Lecture 2: Goals

1
Lecture 2 Goals

• Give you a tour of ABC transporter mechanism
• Give you an example 12 minute talk/presentation
  (or two)
• first a quick recap

2
The ABC of ABC Transport

• ATP
• Binding
• Cassette
• Bind and hydrolyse ATP
• Coupled to the transport of substances across
  cellular membranes.

3
ABC TransportersDiverse Functions in Humans
4
ABC Transporter FamilyShared Modular Design
(NBD-TMD)2
Drug efflux P-glycoprotein (P-gp)
TAP1/2 peptide transporter
5
Mechanism of ABC Transporters

• Key to understanding proteins in disease is
  understanding their mechanism
• Understand domaindomain interactions
• Understanding allostery

TMDTMD NBDNBD TMDNBD
ATP ADP
ATP ADP
6
NBDs Structure, Function and Interaction

• NBD Nucleotide Binding Domain
• 2 NBDs per ABC transporter
• Each NBD ca. 250 amino acids
• ALL NBDs share certain common sequence features

7
NBDs of ABC Transporters are Defined by Five
Sequences
ATP-binding motifs, a.k.a. P loop
Walker B hhhhDE
Walker A GxxGxGKS(S/T)
Conformational motifs
Histidine Loop hhhpH(/-)
Glutamine Loop xxQx
Signature Sequence LSGGQ
8
NBD 3-Dimensional Structure
His-loop
L-shaped structure cylinders are
a-helices arrows are b-sheets ATP loosely bound
by Walker A/B motifs
Signature
Gln-loop
Walker B
Walker A
ATP
9
But, how do two NBDs interact in a functional ABC
transporter?
P
S
S
S
P
P
P
S
back-to-back
sandwich opposite
S
S
P
P
P
S
S
P
embraced
head to head
10
The Sandwich Dimer Provides the Correct
Description of the NBDNBD Interface

• Two NBDs interlock in an ATP-dependent manner
• The importance of the signature motif is
  revealed.
• Communication between domains is possible

P
S
S
P
sandwich dimer
11
Structure of the Transmembrane Domains (TMDs)

• Again, E. coli transporters show the way.
• MsbA transports lipids in bacteria
• Sav1866 transports drugs in S. aureus
• Enabled us to realise some aspects of human ABC
  transporter structure

12
Proposed Atomic Structure of P-glycoprotein
Cell/organelle membrane
2 TMDs, all a-helical
Connecting region (intracellular domain)
Cell cytoplasm
2 NBDs, sandwiching ATP molecules
13
Proposed Cartoon Structure of an archetypal ABC
transporter
two transmembrane domains
intracellular domains
two interlocking NBDs
14
Structure and Mechanism in ABC Transporters
Cross-talk between domains Regulated protein
activity Allostery
ATP ADP
ATP ADP
15
Short TalkStructural and Mechanistic
Examination of an ABC Transporter
16
P-glycoprotein and Drug Transport
cytoplasm
cancer cell
17
Tools to Investigate Pgp Structure and Mechanism

• Pharmacology
• drug binding
• identifies multiple drug binding sites
• Electron microscopy
• identifies structural aspects of proteins
• low resolution cant see atoms, good for the
  overall architecture

18
The catalytic and transport cycle of
P-glycoprotein
Pgp druginside ATP
Pgp drugoutside ADP Pi
But what happens when?
19
Step I Pgp has a high affinity for drugs in the
absence of ATP
Equivalent to saturation of the protein with
drug.
20
Step I Pgp has a circular appearance in the
absence of ATP
(lines are like isobars on a weather map)
electron micrograph of apo form
21
The catalytic and transport cycle of
P-glycoprotein
22
Step II ATP binding reduces drug affinity,
reduction in affinity
23
Step II ATP binding changes protein conformation
ATP binding
apo
ATP bound
Note you cannot interpret this change in
conformation as having a particular function
24
The catalytic and transport cycle of
P-glycoprotein
25
Step III ATP hydrolysis sees a further change in
protein conformation
ATP
ADP.Pi
26
Step III ATP hydrolysis sees no change in
affinity for drug

