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