ABCG5/ABCG8: A Structural View on Sterol Transport

Jyh-Yeuan (Eric) Lee

University of Texas Southwestern Medical Center at Dallas

Dietary Sterols

Cholesterol

Animal (60%) Plant (40%)

ABSORPTION: ~50% < 5%

β-Sitosterol

Campesterol

•  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

Enterocyte

NPC1L1

Chylomicron

SmallIntestine

CholesterolPlant Sterols

G5G8

DIET

Hepatocyte

Liver

G5G8

Bile Canaliculus

Liver/Intestine-Specific Sterol Transporter

Enterocyte

NPC1L1

Chylomicron

SmallIntestine

CholesterolPlant Sterols

G5G8

DIET

Hepatocyte

Liver

G5G8

Bile Canaliculus

Inactivating Mutations ⇒ Sitosterolemia

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:

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

G5G8-Mediated Sterol Transport(Functional Asymmetry Model)

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

?

0 10 20 30 400

2000

4000

6000

Large-scale Purification of Human G5G8Tandem Affinity Chromatography:(Pichia pastoris yeast)

Ni-NTA CBP Gel filtration

CBP: calmodulin-binding peptide

(2-3 mg/L) (1-2 mg/L)

10075

SDS-PAGE:

3C: 3C cleavage site

ATPase activity:(Bile acid stimulation assay)

Liver polar lipids1% cholate(Wang et al, Biochemistry, 2006)

(Johnson et al, Biochemistry, 2010)

(WT/GD: G5WT/G8-G216D)

50

(WT/WT)nm

ol P

i / m

g pr

otei

ns

Optimization of Protein Preparation

1° Ni-NTA 1° CBP

Gel filtration

Shakerculture(4-5days)

MembranePrepara:on

Solubiliza:on(β-DDM)

DDM

MNG

+ Endo H+ 3C protease

Methyla:on(CH3-Lys)

Relipida:on

Synthe:cphospholipids

(Avan:)

±ATPaseinhibitor

Crystalliza:on(manual/robotset-up)

(cholesterol)

Crystalgrowth(2wks–6mths)

Ligands

±Mg/ATP

2° CBP2° Ni-NTA

DDM MNG

DetergentExchange

PD-10(desalting)

Alkyla:on(Cyscapping)

(DoDecylMaltoside) (MaltoseNeopentylGlycol)

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

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 exposure2-5 sec @ APS30 sec @ ALS

•  Radiation damage3-5 frames (< 5°)

•  Signal (I/σ = 1-1.5 at 3.9-4Å)

3.9 Å

3.6 Å

Model Building & Refinement

(Rwork/Rfree = 0.242/0.328)W-SAD

(tungsten single anomalous dispersion)

(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)

Domain Features in G5G8 Heterodimer

RMSD ~ 2Å(~28% sequence identity)

TMD: transmembrane domainNBD: nucleotide-binding domain

ECD: extracellular domainCnH: connecting helixCpH: coupling helix (Lee et al, Nature, 2016)

CpH/CnH/E-helix Triple Helical Bundles(Connecting TMD with NBS)

CnH: connecting helixCpH: coupling helix★: conserved polar residues

TMD Polar Relays(Conserved Polar residues Proximal to NBS)

(Lee et al, Nature, 2016)

: conserved; : not conservedFo - Fc (3.0 σ)Vestibules

Vestibules at TMD-Membrane Interface:Sterol Binding/Entry?

A540

F540＊

＊

LETTER RESEARCH

Extended Data Figure 5 | Vestibules in the membrane spanning region. a, b, Left: cartoon of G5G8 TMDs in transparent surface (orange and blue). Vestibules on opposing faces of the TMDs are highlighted in boxes. Middle: Fo − Fc difference electron density map contoured at 3.0σ showing extended features at the vestibules. TMHs are numbered. Right: highly conserved residues in G5 and G8 (sequence conservation ≥ 7, see Methods) are shown as orange (G5) and blue (G8) cartoon/sticks. c, Left: in vivo functional reconstitution assay using G5G8 KO mice (G5G8−/− mice)19. Biliary cholesterol levels from mice infected with RR

(empty adenovirus), or with adenoviruses expressing human (h)G5:WT and hG8:WT, or with adenoviruses expressing hG5:A540F and hG8:WT are shown. Pictures of the bile are below the graph. Each experiment represents the mean ± s.d. from three infected mice (n = 3) in each group. Right: expression of hG5 and hG8 detected by immunoblotting using anti-hG5 monoclonal antibodies (see Methods). Connexin was used as the gel-loading control. As a positive control for the mouse and a negative control for the human anti-G5 antibody, we also immunoblotted liver membranes from G5G8+/− mice.

© 2016 Macmillan Publishers Limited. All rights reserved

Precursor

Mature

Vestibules at TMD-Membrane Interface:Structure-based Mutagenesis

(Lee et al, Nature, 2016)

ER-escape mutations Non-ER-escape mutations

E146Q

R419H/P

R543S

G574R

R389H

N437KR184H

P231TR263Q

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)

E146Q

R419H/P

R543S

G574R

R389H

N437KR184H

P231TR263Q

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 relayPolar relayE-helix

Sterol exit?

Vestibule

G5G8-Mediated Sterol Transport

Inward movement(CpH/CnH/E-helix bundle)

G5 G8

CnH

CpHE-helix

CnH

CpH E-helix

Upward movement(TM helices)

G5 G8

4

316

4

3

Molecular Dynamics Simulation

Co-Evolution Analysis

(Lee et al, Nature, 2016)

G5G8-Mediated Sterol Transport(Updated Model)

Inward

Upward

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

Vertebrates

Eukaryotic ABCG Exporters(TMD-based CLANS Network)

(Lee et al, Nature, 2016)

ABC2 Exporter Superfamily CLANS Network

(Lee et al, Nature, 2016)

Acknowledgement•  UT Southwestern:

Helen HobbsJonathan Cohen

Daniel Rosenbaum

Zbyszek OtwinowskiDominika Borek

Nick GrishinLisa Kinch

Xiao-Song XieJin 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 HealthAmerican Heart AssociationHoward Hughes Medical Institute