Membrane
Protein
Structure
&
Func2on:
Ac2ve
Transport
Importance
of
conforma-onal
change
in
transporter
ac-vity
Structure
–
Dynamics
–
Func-on
Membrane
proteins
of
known
structure
h=p://blanco.biomol.uci.edu/Membrane Proteins xtal.html
h=p://www.drorlist.com/nmr/MPNMR.html
Outline
•  Basics
of
membrane
protein
structure
and
differences
from
soluble
proteins.
•  What
is
a
transporter?
Requirements
for
transport
ac-vity.
•  Examples
of
mul-drug
resistance
transporter
structure,
dynamics
and
mechanism
–  Known
mul-drug
resistance
transporter
structures
from
each
family,
or
close
rela-ves
–  What
can
we
observe/infer
about
how
these
proteins
move
in
order
to
perform
ac-ve
transport
•  What’s
leP
to
discover?
–  Common
themes
and
differences
between
the
known
structures
and
mechanisms
KcsA
–
First
Crystal
Structure
Doyle
et
al.
(1998)
Science
Vol.
280.
no.
5360,
pp.
69
‐
77
Diacyl
Glycerol
Kinase
–
Largest
NMR
Structure
Van
Horn
et
al.
(2009)
Science
Vol.
324.
no.
5935,
pp.
1726
‐
1729
Rhodopsin:
7TM
helices
Palczewski,
et
al.
(2000)
Science:
Vol.
289.
no.
5480,
pp.
739
–
745
Membrane
Protein
Structure
•Dis-nct
hydrophobic
and
hydrophilic
domains
required
to
interact
with
the
lipid
bilayer
plus
the
aqueous
environment
on
either
side.
•Even
more
important
to
sa-sfy
hydrogen
bonds
in
the
hydrophobic
membrane
environment.
Regular
secondary
structures.
•Curved
and
kinked
helices
(&
charged/polar
residues)
in
TM
domains
play
func-onal
roles.
Gly
and
Pro
residues
oPen
found
in
conserved
mo-fs.
(GXXG,
GXXXG,
etc.)
•Posi-ve
inside
rule:
more
Arg
+
Lys
on
cytoplasmic
side
of
protein.
Challenges
with
membrane
protein
structural
biology:
•  Crystallography
requires
crystalliza-on….
–  OPen
low
resolu-on
–  Crystalliza-on
condi-ons?
Detergents
good
for
crystalliza-on
may
not
be
the
best
for
func-on.
–  Lipid
cubic
phase
tricky
to
work
with
–  Use
of
an-bodies
–
FAB‐fragment
assisted
crystalliza-on
•  Solu-on
NMR
(and
many
biophysical
methods)
require
a
solubilized
sample
–  Detergents
are
non‐ideal
–  Chemically
different,
highly
curved
–  Ideally
want
li=le
bilayer
patches
•  Solid‐state
NMR
can
work
with
oriented
bilayers,
bicelles,
and
vesicles
but
currently
lacks
resolu-on
(problems
with
big,
dynamic
proteins).
Landau
&
Rosenbusch
PNAS
93:14533
(1996).
Solubilizing
Membrane
Proteins
Ac2ve
Transporters:
Crea2ng
and
Maintaining
Gradients
•  Ionic
and
chemical
gradients
across
membranes
are
cri-cal
for
life:
ATP
synthesis,
nerve
cell
polariza-on
and
signal
transmission,
muscle
contrac-on,
maintenance
of
healthy
intracellular
condi-ons,
etc.
•  Energy
required:
–  ATP
hydrolysis
(primary
ac-ve
transporter)
P‐type
ATPases
use
1/3
of
our
total
energy.
Examples:
Na+/K+
ATPases,
H+
ATPases,
Ca+
ATPases
–  Coupling
to
an
electrochemical
gradient
created
by
a
primary
ac-ve
transporter
(secondary
ac-ve
transporter)
•  Common
mo-f:
Dimer
or
pseudo‐dimer
structure
forming
two
halves
of
the
molecule.
