X-ray Protein Crystallography on
Membrane Proteins

Riken Harima Institute/Kyoto
University
So Iwata

All Proteins in Our Body Have Regular 3D
Structures at the Atomic Level

Structural elements of proteinsProteins are made of polypeptide
chains. Depending on the aminoacid sequence of the chain, proteins
can take various local structures likealpha helix or beta sheet.
Alpha helix

beta sheet

Example of proteinsMolecular Anatomy: X-ray Crystallography Helps to Solvethe Mystery of Protein Structure and Function

DNA Cloning

Protein Expression

Purification

Crystallization

X-ray Data Collection Phasing & Electron Density Map Structure Model

Membrane Protein Structures Solved in our Group
Oxygen Respiratory
Complexes

Complex III

Formate Dehydrogenase

Anaerobic
Respiratory
Complexes
テキスト
Complex II:
Complex IV

Complex II

Photosynthesis

Science299 2003
Complex III: Science
281, 1998, N. Engl. J.
Med. 341,1999
Complex IV: Nature
376, 1995, Nature Str.
Biol. 7, 2000

Nitrate Reductase

Formate Dehydrogenase: Science 295
2002
Nitrate Reductase:
Structure 12, 2004
Polysulfide reductase:Nat. Str. Mol. Biol. 15,
2008

Transporters

Photosystem II

LacY

Mhp1

Photosystem II:
Science 299 2003
LacY: Science 301,
2003, EMBO J. 25,
2006, PNAS 104, 2007
Mhp1: Science 322,
2008

Membrane Proteins• Various functions: oxygen respiration, photo

synthesis, signal transduction, moleculartransport and so on.

• Over 30% of proteins in the human genome
are membrane proteins (up to 10,000).

Example Membrane Protein Structures from
Our Group: Lactose Permease (Sugar
Transporter)• Responsible forsugar uptake intothecells.
Homologue of thehuman glucose
transporter, which
dysfunction causes
d i a b e t e s（Abramson et al,
Science 301,610,
2003).

Photosystem II (PS II)•All oxygen in the atmosphere is generated fromwater using light energy by PS II. (Ferreira et al,
Science 308, 2004).

Oxygen Respiratory Enzymes
II: Mutations Cause Tumors and Premature Aging ( Yankovskaya et• Complexal., Science 299 2003).
III: Mutations cause some human genetic disorders ( Iwata et al.,• Complex
Science 281, 1998, Andreu et al, N. Engl. J. Med. 341,1999)
IV: “Burns” nutrients using O2 for energy production (Iwata et al.,• Complex
Nature 376, 1995, Abramson et al., Nature Str. Biol. 7, 2000)
Complex III
Complex II

Complex IVAnalysis of Exercise Intolerance Mutationsof Complex III (Andreu et al, N. Engl. J.
Med. 341,1999）

Protein Crystallography to improve QOL
Reduce Cholesterol Level
Simvastatin (statin): HMG
CoA Reductase Blocker
Lower Blood Pressure
Cozaar: Angiotensin II
Receptor Antagonist
Lisinopril:
Angiotensin
Converting Enzyme(ACE) Blocker

Cholesterol Synthesis(Mevalonate Pathway)

Blood Pressure Control
by Angiotensin

Blood Vessel Constriction

All Three Targets Are Membrane Proteins
Ligands or
Drugs

Angiotesin
Converting
HMG CoA Reductase Enzyme (ACE)MembraneTransmembrane
helix

Angiotensin II
receptor(GPCR)

Here Comes Another Problem...
Androgen
receptorin follicle
cells

Before (2003)

After (2006)

Membrane Proteins are Major Drug Targets受容体
18%Receptors(18%)
GPCRs
30%30%

輸送体
Other enzymes
47%Membrane(inc.
イオンチャンネル
Channels
Proteins)

Transporters 8%
不明
55%

Unknown

ATPases
情報伝達
3%
核膜関連
1%
ミトコンドリア
転写因子
脂肪酸合成
2%
0%
各種酵素
1%
0%
1%
Others 細胞骨格
3%
その他0%
9%

