Refolding of membrane proteins for structural studies

Lars Linden * RAMC 2005

Membrane proteins as drug targets

m-phasys is the only company focussed exclusively on 3D structures of membrane protein targets

m-phasys is the only company focussed exclusively on 3D structures of membrane protein targets

The human genome: 25% of the human genes

encode for membrane proteins

75%25%

membrane proteins

soluble proteins

67% of the known drug targets are membrane proteins

The known drug targets:67% 33%

All known protein structures:~ 28,000

No human GPCR structure solved

GPCR structures: 1(Rhodopsin from bovine retina)

Membrane protein structures: ~ 100(mostly bacterial proteins)

Why ?Why ?

Human GPCR structures: 0

PDB

Barriers in membrane protein structural analysis...

... and how to get around them

... and how to get around them

Expression system

Purification & Crystallization

3D structureDNA

Refolded protein

Expression in Inclu-sion bodies

Detergent Solubilized

protein

crystal

E. coli ?

• Fast• Cheap• High yields• Multiple strains available• Multiple plasmids available• Selenomethionine derivatives

• Less time for expression = more time for crystallization!

• In 2004, 67% of all structures deposited in the PDB were from proteins expressed in E. coli

Percentage of structures from proteins produced in E. coli

Expression of membrane proteins in E. coli can be toxic

• Eukaryotic membrane proteins are not readily inserted into bacterial membranes

• Bacterial insertion machinery becomes jammed

• Protein production stops after 1 min

• Low yields

Possible solution: Prevent membrane insertion

Does in vitro refolding of membrane proteins work ?

Critical issues:• Energy landscape in

micelles?• Non-vectorial insertion• Local vs. Global

minimum?

Does in vitro refolding of membrane proteins work ?

Yes!

• Bacteriorhodopsin• Light harvesting complex LHC2• Mitochondrial transporters• Diacyl glycerol kinase• Olfactory receptor OR5• Potassium channel KcsA• DsbB• Leukotriene receptor BLT1

pGEX2a-GPCR-His

~6000 bp

APrGST

lac I

HisTag

Ptac

ori

rrBT1T2

Protease cleavage site

GPCR

Expression vector for GST-GPCR-(His)6 fusions

•Expression in E.coli•Preparation of inclusion bodies•Typical yields: 2-50 mg / l

How to identify refolding conditions

Inclusion Bodies(Aggregated Protein)

Refolded & nativeMisfolded Re-aggregated

Solubilisation

Solubilised, butmisfolded protein

Detergent exchange

Purification and quality control of GPCRs

Principal analysis Threshold

Purity (SDS-PAGE): > 90%

Monodispersity (SEC) > 90%

Specific activity (arrestin assay*)

> 70%

Concomitant analysis

Light scattering (DLS)

Ligand binding measurement

G protein activation

GPCRs are rigorously testedfor activity and homogeneitybefore crystallization

GPCRs are rigorously testedfor activity and homogeneitybefore crystallization

*) proprietary functional assay applicableto all GPCRs (including orphans)

Arrestin activity assay

• Arrestin mutant binds to GPCRs constitutively • Doesn't require phosphorylation• Affinity depends on ligand binding• Requires folded GPCR

RA A

RA

RA

AR

1. Bind & wash 2. Detect bound arrestin

GPCR properly folded

GPCR not properly folded

G protein activation

Log Interleukin-8 [M]

-2,0 -1,5 -1,0 -0,5 0,0

5000

10000

15000

20000

25000

30000

35000

40000

45000

g

EC50

= 0.1 nM

Bound G

TPS

[dpm

]

Ligand binding

-2 -1 0 1 2 3

1500

2000

2500

3000

3500

Interleukin 8K

D = 5 nM

Log Interleukin-8 [M]

Bound lig

and [

dpm

]

Refolded GPCRs are functionalExample: CXCR1

Refolded GPCR binds ligand and couples to G protein

Refolded GPCR binds ligand and couples to G protein

Conclusion:• Ligand affinity (KD) like

native receptor• > 80% refolded (Bmax)