Moving
Molecules
Across
the
Membrane
Channel:
open
or
closed
Transporter:
Only
open
to
one
side
at
a
2me
Basic
model
of
required
features
for
channels
and
transporters,
first
proposed
in
the
1960s.
Transporters
&
Channels
Channel:
open
or
closed
•No
energy
input:
Only
flow
down
a
concentra-on
gradient
•Free
diffusion
when
pore
is
open
–
fast
flow,
107
ions
per
second!
•Ga-ng
and
selec-vity
control
what
&
when
•Exact
mechanism
of
opening/closing
varies
Transporter:
Only
open
to
one
side
at
a
2me
Transporters
&
Channels
Channel:
open
or
closed
Transporter:
Only
open
to
one
side
at
a
2me
•Energy
required:
Can
move
molecule
UP
a
concentra-on
gradient
•Only
n
molecules
moved
per
transport
cycle
–
rela-vely
slow
(102‐105
molecules
per
second)
•2
sources
of
energy:
1)
primary,
ATP‐driven
2)
secondary,
use
electrochemical
gradient
•Mechanism
of
opening/closing
and
coupling
to
energy
source?
Many
transporters
are
alike…
•4
phylogene-cally
dis-nct
families,
no
obvious
sequence
homology.
•different
func-ons:
transport
drugs,
sugars,
amino
acids.
Mul2drug
Resistance
Transporters
•
•
•
•
•
•
Need
to
extrude
drugs
“uphill”
against
a
concentra-on
gradient
Bacteria:
mostly
secondary
ac-ve
transporters
in
mul-drug
resistance
(proton
gradient
across
inner
membrane)
Eukaryotes:
mostly
primary
ac-ve
transporters
in
mul-drug
resistance
Most
recognize
a
wide
range
of
hydrophobic,
including
drugs
and
lipids.
Also
implicated
in
transport
of
virulence
factors
and
compounds
important
for
quorum
sensing.
Creates
an
ideal
environment
inside
the
cell
(low,
non‐zero
drug
concentra-on)
for
evolu-on
of
other,
stronger
resistance
mechanisms.
Closely
related
transporters
protect
cells
from
toxic
compounds
and
move
lipids
to
the
correct
membrane.
Drugs
enter
the
cell:
•Hydrophobic
drugs
may
diffuse
into
the
cell
•Uptake
through
other
channels
or
transporters?
Transporters
pump
the
drugs
back
out
Mul2drug
Resistance
Transporters
•  ABC:
ATP
binding
casse=e
–  couple
ATP
hydrolysis
–  Common
eukaryo-c
MDRs
–  Responsible
for
chemotherapeu-c
resistance
in
human
cancers
•  MFS:
major
facilitator
superfamily
–  Couple
proton
gradient
to
drug
export
–  25%
of
all
transport
proteins,
12‐14
TM
domains
•  SMR:
small
mul-drug
resistance
–  Couple
proton
gradient
to
drug
export
–  Very
small,
just
over
100
amino
acids,
4
TM
domains
•  RND:
resistance
nodula-on
division
–  Couple
proton
gradient
across
bacterial
inner
membrane
to
drug
efflux
across
outer
membrane
•  MATE:
mul-an-microbial
extrusion
–  Couple
either
proton
or
sodium
gradient
to
substrate
export
Cartoon
Models
of
Each
Family
Outer
membrane
RND
Periplasm
Membrane
fusion
protein
MFS/SMR
ABC
Inner
membrane
ATP
hydrolyzed
RND
or
MFS
Sobzcak
&
Lolkema
Curr.
Op.
Microbiol.
8:161‐167
(2005)
Borges‐Walmsley,
et
al,
Biochem.
J.
376:313
(2003)
Primary
ac2ve
transport:
ATP
driven
•  ABC
family
of
mul-drug
resistance
proteins
2
nucleo-de
binding
domains
Ex.
