High-chloride concentrations abolish the binding of adenine nucleotides in the mitochondrial ADP/ATP carrier family.

Abstract

The ADP/ATP carrier (AAC) is a very effective membrane protein that mediates the exchange of ADP and ATP across the mitochondrial membrane. In vivo transport measurements on the AAC overexpressed in Escherichia coli demonstrate that this process can be severely inhibited by high-chloride concentrations. Molecular-dynamics simulations reveal a strong modification of the topology of the local electric field related to the number of chloride ions inside the cavity. Halide ions are shown to shield the positive charges lining the internal cavity of the carrier by accurate targeting of key basic residues. These specific amino acids are highly conserved as highlighted by the analysis of multiple AAC sequences. These results strongly suggest that the chloride concentration acts as an electrostatic lock for the mitochondrial AAC family, thereby preventing adenine nucleotides from reaching their dedicated binding sites.

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High-Chloride Concentrations Abolish the Binding of Adenine Nucleotides
in the Mitochondrial ADP/ATP Carrier Family
Eva-Maria Krammer,†Ste ´phanie Ravaud,‡Franc ¸ois Dehez,†* Annie Frelet-Barrand,§Eva Pebay-Peyroula,‡
and Christophe Chipot†*
†Equipe de Dynamique des Assemblages Membranaires, UMR No. 7565, Centre National de la Recherche Scientifique-Universite ´ Henri
Poincare ´, Nancy, France;‡Institut de Biologie Structurale, UMR No. 5075, Commissariat a ` l’E´nergie Atomique (CEA)-Centre National de la
Recherche Scientifique-Universite ´ Joseph Fourier, Grenoble, France; and§Laboratoire de Physiologie Cellulaire Ve ´ge ´tale, UMR No. 5168,
Centre National de la Recherche Scientifique/CEA/Institut National de la Recherche Agronomique (UMR No. 1200)/Universite ´ Joseph Fourier,
CEA, Direction des Sciences du Vivant, Institut de Recherches en Technologies et Sciences pour le Vivant, Grenoble, France
ABSTRACT
across the mitochondrial membrane. In vivo transport measurements on the AAC overexpressed in Escherichia coli demonstrate
that this process can be severely inhibited by high-chloride concentrations. Molecular-dynamics simulations reveal a strong
modification of the topology of the local electric field related to the number of chloride ions inside the cavity. Halide ions are shown
to shield the positive charges lining the internal cavity of the carrier by accurate targeting of key basic residues. These specific
amino acids are highly conserved as highlighted by the analysis of multiple AAC sequences. These results strongly suggest that
the chloride concentration acts as an electrostatic lock for the mitochondrial AAC family, thereby preventing adenine nucleotides
from reaching their dedicated binding sites.
The ADP/ATP carrier (AAC) is a very effective membrane protein that mediates the exchange of ADP and ATP
Received for publication 8 July 2009 and in final form 20 August 2009.
*Correspondence: francois.dehez@edam.uhp-nancy.fr or chipot@ks.uiuc.edu
Christophe Chipot’s present address is Theoretical and Computational Biophysics Group, Beckman Institute, University of Illinois at Urbana-Champaign,
405 North Mathews, Urbana, Illinois 61801.
ATP, the energy fuel of the cell, is synthesized from ADP in
the mitochondria, using the proton gradient created by the
respiratory chain. Alteration of the ADP and ATP exchange
across the inner mitochondrial membrane bears various path-
ologicalimplications(1).TheADP/ATPtransportismediated
by a member of the mitochondrial carrier family, e.g., the
ADP/ATP carrier (AAC). In the absence of a membrane
potential,exchangecanoccurinbothdirections(5).Thestruc-
ture of the bovine-heart AAC has been solved in the presence
of the inhibitor carboxyatractyloside (6,7). It consists of six
a-helices forming a compact transmembrane domain on the
matrix side, with a cavity opened toward the intermembrane
space. Two patches of basic residues line this cavity. The
upperpatch,locatedatthemouthoftheAAC,consistsofresi-
duesK91,K95,R104,K106,R187,andK198.ResiduesK22,
K32,R79,R137,R234,R235,andR279formthelowerbasic
patch that delineates the bottom of the cavity. Noteworthily,
several of these residues are involved in carboxyatractyloside
and/or ADP3?binding (6). The latter two basic patches con-
tribute to the unique electrostatic topology of the AAC,
shaped in a funnel conducive to drive the substrate down-
wardstothebottomoftheinternalcavity(6,8,9).Biochemical
experiments havedemonstrated that mutationofspecificresi-
duesabolishesthetransportactivityofAAC(forareview,see
(10)). The function of the protein can also be affected by an
appropriate modification of the ionic concentration. Experi-
ments emphasize that high concentrations of either magne-
sium or chloride ions inhibit the function of the bovine-heart
AAC (11–13). Mg2þis assumed to bind to the substrate,
which can no longer be transported in a complexed form. In
sharp contrast, chloride is believed to affect directly the prop-
erties of the membrane protein (13).