Human Membrane Proteins

1%
1%
2%4%

Other receptors
4%

7%

4%
4%

Nuclearreceptors
Channels

Transporters

Drug targets

Human Membrane Proteins are Major Targets ofStructural Biology• No human GPCR structure

Soluble Protein
All Membrane Proteins
Human/Mammalian Membrane Protei

Coordinates in PDB

90

30,00022,50060

15,000
30

7,500
0

85

90

95
Year

00

04

0

60

75

90
Year

Establishing a Membrane Protein Crystallography Pipeline
Bernadette Byrne
David Drew

Imperial College
London (Membrane
Protein
Crystallography group,
MPC)

Diamond-MPL

Kostas Beis
- Automated crystallization
Alex Cameron - Data collection
Isabel Moraes - Structure determination
So Iwata-Membrane protein
biotechnology-Membrane protein
production- Transporter biochemistry

ERATO-Kyoto Lab

Takuya Kobayashi - Production facilities (Insect
and Mammalian cells)
Takeshi Murata
Tatsuro Shimamura - Antibody technology
Norimichi Nomura - GPCR biochemistryG-protein Coupled Receptors (GPCRs)• Transmembrane receptors that sense hormones, neurotransmitters and othermolecules.
• >30% drugs are targeting GPCRs
Hormones、Neurotransmitters

Extracellular
space
MembraneG-proteinαβγG-proteincoupledreceptor（GPCR）

GTPα
GDP

GTP

Effectors（Adenylate
cyclase、Phospholypase,etc.）

Second messengers（cAMP,
Ca2+etc.）
Activate Signal Transduction
Pathways

Cytoplasm

Modulate Cellular Responses

Structure of
adenosine
A2a receptor
with antibody
Fab fragment

Solute Carrier Family (SLC) Transporters• Membrane transporters including over 300 membersorganized into 47 families (by Human Genome Organization)• Composed of facilitative transporters (passive transporters)
and secondary active transporters (use an ion gradient forenergy).
Ions (H+, Na+...)

Substrate

Extracellularside

Outwardfacing cavity

Ion
pumps

Membrane

SLC

Intracellular
side

Inward-facing
cavityIons (H+, Na+...)

Outward-facing
conformation

Substrate-boundconformation

Inward-facing
内向き
conformation

Membrane Protein Crystallography Pipeline• Method development to address major bottlenecks for membraneprotein crystallography.

Keyparameters
to monitor

Processes

Technical
options/
developments• Activity• Expressionlevel• Integrity• Monodispersity• Thermal
stability• Detergent

concentration• Resolution• Indexing• Scaling andmerging

Expression~400

Purification~100

Crystallisation Data collection~10~30• Construct• Detergent• Crystallisation

design• Expression
systems• Culture
conditions

choice• Purification
methods• Additives

methods• Detergent
choice• Additives• Antibody• Data collection
systems• Samplemounting

Structure
determination
5• Phasingmethods• Model building
and refinement

GFP-based Membrane Protein Expression and
Purification System in S. cerevisiae (1)MP-TEV-GFP-8His

1.Screen membraneprotein-GFP overexpression by wholecell fluo-rescence

TEV 10His

N

GFP

Drew et. al. Nat.
Methods. 2, 2006
Newstead et. al.
PNAS 104, 2007
Drew et al., Nat.
Protoc. 3, 2008

8His
C

2. Isolate crude
membranes

3.Check integrity
using in-gel
fluorescence

1.Detergentsolubilization

2.Incubation ofsupernatant with
Ni-NTA resin

3 . I M A C
purification

Ni-NTA resin

Spin downnondetergentsolubilized proteins

4. Check cellular
localization by
confocal microscopy

5.Check monodisperisty in different
deter-gent byfluorescence sizee x c l u s i o nchromatography

Flow-through4.Dialysis andcleavage of
GFP-8His

5.Reverse IMAC
toremove
GFP-8His andhis-tagged TEV

Concentrationof
membraneproteinWash

Elution

6.Gel filtration ofm e m b r a n e
protein (option)

GFP-based Membrane Protein Expression and
Purification System in S. cerevisiae (2)• Suitable for fast screening of the most stable/expressed constructs
n
tio
n
, rma
R
ctio
o
PC ansf
ele
s
Tr
Ura