Sav1866
(bacterial),
p‐glycoprotein,
MDR1
More
common
in
eukaryotes
‐
drug
resistance
in
chemotherapy
May
be
importers
or
exporters,
roles
in
immune
system,
uptake
of
essen-al
nutrients,
ex:
CFTR
–  Problem:
only
1
structure
of
a
mul-drug
resistance
protein,
don’t
know
the
structures
of
all
relevant
states
of
any
family
member
–
–
–
–
•  P‐type
ATPases
–  Set
up
ionic
gradients
–
use
1/3
of
our
total
energy
–  3
domains
(N,
A,
P)
interact
with
nucleo-de
–  Sarcoplasmic
re-culum
Ca2+‐ATPase:
only
primary
ac-ve
transporter
where
we
have
structures
of
mul-ple
major
intermediates
•  Both
types
have
nucleo-de/motor
domain
dis-nct
from
TM
transport
pathway,
believed
to
have
similar
coupling
between
the
motor
and
TM
domain
conforma-on
changes
ABC
transporters
ATP
binding
Murakami,
Curr.
Opin.
Struct.
Biol.
18:459‐465
(2008)
ATP
hydrolysis
Sav1866
Outward‐facing
conforma-on
open
to
the
extracellular
side
of
the
membrane
Consistent
with
solvent
accessibility
data,
substrate
access,
mutagenesis
and
cross‐
linking
studies
in
ATP‐
bound
state
(note
mixed‐
monomers
in
TM
“wings”,
unlike
lipid
uptake
ABC
transporters)
ADP
bound
But
believed
to
be
the
ATP‐
bound
conforma-on
Dawson
&
Locher
Nature
443:180‐185
(2006)
Opening
extends
into
bilayer
–
hydrophobic
substrate
may
escape
into
outer
bilayer
leaflet.
Sarcoplasmic
Re2culum
Ca2+‐ATPase
conversion
of
ATP
hydrolysis
to
TM
domain
conforma2onal
change
1ATP
hydrolyzed,
2
Ca2+
transported
to
SR
lumen,
2H+
counter‐transported
Dephosphoryla-on,
open
to
cytoplasmic
side,
allowing
Ca2+
binding
TGES
mo-f
of
A‐domain
ATP
binds,
occluded
conforma-on
formed
All
structures
be=er
than
3Å
resolu-on
ATP
hydrolysed,
protein
phosphorylated,
occluded
conforma-on
forms
ADP
leaves,
A‐domain
swings,
pulling
TM
domains
open
to
lumen,
releasing
Ca2+
Olesen
et
al,
Nature
450:
1036‐1042
(2007).
ATP
hydrolysis/protein
phosphoryla2on
N
ADP
released,
A‐domain
(yellow)
swings
in
to
replace
N‐
domain
(red)
P
A
Ca2+‐ATPase:
the
movie
in
snapshots
RND
transporters
Murakami,
Curr.
Opin.
Struct.
Biol.
18:459‐465
(2008)
RND
family:
AcrB
•  Periplasmic
loops
confer
substrate
specificity
•  Structures
solved
by
mul-ple
groups
in
different
crystal
forms
show
same
results
•  Func-onally
rota-ng
mechanism:
three
different
states
in
each
subunit
of
the
trimer,
the
full
mechanism
revealed
in
a
single
crystal
•  Structure
solved
bound
to
different
substrates
–
mul-ple
drugs
recogni-on
revealed
Func2onally
Rota2ng
Mechanism:
AcrB
The
en-re
complex
Murakami,
Curr.
Opin.
Struct.
Biol.
18:459‐465
(2008)
Func2onally
Rota2ng
Mechanism:
AcrB
2.9
Å
resolu-on
access
binding
Seeger,
et
al,
Science
313:1295
(2006)
extrusion
Func2onally
Rota2ng
Mechanism:
AcrB
2D,
1K
AcrB:
Mul2drug
Recogni2on
minocycline
doxyrubicin
Mul-ple
sites
in
a
big
hydrophobic
binding
pocket
Murakami
et
al,
(2006)
Nature
443,
173‐179.