A synergistic experiment-theory study has been carried out
here to explain the effects of chloride concentrations on the
function of the overall AAC family. Transport activities were
measured for Arabidopsis thaliana AAC isoform 1 (AtAAC1)
expressedinEscherichiacolitoprobehowsensitivetheinhibi-
tion of the AAC family is in response to high-chloride concen-
trations.Molecular-dynamics(MD)simulationsofbovine-heart
AAC isoform 1 (bAAC1) were carried out concomitantly to
decipherhowtransport across theAAC is thwartedby chloride
concentrations. In the light of the above concerted investiga-
tions, supplemented by an evolutionary analysis, congruent
conclusionsaredrawnonchlorideinhibitionintheAACfamily.
Groppetal.(13)observedtheinhibitoryeffectofanionson
purified bAAC1 reconstituted in liposomes. To extend this
observation, functional expression of recombinant AACs in
E.coliwasperformedhere,therebyprovidingauniqueframe-
work for investigating in vivo the biochemical properties of
AACs. Transport of [a-32P]ATP assayed on whole E. coli
cells expressing AtAAC1 demonstrates that the carrier is
produced in a functional form in the inner membrane of
Editor: Hassane Mchaourab.
? 2009 by the Biophysical Society
doi: 10.1016/j.bpj.2009.08.047
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E.coli(Fig.1,inset).Thesameexperimentsperformedatlow
NaCl concentration (0.15 M) do not reveal any significant
effect on transport activity, whereas addition of 0.5 M or
0.6MNaClmarkedlyreducestransport(Fig.1).Theseresults
are in line with measurements (13) based on bAAC1 sharing
74%ofsequencehomologywithAtAAC1. Thedirectinvivo
measurements of ATP uptake inhibition reported here conse-
quently suggest that the inhibition potency of high-chloride
concentrations ought to hold for the entire mitochondrial
AAC family.
Atthemolecularlevel,chlorideionsareenvisionedtoshield
the positive charges lining the internal cavity of the carrier
(13).Onthetheoreticalfront,theeffectsofchlorideconcentra-
tions on the transport properties of the AAC were examined
using three different salt conditions as models of low (e.g.,
0.1 M and 0.15 M Cl?) and high chloride concentrations
(e.g., 0.6 M Cl?). For each concentration, MD trajectories of
thebAAC1embeddedinafullyhydratedpalmitoyloleylphos-
phatidylcholine bilayer were generated, with and without
ADP3?. All MD simulations were performed employing the
NAMD program (14) with the CHARMM27 force field
(15,16) and a revision of the latter for lipids (17).
Thenumberofchlorideionsinthecavityisafunctionofthe
chloride concentration in the solvent (see Supporting Mate-
rial). At low NaCl concentration, the halide ions primarily
interact with residues K22, R79, and R279 of the lower basic
patch. Furthermore, a chloride ion transiently binds to R235
(0.1 M NaCl assay) and to R137 (0.15 M NaCl assay). In
both assays, no chloride ion is permanently complexed with
residues of the upper basic patch. In contrast, in the 0.6 M
NaClassay,severalchlorideionsinteractfrequentlywithresi-
dues of the upper patch (i.e., K91, K95, R187, and K198).
They also interact with residues K22, R79, and R279 of the
lower patch, reminiscent of the simulations at low concentra-
tions. In addition, a chloride ion associates with R234, occu-
pyingthesame position foraslongas29.5ns.The significant
persistenceofthischlorideion,togetherwiththesaturationof
the two basic patches,results at ahigh-chlorideconcentration
in a less accessible binding site for ADP3?.
As was highlighted previously (8,9), the protein exhibits
a funnellike electrostatic pathway. A cross-sectional view of
the three-dimensional map of the electric field (Fig. 2) indi-
cates that the distribution of chloride ions affects the electro-
static signature of the protein (see Supporting Material). At
low-salt concentration, the electrostatic funnel ends at the
bottom of the cavity, where the electric field points down-
wards. Under such circumstances, ADP3?has been shown
to reach spontaneously its dedicated binding site (8,9). At
0.6MNaCl,thetopologyoftheelectricfielddiffersmarkedly
above the region formed by the lower basic patch, where its
directionnowpointsdownwards.Inthisscenario,anegatively
chargedsubstrateispreventedfromreachingthebottomofthe
cavity. Thus, even though an electrostatic funnel prevails at
a high-chloride concentration, it does not end as deep in the
cavity as it would at low-salt concentration.