• Still low affinity site

27
The catalytic and transport cycle of
P-glycoprotein
Drugin
ATP
D?P-gp
step I
step II
Drugout
P-gp
ATP
step III
P-gp
ADP.Pi
28
Step IV Phosphate release sees gain of high
affinity drug binding
29
Step V ADP release sees reversion to apo
enzyme structure
Apo
ADP.Pi
30
How does structure relate to function in
P-glycoprotein?
Drugin
ATP
D?P-gp
step I
P-gp
step II
Drugout
steps IV, V
P-gp
ATP
ADP
step III
Pi
P-gp
ADP.Pi
White high affinity Yellow low affinity
31
Pgp Catalytic and Transport Cycle Summary

• Changes in drug affinity linked to ATP
  hydrolysis.
• Transmembrane domains (drug-binding regions) must
  be allosterically coupled to NBDs (ATP
  hydrolyzing regions).
• Decoupling would be an effective anti-Pgp
  treatment to reverse drug resistance.

32
references for this mini-talk

• 1 Rosenberg, M.F. et al. (2001) Embo J 20,
  5615-25.
• 2 Martin, C., Berridge, G., Mistry, P.,
  Higgins, C.F., Charlton, P. and Callaghan, R.
  (2000) Biochemistry. 39, 11901-11906.
• 3 Martin, C., Higgins, C.F. and Callaghan, R.
  (2001) Biochemistry 40, 15733-42.
• 4 Stenham, D.R., Campbell, J.D., Sansom,
  M.S.P., Higgins, C.F., Kerr, I.D. and Linton,
  K.J. (2003) FASEB J. 15, 2287-2289.

33
Short TalkAnti-cancer drugs and the evidence
that ABC transporters export them
34
Commonly used drugs in chemotherapy

• alkylating agents, anthracyclines
• prevent DNA replication
• vinca alkaloids, taxanes
• disrupt microtubule assembly
• etoposide
• inhibits topoisomerase II
• methotrexate
• prevents purine synthesis

35
Is there a common chemistry?
methotrexate
etoposide
36
How can you prove that an ABC protein transports
drugs?

• in vitro
• bind drugs
• causes cells to become resistant
• show transport of labelled drugs
• in vivo
• show that transporter expression correlates with
  drug resistance
• functional imaging

37
fact drugs do bind to ABC proteins
3H-drug P-gp enriched membranes
incubate
filter
count radioactivity
38
fact ABC proteins can interact with multiple
drugs
Add initial drug at increasing concentrations
39
fact-ish a map of drug interactions sites exists
I vinblastine
IV nicardipine GF120918
II XR9576 Hoescht33342
III paclitaxel
40
fact cells expressing ABC proteins are drug
resistant
incubate drug cells not/expressing ABC pump
cell death assay
41
fact functional and reversible drug efflux can
be demonstrated
normal cells
P-gp expressing
P-gp inhibitor
P-gp expression level
drug concentration
Stenham et al, Faseb J 2003
42
fact ABC proteins affect drug distribution in
tumour models
Dt accumulation of drug in tumour spheroid
culture
non ABC expressing cells
ABCB1/Pgp expressing cells
non ABC expressing cells
ABC non-expressing cells
43
fact functional drug redistribution can be
observed in vivo
functional imaging of 99Tc-drug distribution in
live animals.
altered distribution if drug is given with an
ABC transporter inhibitor
inhibitor
no inhibitor
44
fact expression of pumps and clinical
correlation in leukaemia
45
fiction? expression of pumps and clinical
outcome in solid tumours

• thats presentation 3

46
references for this mini-talk

• 1 Martin, C., Berridge, G., Mistry, P.,
  Higgins, C.F., Charlton, P. and Callaghan, R.
  (2000) Biochemistry. 39, 11901-11906.
• 2 Seelig, A. (1998) Eur. J. Biochem. 251,
  252-261.
• 3 Stenham, D.R., Campbell, J.D., Sansom,
  M.S.P., Higgins, C.F., Kerr, I.D. and Linton,
  K.J. (2003) FASEB J. 15, 2287-2289.
• 4 Martin, C., Walker, J., Rothnie, A. and
  Callaghan, R. (2003) Br J Cancer 89, 1581-9.
• 5 Bates, S.E. (2003) in ABC Proteins From
  Bacteria to Man, pp. 359-391 (Holland, I.B.,
  Cole, S.P.C., Kuchler, K. and Higgins, C.F.,
  Eds.) Academic Press, New York.