MFS,
SMR
&
MATE
transporters
Murakami,
Curr.
Opin.
Struct.
Biol.
18:459‐465
(2008)
MFS,
SMR
&
MATE
families
•  Alterna-ng
access
model
•  Mostly
TM,
no
big
soluble
domains
–  SMR:
4
TM
helices,
≈110
amino
acids
–  MFS:
12‐14
TM
helices
(2
bundles
of
6
TM
helices
form
core
structure),
≈400
amino
acids
–  MATE:
12
TM
helices
(2
bundles
of
6
TM
helices)
similar
to
MFS
family
in
size
•  Very
common
–
not
just
mul-drug
resistance,
but
MFS
proteins
transport
sugars,
amino
acids,
etc.
–  Only
two
MFS
structures
known:
LacY
(lactose
permease),
GlpT
(glycerol‐3‐
phosphate
transporter)
•  First
MATE
structure
published
in
October
2010
–  Export
secondary
metabolites
important
in
host
defense
as
well
as
xenobio-cs.
–  Widespread
like
MFS
family.
Think
about
the
energeCcs
involved
in
driving
transport
in
this
way
Secondary
transport:
symport,
an2port,
and
uniport
Forrest
&
Rudnick
(2009)
Physiology,
24,
377‐386.
Uniport
A
A
A
net
transport
A
Symport
Only
doubly‐substrate‐bound
and
apo
states
undergo
conforma-onal
exchange
B
A
B
A
B
net
transport
A
A
A
An2port
Only
single‐substrate‐bound
states
undergo
conforma-onal
exchange
A
A
A
net
transport
B
B
B
MFS
family:
EmrD
•Not
really
open
to
either
side
of
the
membrane.
•True
intermediate
state?
Or
just
trapped
by
crystal
packing?
Yin,
Science
312:741
(2006).
Alterna2ng
access
–
an2port
:
GlpT
Inward‐facing
conforma-on,
no
substrate
bound,
2
Arg
face
in
toward
ac-ve
site,
3.3
Å
resolu-on
Lemieux,
et
al.
Curr.
Opin.
Str.
Biol.
14:405‐412
(2004)
GlpT:
What’s
the
other
state?
Two
halves
rocked
rela-ve
to
each
other
to
predict
the
open‐out
conforma-on
Lemieux,
et
al.
Curr.
Opin.
Str.
Biol.
14:405‐412
(2004)
Alterna2ng
access
model:
LacY
Proton/lactose
symport
Abramson
et
al,
Science
301:610‐615
(2003)
Apo
LacY
at
3.0
Å
resolu2on
All
structures
are
of
C154G
mutant,
WT
does
not
readily
crystalize
–
C154G
stabilizes
inward‐facing
conforma-on
All
known
MFS
crystal
structures
are
of
inward‐facing
states
or
intermediate
states.
LacY:
what’s
the
other
state?
Crystal
structure
with
sugar
bound
(lactose
analog
TDG)
at
3.6
Å,
yellow
residues
show
increased
NEM
labeling
upon
substrate
binding
Abramson
et
al,
Science
301:610‐615
(2003)
Model
based
on
60
degree
rota-on
(rocking)
of
curved
helices,
compa-ble
with
accessibility
&
cross‐linking
data
for
other
state
LacY:
what’s
the
other
state?
DEER
(four‐pulse
double
electron‐electron
resonance)
measurements
of
distances
in
spin‐
labeled
LacY
Smirnova,
et
al,
PNAS
104:16504‐16509
(2007)
Distance
changes
upon
sugar
binding
Blue/green
–
no
sugar
Red/purple‐
sugar
LacY:
what’s
the
other
state
model
based
on
DEER
measurements
Open‐inward,
no
sugar
bound
Smirnova,
et
al,
PNAS
104:16504‐16509
(2007)
Open‐outward,
sugar
bound
MATE:
NorM
from
Vibrio
cholerae
50Å
50Å
It
looks
like
an
MFS
transporter!
cytoplasm
cytoplasm
He,
et
al.
