
β2-AR + BI187107
Folded β2-AR
+ Carazolol
M1 Nb65 Nb67 Nb69 Nb71 Nb72 Nb80 Nb84 Nb60 Nb68 Nb74
Nbs bind exclusively to the agonist
bound receptor
Elution volume (ml)10 20
UV
28
0 a
bso
rbti
on
Size exclusion
Nb80
β2-AR + BI187107
β2-AR + carazolol + Nb80
β2-AR + BI187107 + Nb80
Nbs with G protein-like behaviour?
G protein G protein
Full agonist
Full agonist: isoproterenol
Nbs with G protein-like behaviour?
Basal statesAgonist
G protein G protein
Inverse
agonist
Log ICI-118551 conc. (M)
β2-AR HDL
Inverse agonist: ICI-118551
Nbs with G protein-like behaviour
Zie Yao, X. et al. (2006) Nat. Chem. Biol. 2, 417-422
TM6
TM3
Nbs as crystallization
chaperones for β2-AR
Turning on
a GPCR
Carazolol structure
BI187107-Nb80 structure
Ionic lock?
G-protein binding site
Opsin-transducinb2AR-Nb80
Holy grail of signal transduction
• Nucleotides
• Detergents
Generate 2-AR:Gs specific
Nanobodies
Complex+
GERBU
Complex+
GERBUComplex Complex
Day 1 Day 14 Day 28 Day 42
Selection by panning
Selection by panning
Apyrase
Str
epta
vid
in c
oate
d s
urf
ace
GDP bound
Nb35 protects to complex from
dissociation by GTP
T4L
2-AR
GS
G
G
Nb35
Crystallization of the 2AR-Gs complex
2-AR
GS
G
G
Nb35
Nb35 binds the GSG interface
-helical domain
Ras like
GTPase domain
Rigid body movement of -helical domain
GTPS-bound Gs
Carazolol:2-AR
BI:2-AR:Nb80
BI:2-AR:Gs
Active vs inactive GPCR
conformations
GTPS-bound Gs (PDB ID: 1AZT)
In conclusion
Nbs recognize conformational epitopes on GPCRs
Nbs selectively stabilize active conformations of
GPCRs
Nbs can mimic the effects of GPCR ligands, including
the G-protein
Nbs serve as crystallization chaperones for GPCRs
Conformational Nbs have reciprocal allosteric effects
on agonist vs inverse agonist binding
Nbs can stabilize transient PPIs like the GPCR-Gs
complex
Thermal stabilization of 2-AR
Thermal stabilization of 2-AR
2-AR+ISO
2-AR+ISO+Nb80
2-AR+Nb80 (No ISO)
Kobilka lab
Stanford University
Steyaert lab
Vrije Universiteit Brussel• Els Pardon
• Nele Buys
Composite view of
transmembrane signaling
Parts of multiprotein complexes
Secretion system II
E. coli
Multidomain proteins
Intrinsically disordered proteins
MazE-Nb complex
Amyloidogenic proteins
2-microglobulin
Chaperone-assisted X-ray crystallography
with nanobodies: Xaperones.com
Xaperones are antigen binding fragments from
heavy chain-only antibodies that:
• Increase the stability of soluble proteins and
solubilized membrane proteins
• Reduce the complexity in conformationally
rich proteins and protein complexes
• Allow to affinity-trap active protein
• Increase the polar surface enabling the
growth of diffracting crystals
• Bind cryptic epitopes and lock proteins in
unique native conformations
Chaperone-assisted X-ray crystallography
with nanobodies: Xaperones
IDP
Crystal lattice
Chaperone-assisted X-ray crystallography
with nanobodies: Xaperones
Multidomain
protein
Reduced conformational flexibility
Chaperone-assisted X-ray crystallography
with nanobodies: Xaperones
Amyloidogenic
monomer
Steric hindrance
HTS or fragment based
Applications of conformational
nanobodies
HTS or fragment based
Applications of conformational
nanobodies
Drugable conformations• GPCRs
• Channels
• Kinases
• Nuclear receptors
Boni• Crystallization chaperone
• Purification
• Biophysics
• Biology
Nb affinities
Phage selectedBacterial 2 Hybrid
10-4
10-3
10-2
10-1
103
104
105
106
BMInt71
BMInt88
BMInt67
BMInt64
BMInt62
BMInt53
BMInt51
BMInt47
BMInt6
BMInt58
BMInt4
cAbInt9
cAbInt7
cAbInt2
cAbInt8
cAbInt6
cAbInt5
cAbInt11
cAbInt3
cAbInt1cAbInt10
cAbInt12
cAbInt4
ko
n (
1/M
s)
koff
(1/s)
10 µM
1 µM
100 nM10 nM1 nM
CB2 bindersReconstituted receptor
+
Agonist CP55940
Receptor+
GERBU
Receptor+
GERBU
Receptor+
GERBUReceptor Receptor
Day 1 Day 14 Day 28 Day 42 Day 56
CB2 binders
• 70 unique sequences
– 50 can be grouped in 8 sequence families
– 20 represent seperate unique sequence families
28 different paratopes
Klaus Gawrisch, NIH, Bethesda, Washington
CB2 + agonist
CB2 + inv. agonist
CB2 + agonist
CB2 + inv. agonist
Immune system seems the explore different conformations
of the immunized GPCRs
CXCR4 binders
6 X
CHO background
+
HEK background
Nanobody family # sequences/family # unique representatives
1 27 13
2 3 3
3 2 1
4 19 8
5 16 5
6 1 1
7 1 1
8 1 1
CXCR4 binders
Nb CA4142Marc Parmentier, ULB
CXCR4
CXCR4 cl 44717
Control
Nb CA4137 Nb CA4138 Nb CA4139 Nb CA4140 Nb CA4141
Nb CA4142 Nb CA4143 Nb CA4144 Nb CA4145 Nb CA4146
CXCR4 antagonistsLum
inescence
Log [SDF-1a]
Aequorin-based calcium mobilization assay (CHO-CXCR4 cells)
Log [AMD3100]Log [Nb CA4143]Log [Nb CA 4145]
Rearrangements of transmembrane
segment packing interactions
D3.49
R3.50
Y3.51
12Å
Whorton et. al. (2007) PNAS 104, 7682.
Nbs with G protein-like behaviour?
G protein G protein
Full agonist
R:G ratio
β2-AR HDLs
GPCRs are allosteric switches
Ortosteric, allosteric or biased ligands
G proteins, β arrestins, GRKs
Heavy-chain antibodies
Tylopoda Camelidae
Camelus dromedariusCamelus bactrianus
Lama glamaLama guanocoLama alpacaLama vicugna
GPCR transmembrane signaling
Nbs with G protein-like behaviour?
Partial agonist: salbutamol
Versatility of the Nb fold
Amyl-B7 Amyl-D9Amyl-D10
Amyl-D9
framework
Porcine amylase