S. cerevisiae

11~13 days
n

ctio

n
ele
es
atio lection
Vector
n
i
m
c
r
i
net
construction ransfo MD seGe
T

P. pastoris

19~23 days
Vectorconstruction

Bacmid.

nctio
sfe P1 virusnTra

P2 virus

30~40 days

Titer check

Easy to transfer tothe systems to
produce better
proteins

Insect cells
10

5

20

15

25

30

35

Days

• Easy to construct variants by combining multiple fragments
Primer 1 & 2
Primer 3 & 4
Primer 5 & 6+

Sugawara et. al.
BBRC. 704, 2009

N5Q, N18Q
1-29

pDDGFP-2 vector
F116W

Name

Deletions and mutations

HRH1-WL

HRH1 whole length

HRH1-Nd-i3dHRH1(20-487), i3d(229-397)

HRH1-Nd-T4L

HRH1(20-487), T4L

HRH1-Nd-F116W-i3d

HRH1(20-487), F116W, i3d(229-397)

HRH1-Nd-L123W-T4L

HRH1(20-487), F116W, T4L

472-487
20

222

404

T4L
HRH1-Nd-T4L487

i3d (229-397)

Examples: Crystallisation of SLCs
• Crystals from 16 SLC proteins and their orthologues were obtained• Only six crystals diffracted better than 4.0 Å, one better than 3.0 Å.
• Problem: extremely difficult to obtain high-quality crystals.
Substrate(s)Glucose transporters (SLC2)
1 GLUT5 Rat
Fructose, Glucose
2 GLUT7 Human
Fructose, Glucose
3 LacY
Bacterial
Lactose4 FucP
Bacterial
FucoseBicarbonate transporters (SLC4)5 Band3
Human
Bicarbonate/Cl6 BOR1
Rice
Borate/?
7 BOR1
Arabidopsis
Borate/?
Sodium/proton exchangers (SLC9)8 NhaA
Bacterial
Na+/H+
9 NapA
Bacterial
Na+/H+
Sodium/bile salt cotransporters (SLC10)
10 NtcP
Rat
Bile salt/Na+
11 NtcP
Bacterial
Bile salt/Na+
Proton/oligopeptide cotransporters (SLC15)
12 PepT
Bacterial
oligopeptide
13 PepT
Bacterial
oligopeptide
14 PepT
Bacterial
oligopeptide
Mammalian

Plant

Bacterial

Res.
(Å)

1

9

4

11

5.0
3.6
10.0

5

12

4.5
3.3

6

13

7

14

5.0
10.0
3.5
3.8

10.0
2.8
3.8
3.0
6.5

Crystallization of Membrane Proteins using
Antibody Fragment or Artificial Binder
Antibody or Binder
Detergent
MicelleMembrane
Protein

Detergent solubilisedm e m b ra n e p r o t e i n s(difficult to crystallize
because of thedetergent micelle).

M e m b r a n e
protein Antibody/binder complex

Crystal of themembrane protein antibody/binder
complex

Antibody fragments for membrane protein crystallisation
Fv

yeast
cytochrome bc1
complexbacterial
cytochrome coxidase
Iwata, Michel et al. (1995)

Fv

Hunte, Michel et al. (2000)

Fab

Fab

Fab

bacterial
K+ channel(KcSA)
MacKinnon et al. (2001)

bacterialchloride channel(ClC) MacKinnon et al. (2003)

human β2adrenergic
receptor
Kobilka et al. (2007)

FabAntibodies for Membrane Protein Crystallization

Antibody
NOT suitableforcrystallisation
recognizing asequential
epitope

Ab
tag

Ab

Antibody
suitable forcrystallisation
recognizing a
conformational
epitope

Problem: extremely difficult to raise antibodies against native mammalian
membrane proteins

KcsA K-channel

CLC CL cannel

Cytochrome bc１complex

KvAP K-channelImproved Immunization Strategies
purified protein

protein-liposome+ Adjuvant(pertussis toxin)+ Adjuvant(Lipid A)

MRL/lpr or BALB/c mouse

Hybridoma / phage display

protein-baculovirus

Comparison of Immunization Strategies

BV/sgp64Tg
Immunizatio
n methods
u s i n g
liposomes
are most
efficient.