Nature
467:
991
(2010).
NorM
from
Vibrio
cholerae
Note
cleP
open
to
the
outer
leaflet
of
the
bilayer.
He,
et
al.
Nature
467:
991
(2010).
NorM
mechanis2c
model
Proposed
transport
mechanism.
In
the
outward‐facing
conforma-on,
a
ca-on
(yellow)
binds
at
a
conserved
site
(blue
oval;
step
1).
Ca-on
binding
induces
structural
changes
to
the
inward‐
facing
conforma-on
(step
2),
which
is
competent
to
bind
substrate
(organic
ca-on
in
green)
from
the
inner
membrane
leaflet
or
cytoplasm.
Substrate
binding
causes
structural
changes
back
to
the
outward‐facing
conforma-on
(step
3),
allowing
export
and
ca-on
binding.
He,
et
al.
Nature
467:
991
(2010).
EmrE:
SMR
family
•  Small,
minimal
transport
model
•  Single‐site
alterna-ng
access
model
–  Glutamate
residue
cri-cal
for
ac-vity,
E
to
D
mutant
alters
pKa
of
transport
–  Aroma-c
residues
in
ac-ve
site
key
to
aroma-c
ca-on
transport
• 
Accessibility,
cross‐linking,
mutagenesis
studies
used
to
create
mode
• 
An-porter:
1
polyaroma-c
ca-on
per
2H+
EmrE:
the
structure?
An-parallel
homodimer?
3.5
Å
resolu-on,
crystallized
from
nonylglucoside
Asymmetric,
even
in
lipids
Chen,
Yen‐Ju
et
al.
(2007)
Proc.
Natl.
Acad.
Sci.
USA
104,
18999‐19004
7Å/16Å
resolu-on,
DMPC
Ubarretxena‐Belandia,
et
al
EMBO
J
22:6175
(2003)
FRET
confirms
an2parallel
topology
within
the
dimer
Spectrum
of
EmrE
Saturated
with
the
Tight‐Binding
Substrate
TPP
45
°C
pH
7.0
DMPC/DHPC≈0.33
Total
lipid
≈
400mM
15N
(ppm)
0.5mM
EmrE
1.5mM
TPP+
1H
(ppm)
20m
NaCl,
20mM
potassium
phosphate
TPP+
Transport
by
EmrE
Is
peak
doubling
due
to
assymetric
structure
or
slow
interconversion?
+
+
Characterizing
Slow
Conforma2onal
Exchange
by
NMR
ZZ‐exchange
spectroscopy
1.
Label
ini-al
chemical
shiP
2.
Mixing
-me:
wait
tens
of
milliseconds
Did
the
protein
convert
from
state
A
to
state
B?
3.
Label
final
chemical
shiPs
Determining
the
Exchange
Timescale
Starts
and
ends
in
same
state
Conforma-on
changes
kf
=
kr
=
5
±
0.5
s‐1
EmrE:
the
Movie
What’s
lei
to
study?
•  Biochemical
data
(cross‐linking,
solvent
&
substrate
accessibility)
shows
big
conforma-onal
changes
during
transport
process.
•  Both
structure
and
dynamics
of
transporters
needed
to
really
understand
how
they
func-on:
we
need
be=er
movies,
not
just
morphed
structures.
•  Limited
resolu-on
of
known
structures.
•  Not
all
conforma-onal
states
crystallized
for
most
cases.
•  Very
li=le
quan-ta-ve
dynamics
-ed
to
structural
data
–  how
fast
is
interconversion
(apart
from
substrate
on/off)
–  how
is
interconversion
triggered
(molecular
mechanism)
–  Lack
of
structural
data
makes
it
difficult
to
design
and
analyze
quan-ta-ve
dynamics
experiments
in
terms
of
structural
change
•  We
want
a
detailed
mechanism
–
how
can
we
target
mul-drug
efflux
transporters
to
avoid
an-bio-c
resistance
and
chemotherapy
resistance?