Download Structure-function of the ADP/ATP carrier

Membrane Transport
Structure-function of the ADP/ATP carrier
Martin Klingenberg
Institute of Physical Biochemistry, University of Munich, Goethestrasse 33, 8000 Munich 2, Federal Republic of
Germany
‘I’he A1 )P/A’I’P carrier (AAC) is an important
member of the family of mitochondrial solute
carriers. I hese transport prirnarily solute anions
between the cytosol and the mitochondrial matrix.
Although the proteins are of similar size, they can
handle a very wide range of solute sizes, from the
slidlest ones such as 0 1 I or I I in the uncoupling protein of brown fat adipose tissue to about the
largest solutes involved in well defined transport,
such ;IS A1 11’ and ATP.
Our ideas of the structure of the mitochondrial
carriers are derived from the priinary structures 11 I
and from probing the carriers with membrane
imperrneant or permeant agents [2J with substrate
analogues and from reactions with antibodies [ 31.
I:urthermore, ultracentrifugal studies on the isolated
AAC and other proteins have shown that these proteins have a large hydrophobic surface which binds
an extensive detergent micelle [41.Further crude
structural information corms from circular
dichroism studies, from the tumbling rates in the
rnernbrane of spin-labelled carriers etc. [ 51.
‘I’he evaluation of the primary sequence for
hydrophobic transmembrane helices is, in the case
of the AAC carrier, quite equivocal because of the
widely dispersed ionic residues. 1 Iere, the finding of
internal repeat structures within the mitochondrial
carriers permits a more detinite attribution of the
hydrophobic regions. Internal homology analysis
first tor the AAC and subsequently for all other
mitochondrial carriers for which the structure is
known showed that the whole sequence of around
300 residues is divided up into three similar repeats
each of 100 residues IO-XI. Although the degree of
sequence similarity between these repeats is very
low, by appropriately inserting deletions and
arranging these repeats, striking conservations o f
certain residues were found. I n particular, some
obviously critical glycine, proline and acidic residues were conserved. Additionally, by the overlay
of the three repeats, the hydrophobic regions
become more distinctly visible. In each repeat two
hydrophobic stretches of about 20 residues can be
attributed to transmembrane helices. The first helical region is more hydrophobic than the second one
\vithin each repeat. T h e triplicate structure suggests
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an intraprotein symmetry based on three sirnilar
building blocks.
Each building block traverses the membrane
twice. Two putative transmembrane helices are
separated by hydrophilic stretches of 40-50 residues. In these central sections of each domain about
10- 15 polar residues are located. ‘I’he connections
between the three repeat domains are considerably
shorter consisting o f only about 12-1 5 residues.
They are also quite hydrophilic and contain
between three and six polar residues. In general, the
threefold interdomain conservation of residues is
inore pronounced in the central hydrophilic regions
than between the helical stretches. The conserved
localization of charged residues which often occur
in clusters is quite striking. I n contrast, in the connecting regions the repetition of charged residues is
less distinct.
The membrane topography of the nucleotide
carrier is deduced from different types of evidence.
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1 he definition of putative trans-helical segments is
strengthened first, by the repetition in the three
domains, second, by probing certain residues with
membrane impernieant 121 or permeant agents, and
third, by using antibody probes in particular t o the
N-terminus [ 3). Proteases were not useful because
no clearly definite cleavage sites could be discerned.
‘I’he location of the C- or N-terminus is still uncertain as the terminal segments are not accessible t o
specific probes or proteases. For the A1 )l’/Arl’l’
carrier the N-terminus was shown to protrude t o
the cytosolic side by use of peptide-directed aritibodies [ 3 ] .It remains controversial whether there is
an even or uneven nurnber of transmembrane segments. No direct evidence for the location of the Cterminus of the AAC has been produced so far.