Purified protein/lpr
Pichia membrane/
Balb/cPichia membrane/
lpr
liposome/Balb/c
liposome/lpr
liposome/
antagonist/Balb/c
liposome/
antagonist/lpr

Times of immunization

Antibody Screening for Membrane Protein
Crystallization•Combining ELISA with native membrane proteins and the Western/DOT
blots with denatured membrane proteins.
Antibodies suitablefor crystallisation:
recognizing
conformational
epitopes

Membrane
Protein

ELISA +, WB/DOT -Antibodies NOT
suitable forcrystallisation:
recognizing
sequential epitopes

Membrane
Protein

ELISA +, WB/DOT+ELISA -, WB/DOT +Liposome ELISA (L-ELISA)

membrane protein
denaturation - causes false positives on the ELISA plate.

Lipids with 16:0 Biotinyl CAP PE

Antigen reconstituted into
proteoliposome

Testing culture sup.

Y

•P r e v e n t

HRPAntibody Screening using L-ELISA
and DOT blot

Conformational epitope
Sequential epitopeAffinity Test using Biacore• Select antibodies with a low Kd (high affinity)
and a slow koff (slow dissociation)
Antibody
captureTargetprotein
capture/release

抗GPCR抗体!
抗ターゲット抗体
Anti-membraneproteinantibody

抗マウスFc抗体!
Anti-mouse
Fc
antibody

GPCR(アナライト)!

AntigenmembraneproteinSensor プ!
chip
センサーチッslow dissociation-1-8 M
koff < 10-4 ,sK
d< 10

fast dissociation-1 ~ 10-6 M
koff ~ 10-2 ,sKdpoor affinity

Membrane Protein - Antibody Fab Fragment
Complex Preparation
Adenosine A2a
Fab

Fab+A2aA2a＋FabA2a with
variousamountFabfragment

A2a

Fab
A2a
lipid？
Complex crystals

Human Anion Exchanger

Antibody significantly improves
the thermostability of A2a
Fab

Band3＋Fab

Band3

CPM assay

Band3
＋Fab
Fab

+Fab
-Fab

Applications for Functional/Therapeutic
Antibodies and for Imaging-Fab2838 locks adenosine A2a receptor
into the inactive conformation-Cell imaging using antibodies against
adenosine A2a receptor

NECA(Agonist, only binds tothe active form)

Cultured cells

GFP(control)

ZM241385(Antagonist, canbind to the inactive form)

C40010+2nd Ab(+Alexa546)

Mouse brain

with 1ry antibody

ControlPlan of the MPL Facility

Automated Crystal
Optimisation at Diamond
Membrane Protein
Laboratory (MPL)

Fully integrated sitting-drop Rhombix crystallisation robot

Diamond-MPL

Liquid Handler

Robotic arm forplate movement
Fluidigm Toparz
microfluidics system

Crystal hotel/imager

Nanodrop
crystallisation robot
Cubic phasecrystallization robot

Remote access databaseand control system
PX scanner in-plate Xray diffraction system• Funded by Wellcome Trust/Japan Science and Technology
Agency/BBSRC• Imperial/Diamond joint project• Methods development for
automated membrane protein
crystallisation and high quality
X-ray data collection• User training and researchfacility for membrane protein
crystallisation and datacollection

X-ray Data Collection at the Diamond Microfocus
Beamline I24

Beamline I24 at Diamond. A.
Beamline optics. B. Beam focus
options. C. Sample environment withIrelec CATS robotic sample changer,
grid scanning system (left) and theplate gripper (right). D. Aperture array.

