The ATP binding cassette (ABC) transporter ABCG5/ABCG8 mediates sterol excretion in liver and intestines. Mutations inactivating either ABCG5 or ABCG8 cause sitosterolemia, a rare autosomal recessive genetic disorder characterized by hypercholesteromelia and premature atherosclerosis. We crystallized human ABCG5/ABCG8 in lipid bilayers and solved an X-ray structure. ABCG5/ABCG8 is the first ABCG transporter structurally characterized at atomic resolution and presents a new transmembrane fold for the ABC transporter superfamily. It shows the asymmetric nucleotide-binding sites interacting with the transmembrane domain through a conserved polar network, where the coupling-connecting helices are structurally asymmetric. The structure reveals new features of sterol transport at the bilayer-transporter interface, allows us to propose a sterol transport model and provides a new framework to study active lipid transport and to model other ABC transporters. This presentation share slides that describes the background of the physiology, protein purification, structural determination and interpretation of human ABCG5/ABCG8.

1. ABCG5/ABCG8: A Structural View on Sterol Transport
Jyh-Yeuan (Eric) Lee
University of Texas Southwestern Medical Center at Dallas

2. Dietary Sterols
Cholesterol
Animal (60%) Plant (40%)
ABSORPTION: ~50% < 5%
β-Sitosterol
Campesterol

3. • Autosomal recessive genetic disease (50-120x plasma sitosterol)
• Heterodimeric ATP-binding cassette (ABC) transporters
• Amino acid sequence identity: ~28%
Sitosterolemia v.s. ABCG5 & ABCG8
(Bhattacharyya & Conner, JCI, 1974)
(Berge & Tian et al, Science, 2000)
(Lee et al, Nat. Genet., 2001)
(Lu et al, Am. J. Hum. Genet., 2001)
(2p21)
A: Walker A motif
(GxxGxGKS/T)
B: Walker B motif
(ϕϕϕϕDE)
S: ABC signature motif
(ϕSGGQ/E)
ϕ: hydrophobic amino
acids
Transmembrane domain
Nucleotide-binding domain

4. Enterocyte
NPC1L1
Chylomicron
Small
Intestine
Cholesterol
Plant Sterols
G5G8
DIET
Hepatocyte
Liver
G5G8
Bile Canaliculus
Liver/Intestine-Specific Sterol Transporter

5. Enterocyte
NPC1L1
Chylomicron
Small
Intestine
Cholesterol
Plant Sterols
G5G8
DIET
Hepatocyte
Liver
G5G8
Bile Canaliculus
Inactivating Mutations ⇒ Sitosterolemia

6. Animal Model & in vivo Reconstitution
(Yu et al, PNAS, 2002)
+/+-/-
G5G8-/-
G5 & G8 restore biliary sterol secretion:
(obligate heterodimers)
mG5:
mG8:
Calnexin:
Precursor
Mature
(Graf et al, JBC, 2003)
bile extraction
G5G8-/- mice develop sitosterolemia:

7. Asymmetric Nucleotide-Binding Sites (NBS)
A: Walker A
S: ABC Signature
GSSGSGKT
LSGGE
GSSGCGRA
ISTGE
B: Walker B
NBS1
NBS2
(Zhang et al, JBC, 2006)
(Wang et al, JBC, 2011)
Sterol Transport
(mouse model)
(inactivating mutations)
VMLFDE ILILDE

8. G5G8-Mediated Sterol Transport
(Functional Asymmetry Model)

9. TMD
NBD
Questions:
• How is the ATPase activity coupled to sterol transport?
• How does TMD move sterols across the membranes?
Molecular Mechanism of G5G8
?
?
ABCB exporters nucleotide-free Nucleotide-bound
ATP
Sterols
?

10. 0 10 20 30 40
0
2000
4000
6000
Large-scale Purification of Human G5G8
Tandem Affinity Chromatography:
(Pichia pastoris yeast)
Ni-NTA CBP Gel filtration
CBP: calmodulin-binding peptide
(2-3 mg/L) (1-2 mg/L)
100
75
SDS-PAGE:
3C: 3C cleavage site
ATPase activity:
(Bile acid stimulation assay)
Liver polar lipids
1% cholate
(Wang et al, Biochemistry, 2006)
(Johnson et al, Biochemistry, 2010)
(WT/GD: G5WT/G8-G216D)
50
(WT/WT)
nmolPi/mgproteins

11. Optimization of Protein Preparation
1° Ni-NTA 1° CBP
Gel filtration
Shaker culture
(4-5 days)
Membrane
Prepara:on
Solubiliza:on
(β-DDM)
DDM
MNG
+ Endo H
+ 3C protease
Methyla:on
(CH3-Lys)
Relipida:on
Synthe:c
phospholipids
(Avan:)
± ATPase
inhibitor
Crystalliza:on
(manual/robot set-up)
(cholesterol)
Crystal growth
(2 wks – 6 mths)
Ligands
± Mg/ATP
2° CBP2° Ni-NTA
DDM MNG
Detergent
Exchange
PD-10
(desalting)
Alkyla:on
(Cys capping)
(DoDecyl Maltoside) (Maltose Neopentyl Glycol)