I Iowever, in the similar uncoupling protein o f
brown adipose tissue iiii extended hydrophilic Cterminus was clearly shown t o be located on the
cytosolic side. both by proteolytic digestion and by
specific fluorescent SI I-probes [9]. These findings
strongly suggested that the C-terminus in A A C is
also on the cytosolic side. T h e location of both the
C- and N-terminus o n the cytosolic side would also
concur with the even-numbered transmembrane
helices in the three segments.
Probing \vith n~cmbr;inr-imperme;int rcagelits, such ;is ~,!.ridox;ilpliosptiate, showed that
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Biochemical Society Transactions
548
visualized that a translocation channel exists within
each subunit formed by the three repeat domains
along the pseudo threefold symmetry axis. Another
model depends on the dirneric structure of these
carriers. T h e carrier homodirners have been shown
to have only one binding site for specific inhibitors
or substrates. This suggests that there is one central
channel formed by the two dimers along the twofold symmetry axis [ 1 1 I. In this case the channel
would be surrounded by the six domains of the two
subunits. This model would be inore accorninodating to translocating very large solutes. such as A1 11’
and A‘I’P.
An essential part of the single-binding centregated pore model of carrier action are the gates on
both sides of the translocation channel (reviewed in
[12]). T h e model stipulates that either the inner or
outer gate is closed in the external or internal
carrier state. Further conceptual and experimental
developments of the gated pore model address the
problem of how the catalytic energy for the transport is generated [ 13, 141. ‘1’0 put it in simpler
terms: how is the activation barrier for the solute
translocation across the membrane decreased by
the solute-protein interaction? In an attempt t o
answer this question, we conceived the ‘induced tit
transition state’ mechanism in which we postulate
that during the translocation a transition state
occurs in which the binding centre has a conformation that exposes a maximum interaction with the
some lysine groups in the hydrophilic intradomains
are also accessible from the cytosolic side [2]. This
accessibility from the outside and inside was
dependent on the functional state of the carrier. The
accessibility from the cytosolic side of the central
regions seemed at first to contradict their localization on the matrix site according to the six-helical
folding models. Ilowever, both types of data may be
reconciled by assuming that a loop of each of these
central portions is lining the hydrophilic translocation path Mithin the carrier molecule [ I ] . The
lysines in these loops are proposed to be accessible
through the hydrophilic transport channel depending on whether the external o r internal gate is open
or closed (1:ig. 1).
1;urther support of this model is derived from
studies on the localization of the nucleotide- and
inhibitor-binding sites. With 2-azido- or X-azidoATP the central region of the second domain was
labelled 110, 10aJ.T h e nucleotides are incorporated
to the same position in intact mitochondria as in the
isolated protein. This means that they probably gain
access from the cytosolic side to the same binding
site as from the membrane side. In fact, according
to the single-binding centre-gated pore model the
nucleotide-binding region should be accessible
from both the outside and inside.
The three-dimensional folding model is critically dependent on assumptions about the localization o f the translocation channel. Thus, it can be
Fig. I
The proposed charge distribution in the folding of the ADP/ATP carrier
(AAC-2) from yeast
The three-repeat-domain structure i s stressed A hydrophobic loop extends into the
membrane region from the intradomain central matrix section containing some polar
residues These loops are visualized to line the translocation channel The loop of the
second domain contributes t o the nucleotide-binding site
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Membrane Transport
Fig. 2
Fig. 3
The role of the gates in the induced-fit mechanism of
the single-binding centre-gated pore model
The charge-oscillating model of gating in the singlebinding centre-gated pore mechanism
In the transition state the binding centre i s best fitted t o the
substiate The released binding energy results in a conformational-labile transition state, in which both gates around the
binding centre are open t o permit easy substrate transfer t o
either side of the membrane
For details see text. A - , anionic subrtrate.
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/ Closed \
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(Half) open
solutes (Fig. 2). Whereas in the external and internal
states the binding centre is postulated not t o display
substrate-like conformations, these are induced by
the solute-protein interaction in the transition state.