Binding numerical experiments, where ADP3?is initially
locatedatthemouthofAAC,haverevealedthatthissubstrate
binds spontaneously to the protein, independently of its start-
ing position (9,8). Simulations featuring three different
ligand-association assays carried out at 0.15 M NaCl led to
the association of ADP3?(see Supporting Material), consis-
tentwiththeexperimentaldata(Fig.1),aswellastheprevious
findingsofDehezetal.(9).Consideringninedifferentstarting
positions for ADP3?, association was, however, never
observed at 0.6 M NaCl (Fig. 3). Nine similar experiments
weresubsequentlyrepeatedintheabsenceofanionicconcen-
tration, while keeping the same initial position of the
substrate. All these simulations invariably led to the associa-
tion of ADP3?(Fig. 3). Put together, these ligand-association
and transport assays remarkably illustrate the crucial role
played by chloride concentration on AAC activity.
Inhibition does not appear to be specific to bAAC1. These
experiments suggest, on the contrary, that abolition of trans-
port by chloride ions is characteristic of the AAC family.
FIGURE 1
AtAAC1. (Inset) Time dependence of [a-32P]ATP uptake into
intact IPTG-induced E. coli cells harboring plasmid encoding
mature AtAAC1 (squares) or the control plasmid (circles).
The data shown is the mean of four independent experiments
(SE < 10% of the mean values; see Supporting Material).
Inhibitory effect of NaCl on the [a-32P]ATP uptake by
FIGURE 2
tion of the salt concentration. (A) Orientation of the bAAC1. A
molecular-surface rendering and a ribbon representation are
used for regions of the protein located, respectively, behind
and in front of the plane in which the electric field is shown. The
basic patches are highlighted in cyan. The cross-sectional view
of the electric field after 30 ns differs between (B) 0.1 M and (C)
0.6 M assay. Maps of the electric field were obtained using the
PMEPot (18) module of VMD (19) and OpenDX (http://www.
opendx.org).
The electric propertiesof apo-bAAC1 differ as a func-
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Multiple-sequence alignment was, hence, carried out, based
on 74 AAC sequences. Analysis of the alignment reveals
a conservation of those residues directly involved in the
binding of halide ions (see Supporting Material). It should,
therefore, be expected that shielding of dedicated basic resi-
dues by chloride ions is a common phenomenon likely to
occur in all mitochondrial AACs. The small size of these
ions allows each single amino acid essential to the transport
to be targeted with optimal accuracy, regardless of the local
structure of the carrier.
In this work, the effect of high-chloride concentration on
the activity of the mitochondrial AAC family has been un-
raveled by combining synergistically experimental and theo-
retical investigations. Uptake experiments of radioactively
labeled ATP4?have demonstrated that transport in the plant
AtAAC1 is also inhibited at high-chloride concentrations.
Molecular detail of the inhibition is unveiled by MD simula-
tions performed on the bAAC1 structure. As a function of the
concentration, a large number of ions are prone to gush into
the internal cavity of the protein. In turn, ionic saturation of
those residues pertaining to the two basic patches of the AAC
strongly modulates the topology of the internal electric field,
thus, locking the carrier in a state unfavorable to substrate
binding. A conservation analysis performed over a large
set of AAC sequences shows that the key residues shielded
by Cl?are conserved. This observation strongly suggests
that inhibition at high-chloride concentrations can occur
independently of the AAC sequence. Chloride concentration
may, therefore, be viewed as an electrostatic lock for the
mitochondrial AAC family, preventing the adenine nucleo-
tide from reaching its target-binding site. Our results empha-
size that in the context of functional assays, particularly for
experimental nucleotide binding assays, chloride concentra-
tion constitutes an important parameter that ought to be opti-
mized carefully.
SUPPORTING MATERIAL
Eleven figures and three tables are available at http://www.biophysj.org/
biophysj/supplemental/S0006-3495(09)01430-1.
ACKNOWLEDGMENTS
The authors are indebted to the Grand Equipement National de Calcul Inten-
sif and the Centre Informatic National de l’Enseignement Supe ´rieur for
provision of computer time.
ThisworkisfundedbytheL’AgenceNationaledelaRecherche,theCommis-
sariata ` l’E´nergieAtomique,theCentreNationaldelaRechercheScientifique,
and the European Drug Initiative on Channels and Transporters program.
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FIGURE 3
results in the binding of ADP3?only after removal of the ions. (A)
The different initial positions of ADP3?are depicted as colored
tubes. A molecular-surface rendering is used for the protein
(white), the upper (light-blue), and the lower patches (ice-blue).
Chloride ions are shown as green van der Waals spheres. (B)
Association experiment C. The starting position of ADP3?and its
final positions at 0.6 M NaCl and without salt are shown, respec-
tively, as van der Waals spheres, and green and orange tubes.
Image rendering using VMD (19).
The series of ligand-association assays at 0.6 M NaCl
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