Conclusion:• Couples to Gi/o

• EC50 like native receptor

Refolded GPCRs are homogenousExample: CXCR1

SDS-PAGE

1 2 3

- GPCR dimer

- GST-GPCR fusion

- GPCR monomer

1. Inclusion body fraction2. Ni chelate purified3. SEC purified

Refolded CXCR1 is >90% pure and monodisperse

Refolded CXCR1 is >90% pure and monodisperse

Conclusion:• 95 % pure on SDS gel

Conclusion:• 85 % pure by SEC

analysis

SEC

Abso

rpti

on

Volume [ml]

8 10 12 14 16-0,02

0,00

0,02

0,04

0,06

0,08

0,10

0,12

Superdex 200

Refolded GPCRs are homogenousExample: GPR3

Analysis Result

Purity (SDS-PAGE): 95 %

Monodispersity (SEC) 90 %

Specific activity (arrestin assay)

80 %

8 10 12 14 16-0,02

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

A

bso

rpti

on

Volume [ml]

Refolded GPCRs form crystals

CrystallizedPipelineCrystallized

Pipeline

Rhodopsin family

Optimization of crystallization conditions: strategy

• Truncated mutants (N- and C-termini, long loops)

• Co-crystallization with ligands (agonists, antagonists, inverse agonists)

• Co-crystallization with binding proteins (ß-arrestin, G proteins, antibody fragments)

• Stabilization with lipids

• Variation of crystallization method: vapour diffusion, microbatch, lipidic cubic phases, free interface diffusion

• Selection for more thermostable mutants

Anti-GPCR monoclonal antibodies

• Successful programs with antibody companies and academic

groups

• Refolded GPCRs used as immunogen or panning target

• Antibodies obtained from mice (IgG) and phage display systems

(scFvs and Fabs)

• Antibodies recognize native GPCRs (FACS)

• Affinity from 1 nM to 1 µM

• Some are antagonistic

• Some have conformation-specific epitopes

Apart from their use in co-crystallization, antibodies might be

used as diagnostic tools or therapeutics

m-fold CXCR1-antibody complex formation

• Immunization with CXCR1 Liposomes• Monoclonal IgG, FACS and ELISA positiv

• Ligand (IL-8) is displaced by antibody (IC50 = 0,33 nM)

• CXCR1 receptor and 9D1 antibody form a stable complex• scFv cloned, expressed and purified -> Co-crystallisation

0.0

10.0

20.0

30.0

mAU

6.0

8.010.0

12.014.0

ml

CXCR1

B: anti-CXCR1 mAB 9D1A: CXCR1-receptor

0

20

40

60

80

100

mAU

6.0

8.010.0

12.0 14.0 ml

9D1

BSA

Aggregate

C: co-complex

0

20

40

60

mAU

6.0 8.0 10.0 12.0 14.0 ml

CXCR1 + 9D1

Bacterial and human ion channels

• Potassium Channels :

voltage gated KvLQT4

hERG

Kv1.3

VIC (Salmonella t.)

MJKch (Methanococcus j.)

Ca2+ activated KCa4

• Cloning and expression of different constructs of hERG, Kv1.3, KCa4 transmembrane region S1-S6

Bacterial and human ion channels

• Ion channels are easily purified

• Refoldung screen for hERG, Kv1.3, KCa4, VIC and MJKch

• Tetramerisation can be detected on modified SDS or blue native Gels

VIC

tetramer

monomer

hERG

tetramer

66

132

11666

4535

25

116

66

45

35

25

1818

Potassium channel can be produced with M-FOLD™

Refolding works for K channels

Refolding works for K channels

116

66

4535

25

18

unfo

lded

116

66

4535

25

18re

fold

ed

Conclusion:• Refolded K channel

forms tetramer• > 95 % refolded

Refolded K channels reconstituted into planar bilayer (BLM)

K channel crystals

Ion channel crystals diffract to 12 Å

Acknowledgement