This ‘induced fit’ is the key element of transport
catalysis in carriers. In contrast, in enzymes an
induced fit is catalytically counterproductive and
may only enhance specificity.
Recause the gating is an essential part of the
conforrnational changes during the translocation
process, we have to ask how the gates are configured in the transition state. An approximately
symmetrical state is probable. Originally we
assumed closed gates around the substrate. I lowever, for considerations of catalytic efficiency we
now favour that both gates are open (Fig. 2). ‘I’he
gating and its control is suggested to involve alternating formation and breaking of ionic pairs. The
control conies from the binding of the anionic substrate t o one or more positive charges in the binding centre. This case is illustrated in Fig. .? as what
we call the ‘charge oscillating model’. Negative residues on both sides of the binding centre are postulated to be rnobile and can pair either with the
positive residue of the binding centre or, by moving
outwards, with the positive residues of the gates.
Without a solute the central positive charge is free
and can pair with either the internal or external
mobile negative charge. Hy neutralizing one mobile
negative charge it releases one positive charge at a
gate whic*h can now form an ion bond across the
channel ; i d then close the gate. At the same time,
the opposite gate is open because its positive residue is neutralized by the mobile negative charge.
On binding of the anionic solute the central positive
residue is sequestered and the released mobile
negative charge moves towards the outside and
pairs with the positive charge of the gate. Thus,
binding of a substrate is propagated to the gates by
the two mobile negative residues such that both
gates are open in the transition state. If both gates
were closed. too much binding energy might be
consumed and the energy level of the transition
state would form a trap for the substrate from which
i t would dissociate too slowly. The open-gated
transition state induces t o the carrier a highly labile
state. It can flip either into the external or internal
state from which the substrate is readily released.
‘I’he model requires the proper arrangement
of charges along the translocation channel. As seen
in 1:ig. 1, a sufficient number of charges is available,
particularly in the three central domain regions
which are thought to loop into the translocation
channel. It would be most intriguing to investigate
by site-directed rnutagenesis which of these charges
interrupt the gating process. This is n o w in progress for the AAC-2 of Saccharomyces cereokiae.
1. Klingenberg, bl. ( 10x0) Arch. Hiochem. I3iophys. 270,
1-14
2. Hogner. W., Acluila. i 1. & Klingenberg. M. (10x0)
E u r . J. I3iochcm. 161. 01 1-020
3 . Ihandolin, (;., Houlay, I:., I);ilbon, 1’. 8( Vigiuis, 1’. V.
(10x9) Hiochemistry 28, 1003-1 100
4. I lackenberg, 11. & Klingciiberg, M. (10x0) Hioc~hemistry19, 548-555
5. I lorwath, I,. I., Munding, A,, I k y c r , K., Klingenberg,
\I. 8( Marsh, I ) . (19XO) Hiochemistry 28, 407-41-1
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Biochemical Society Transactions
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6. Sarastc. M. It Walker, J. 1.: (10x2) FEHS 1,ett. 144,
2 5 0-2 5 4
7 . Aqiiila, 11.. Idink, T. A. It Klingenberg. IL1. (1085)
EMHO J. 4. 2.700-2.77f)
8. Acluila, 11.. ],ink. T. A. rU tilingenberg, hl. (1087)
F m S I d t . 212, 1-0
0 . Klingcnberg, XI. It Appcl, M.(1080) Eur. J. Hiochcm.