12. 100 nm
Analytical gel filtration: Negative-stained TEM single particles:
(FEI Tecnai G2)
ABCB1
Void G5G8
G5G8
(Lee et al, J Biol Chem, 2002)
Stable and Monodisperse G5G8

13. Bicelle Crystallization (Lipid Bilayers)
G5G8 bicelle preparation
Bilayer
(DMPC +
cholesterol)
Micelle
(CHAPSO)
(DHPC)
Bicelle
G5G8
G5G8
(MNG)
Crystal growth & X-ray diffraction
100 µm • Long exposure
2-5 sec @ APS
30 sec @ ALS
• Radiation damage
3-5 frames (< 5°)
• Signal (I/σ =
1-1.5 at 3.9-4Å)
3.9 Å
3.6 Å

14. Model Building & Refinement
(Rwork/Rfree = 0.242/0.328)
W-SAD
(tungsten single anomalous dispersion)

15. (Hollenstein et al, Curr Opin Struct Biol, 2007)
(Rees et al, Nature Rev Mol Cell Biol, 2009)
New TMD Fold for ABC Transporters
(Lee et al, Nature, 2016)

16. Domain Features in G5G8 Heterodimer
RMSD ~ 2Å
(~28% sequence identity)
TMD: transmembrane domain
NBD: nucleotide-binding domain
ECD: extracellular domain
CnH: connecting helix
CpH: coupling helix
(Lee et al, Nature, 2016)

17. CpH/CnH/E-helix Triple Helical Bundles
(Connecting TMD with NBS)
CnH: connecting helix
CpH: coupling helix
★: conserved polar residues

18. TMD Polar Relays
(Conserved Polar residues Proximal to NBS)
(Lee et al, Nature, 2016)

19. : conserved; : not conservedFo - Fc (3.0 σ)Vestibules
Vestibules at TMD-Membrane Interface:
Sterol Binding/Entry?

20. A540
F540
＊
＊
Precursor
Mature
Vestibules at TMD-Membrane Interface:
Structure-based Mutagenesis
(Lee et al, Nature, 2016)

21. ER-escape mutations Non-ER-escape mutations
E146Q
R419H/P
R543S
G574R
R389H
N437K
R184H
P231T
R263Q
L501P
G574E
L596R
G5 G5G8 G8
Missense Mutations in Sitosterolemia
(Graf et al, JBC, 2004)
: conserved (multiple sequence alignment (MSA) value ≥ 7)
: less/non-conserved (MSA < 7)

22. E146Q
R419H/P
R543S
G574R
R389H
N437K
R184H
P231T
R263Q
L501P
G574E
L596R
G5 G5G8 G8
Missense Mutations in Sitosterolemia
(Graf et al, JBC, 2004)
: conserved (multiple sequence alignment (MSA) value ≥ 7)
: less/non-conserved (MSA < 7)
Polar relay
Polar relay
E-helix
Sterol exit?
Vestibule

23. G5G8-Mediated Sterol Transport
Inward movement
(CpH/CnH/E-helix bundle)
G5 G8
CnH
CpH
E-helix
CnH
CpH E-helix
Upward movement
(TM helices)
G5 G8
4
3
1
6
4
3
Molecular Dynamics Simulation

24. Co-Evolution Analysis

25. (Lee et al, Nature, 2016)

26. G5G8-Mediated Sterol Transport
(Updated Model)
Inward
Upward

27. Summary
• G5G8 bicelle crystal structure at 3.9 Å
(lipid bilayer-bound)
• New TMD fold for ABC transporters
• Cpn/CnH/E-helix bundle connects NBS to TMD
• Asymmetric interaction between CnH & CpH
• Conserved polar residues proximal to NBS
(TMD polar relays)
• Updated model for G5G8-mediated sterol transport
• Structure-based mutagenesis at sterol-interacting site
• Structure-based interpretation for disease-causing mutations

28. Vertebrates
Eukaryotic ABCG Exporters
(TMD-based CLANS Network)
(Lee et al, Nature, 2016)

29. ABC2 Exporter Superfamily CLANS Network
(Lee et al, Nature, 2016)

30. Acknowledgement
• UT Southwestern:
Helen Hobbs
Jonathan Cohen
Daniel Rosenbaum
Zbyszek Otwinowski
Dominika Borek
Nick Grishin
Lisa Kinch
Xiao-Song Xie
Jin Wang
Junmei Wang
Structural Biology Lab (SBL)
• Texas Tech HSC:
Ina Urbatsch
• Beamlines:
APS 19ID, 23ID (Argonne)
ALS 821, 822 (Berkeley)
• Funding:
National Institute of Health
American Heart Association
Howard Hughes Medical Institute