•Membrane protein
crystals are often weakly
diffracting, small andradiation sensitive.
• Diamond beamline I24
is designed to collecthigh quality data frommicro crystals (5-50µm).
• Developing a new
crystal mounting systemand software tocomplete a dataset frommultiple crystals to avoid
radiation damage.
• Collaboration with Dr.
Evans, I24 principal
beamline scientist

Adenosine A2a Receptor-Fab Complex Crystal• Maximum resolution < 2.5Å• Difficult to freeze. A large mosaicity.
Standard size
Beam (50µ x
50µ)Micro Beam
(10µ x 10µ)6

Standard Size Beam vs Micro Beam
2.7ÅStandard size beam
(IO4: 50µ x 50µ,50% transmission,
5 sec)

2.5Å

Micro Beam (I24:
10µ x 10µ,30%transmission, 1sec)

7

1 2

3

4

6
9

5

7 8

2

5

8

8

!"

%#

Membrane transporters & respiratory enzymes
(")

("&

*

+

%"

$

!#

)

,

&'

"

Mhp1 hydantoin transporter(Science 2008, Science 2010)

In
Oligopeptide
transporter (EMBO J.
2010)
B.

ASBT bile acidtransporter (Nature
Core
Pa
2011)
#$+&,(-!

Out

In
C.

NO reductase- Fab complex(Science 2010)C

Human anion exchanger - Fab complex

Core

Panel!

Core

G-protein coupled receptors
Human
Histamine H1R
a(Nature
2011)
ECL3
ECL2

S-S

Human Adenosine A2aR-Fab
complexIV
III(Nature in press)

b

ECL1

ECL1

ECL2

N
S-S

PO43-

Antagonist
Doxepin(ZM241385)
I
ECL3

III
D107
W428

VII II

V VI

VIII

V

Doxepin
CWxP

II

VI

I
c

IV

NPxxY

D(E)RY

VII

ICL2

ECL3

i3-loop
VI VII

ICL1

III

A2a
ECL1
ECL2
receptor VIII
Doxepin

VIII

C

H 1R N
!2AR

IV

V
C

ICL3

Human Muscarinic acetylcholine
M2R (Nature in press)

C

II
I
N
H 1R
D3R

Fig.1

Antib
ody
Fab
Frag
ment

Antihistamine (Doxepin)

CDR-H3InverseagonistHistamine H1 receptor
Collaborators
Imperial College London/ Diamond Light Source (MPL)/ Kyoto University (ERATO)
Tatsuro Shimamura(ERATO)
Mitsunori Shiroishi(ERATO)
Hirokazu Tsujimoto (ERATO)
Simone Weyand (MPL)
Takuya Kobayashi (ERATO)
So Iwata
Diamond Light Source
GraemeWinterThe Scripps Research Institute
Vadim Cherezov
Wei Liu
GyeWon Han
Raymond C. Stevens
University of California, San
Diego
Vsevolod Katritch
Ruben Abagyan

Allergic reactions and histamine
Capillary
AllergenAllergen likepollenstimulates Tcells
Allergen
花粉

Allergen花粉

IL-4

inflammation
Sensory nerve
stimulationsneezeHistamine
ヒスタミン

T-cells (Th2)release IL4
IgE

IL-4 stimulates
B-cells to
produce IgE
molecules

vasodilati
on,increased
permeability

IgE molecules
attach themselves tothe mast cells.

Allergenstimulates themast cells
attached with theIgE molecules toreleasehistamine andother chemical
mediators

Mucous tissue
increased secretion
runny
nose
Smooth muscle
contraction
Asthma,diarrhea

Histamine H1 Receptor (H1R)
Allergen
Histamine Vasodilation

•

•

•

Expressed in smoothmuscles, on vascularendothelial cells, in theheart.
Cause vasodilation,
along with increased cellpermeability when
activated.
In CNS, involved inhistaminergic neuron
activation.

Increased cellpermeability

Mast cells

Inﬂammation

Vascularendothelial
cells

Neutrophil
granulocyte
Histamine

CNS（histaminergic
neurons ）Histamine H1 Receptor Antagonists(Inverse agonists, Antihistamines)
Histamine•••

Inhibit H 1 R to alleviate
allergic reactions.
1st gen. drugs show side
effects (sedation, dry mouth,
arrhythmia) because of BBB
permeability and low H1R
selectivity.
2nd g e n . d r u g s w i t h acarboxylic moiety show
reduced BBB permeability
and higher H1R selectivity.