180,123-131
10. Mayingw. I’.. M’inklcr. li, It Klingenberg, M. (1080)
1;EHS I,ctt. 244, 421-420
10a. Ihlbon. I)., Hrandolin, C.. Houlay. F. et al. (1088)
Hiochemistry 27. .5 141- 5 140
11. Klingcnlwrg, hl. (1081) Nature ( I m ~ d o n ) 290.
440-45 4
12. Klingciibcrg. M. ( 1070) T h e Fnzymes of 13iologic;il
Menilmrics; !Llemhranc 7’r;Irlsport (Akrrtonosi,A. N..
ed.). Vol. 3 . pp. 3x3-438, I’lcnum I’ublishing C‘orp,
New York/l.ondon
1.3. Klingenbcrg, hl. (1085) Ann. N.Y. Ac-ad. Sci. 456,
270-288
14. Klingenbcrg, 14. (1001) A Stud!. of linzymcs;
Mechanism of Enzyme Action (Kuby, S.A,, cd.).Vol.
11. pp. 307-388. CKC I’rcbss, 1ioc;i K;rton
Received 28 April 1092
Studies of the structure and function of sarcoplasmic reticulum (CaZ -Mg2 )ATPase using immunological approaches
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J. Malcolm East, Ian Matthews, Richard E. A. Tunwell,* Ana M. Mata,t and Anthony G. Lee
SERC Centre for Molecular Recognition, Department of Biochemistry, University of Southarnpton, Southarnpton
SO9 3TU. U.K.
Introduction
Amino acid sequence data and site-directed mutagenesis studies have contributed greatly to our
understanding of the uay in \bhich the P-type cation
transporters operate I 1. 2 1. In particular, sitedirected rnutagenesis experiments carried out on
the (<’a’ -Mg’ )-A‘l’Pase of sarcoplasmic reticulum (SK) have provided invaluable information
about the location of the Ca”-binding sites 1.3 I, the
nature of the nucleotide-binding domain [-I
I and the
function of the P-strand domain 15 I, as \\ell as highlighting critical proline residues in the transmembraneous domain 16 I. I kspite these recent
advances, information about the molecular events
which characterize membrane transport and high
resolution structural information about such transporters is still lacking.
The (<’a’ -Mg’+ )-ATI’ase of SR is one of the
most studied of the P-type cation A‘I’Pases [ 7 1. It is
available in large amounts and ;it high purity from
skeletal muscle, making it ;in obvious candidate for
study. The 1’-type ATPases also include a number
of other physiologically important cation transporters such as the plasma membrane (Ka K )-A‘l’l’ase and the 1 I -A‘l’Pase from stom;ich.
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Members of this class of A‘l’l’ases have similar
amino acid sequences and are thought to pump
their respective ions by ;in analogous mech;inism
[ 1 1. Thus. any information gained about the structure-function relationships of the (Ca’+-Mg”)ATPase mill provide important insights into the
working of other members of this family of transporters.
I he series of events which lead t o cation
translocation has been elucidated in some detail for
a number of these transporters and similar pathmays appear t o be folloued in each instance I X I. In
the case of (Ca”-1Llg’+)-A‘l‘l’;tse this involves the
sequential binding of 2C;i’+ and Nl’P t o the El
form of the ATI’ase, which has two high-;iffinity
calcium-binding sites exposed t o the cytopl;ismic
environment, to give E 1 .X’;i’ .ArI’I’. 1;ollouing
phosphorylation of the A‘l’l’use t o give 1: 1 I’X‘;i’+
the A‘I’I’ase undergoes a conform;itioiial cliange t o
E21’.2Ca’+ . In the E2 conforination the <’a’+ -binding sites face the lumen and are of IOU affinity. A s a
consequence the 2Ca’
dissociates from the
Al’Pase and the Al’l’ase is depIios~~lioryl;itedto
give E2 which can then rindergo ;I confi)rm;ition
change back t o E l so that the cycle can continue. In
contrast to our understanding of the reactions
which make up the catalytic cycle of the AW’ase,
relatively little is knou 11 about the molecular corrclates of these events.
Although procedures tor the cr
the (Ca’+-Mg’+)-ATPascin three-dimensioiis are
riou being developed 10- 1 1 ] and some structural
information is becoming available from these crystals [ 121. most of our current understanding about
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