1st generation antihistamines

2nd generation antihistamines

のStructure of H1R
a

b
ECL3

ECL2

c

ECL2

S-S

VIII

V

II

Doxepin
CWxP

ECL3

D107

VII II

V VI

VI

I
c

IV

VII

IV

Comparison
with
β2-adrenergic
receptor (β2 AR)

III
ECL1

VIII
V
C

Doxepin

VIII
ICL2

I
H 1R N
!2AR

ECL2

NPxxY

D(E)RY

ICL3

C

Doxepin

III
W428

next
slide

ECL1

N
PO43-

III

ECL1

S-S

a

IV

Histamine H1R

VI VII

Fig.1

II

Comparison

ECL3

ICL1

C

I
N with
H 1R
dopamine D3
D3R
receptor
1(D3R)

Binding interactions of doxepin
a II Y458
D107

7.43

VII

3.32

NH+

PO43-

W4286.48

Y1083.33

F4246.44

VI

T1123.37
I1153.40

IV N1985.46cY4316.51

Y4316.51
W4286.48

W1584.56

Y4587.43

D1073.32

DoxepinIII
Doxepinbinding site
(1st gen.
antihistamine)b

V F432

T1945.43

ECL2

6.52 F4356.55

F4246.44

PO43-

O

T1945.43
W1584.56

Doxepinbinding
interactions

I1153.40 T1123.37 N1985.46

dK179ECL2
Doxepin

S1113.36

F4326.52

A1955.44

VII

Y1083.33 F4356.55

Carazolol

Eticlopride
ZM241385

H4507.35

III
VI

Y4316.51

VI
Anionbinding
region with
PO43-

ECL3

V

Doxepin

K1915.39

V

W4286.48

Ligandbinding
positionsin GPCRs

Fig.3

ECL3

III

V

K1915.39

Y4316.51

PO43-

VII

VI

H4507.35

Y4587.43

cY1083.33

Z-olopatadine
III
T112
S1113.36

levocetirizine

VI
K1915.39

3.37 I1153.40

H4507.35

V

K1915.39

V

H4507.35

F4326.52

W4286.48

Doxepin

Fig.3
Eticlopride
Carazolol

ZM2

4.56

T194

Y1083.33 F4356.55

POY4587.43Y4316.51

NH+

W158
O
F4246.44
I1153.40 T1123.37 N1985.46

d

PO43-

ECL3

Y4316.51

PO43-

K179ECL2VII

D1073.32

W4286.48 S1113.36

a
VI

VII

III

Doxepin

ECL2

T1945.43

W4286.48

A1955.44

V

VI

VI

F4326.52 F4356.55

I1153.40

b

ZM241385
Y4316.51

DoxepinF4246.44

Doxepin
W4286.48

VII
Eticlopride

4.56

1st gen. antihistamine(doxepin)

K179ECL2c

IV N1985.46

W1584.56

V

T1123.37

Y1083.33

III

D1073.32

7.43

W158
O
F4246.44
I1153.40 T1123.37 N1985.46

a dII Y458Carazolol

6.52 F4356.55

A1955.44

V F432

T1945.43

I1153.40

Doxepin

ECL2

K179ECL2c

IV N1985.46

W1584.56

Interactions of 2nd generation antihistamines
with H1R

2nd gen. antihistamines (models)bA1955.44

N1985.46

IV

D178ECL2

fexofenadine

Y4316.51

V
K191

K179

D1073.32
W1584.56

acrivastine

d

K191
K191

Residues in PO43binding site, Lys
179, Lys 191, Tyr
431 are unique to
H1R.

T1945.43

They are involvedin the 2nd gen.
antihistamine
binding.

histamine H1
K191

histamine H2
G

serotonin
T/S

acetylcholine
T/S

α adrenalineVβ adrenalineA

D3 dopamine
V

Fig.4

Action mechanisms of 1st and 2nd
generation antihistamines
Antihistamines(Inverse agonists)

histamine(agonist)ーExtracellularspace

1st gen.

2nd gen.

ーXー＋

Cytoplasm G-protein
Active H1RInactive H1R

Other aminergic receptors(inactive forms)