Phenylalanine-508 mediates a cytoplasmic–membrane
domain contact in the CFTR 3D structure crucial
to assembly and channel function
Adrian W. R. Serohijos*†‡, Tama ´s Hegedu ˝s*§, Andrei A. Aleksandrov§¶, Lihua He*§, Liying Cui*§,
Nikolay V. Dokholyan*‡?, and John R. Riordan*§?
Departments of *Biochemistry and Biophysics,†Physics and Astronomy,¶Biomedical Engineering,‡Molecular and Cellular Biophysics Program, and
§Cystic Fibrosis Center, University of North Carolina, Chapel Hill, NC 27599
Communicated by Aziz Sancar, University of North Carolina School of Medicine, Chapel Hill, NC, January 10, 2008 (received for review November 26, 2007)
Deletion of phenylalanine-508 (Phe-508) from the N-terminal nu-
cleotide-binding domain (NBD1) of the cystic fibrosis transmem-
brane conductance regulator (CFTR), a member of the ATP-binding
cassette (ABC) transporter family, disrupts both its folding and
function and causes most cystic fibrosis. Most mutant nascent
chains do not pass quality control in the ER, and those that do
remain thermally unstable, only partially functional, and are rap-
idly endocytosed and degraded. Although the lack of the Phe-508
peptide backbone diminishes the NBD1 folding yield, the absence
of the aromatic side chain is primarily responsible for defective
CFTR assembly and channel gating. However, the site of interdo-
main contact by the side chain is unknown as is the high-resolution
3D structure of the complete protein. Here we present a 3D
structure of CFTR, constructed by molecular modeling and sup-
ported biochemically, in which Phe-508 mediates a tertiary inter-
action between the surface of NBD1 and a cytoplasmic loop (CL4)
in the C-terminal membrane-spanning domain (MSD2). This crucial
cytoplasmic membrane interface, which is dynamically involved in
regulation of channel gating, explains the known sensitivity of
as NBD1 and provides a sharply focused target for small molecules
to treat CF. In addition to identifying a key intramolecular site to
CFTR structure and function and provide a platform for focused
biochemical studies of other features of this unique ABC ion
ABC transporter ? cystic fibrosis ? domain interactions ? modeling ? protein
transport, providing a rate-limiting step in the regulation of salt
secretion and reabsorption (1). Secretory diarrhea results from
persistent activation of CFTR by enterotoxins (2). When CFTR
is absent or defective in humans, normal salt and water ho-
meostasis in epithelial tissues cannot be maintained, resulting in
the accumulation of macromolecular secretions and chronic
infection and inflammation of the airways (3). At the biochem-
ical and cell biological levels, CFTR is distinguished primarily by
two characteristics. First, belonging to the human C subfamily of
ATP-binding cassette (ABC) transporters, CFTR is unique as
the only member of the entire family known to function as an ion
channel (4). Progress toward understanding how fundamentally
similar structures accomplish rapid bidirectional ion permeation
in the case of an ion channel, and much slower vectorial active
transport by transporters, is beginning to shed new light on the
mechanism of each (5). The second feature of CFTR that is of
fundamental importance is the major CF-causing mutation,
deletion of Phe-508 (?F508), which results in misfolding and
misassembly of the complex multidomain protein (6).
The in-frame deletion of the single Phe-508 codon present in
?90% of CF patients prevents conformational maturation in the
he cystic fibrosis transmembrane conductance regulator
(CFTR) plays a fundamental role in metazoan epithelial ion
endoplasmic reticulum (ER). Any mutant protein that reaches
the cell surface remains thermally unstable, capable of only
partial chloride channel activity, and is rapidly endocytosed and
degraded (7). In vitro experiments with the isolated first nucle-
otide-binding domain (NBD1) reveal that the ?F508 mutation
decreases folding yield (8). However, other than the absence of
the Phe-508 residue from its surface, the overall 3D structure of
the domain is little altered (9–11). This observation led to the
hypothesis, for which there is some support (12), that Phe-508
may mediate a critical interaction between NBD1 and a site
elsewhere in the protein. As yet, the hypothesis has not been
confirmed nor has the site been identified. There are indications
understanding of both the impact of the ?F508 and the utiliza-
tion of the ABC architecture to generate ion channel activity has
been hampered by the lack of 3D structural information. Al-
though the crystal structure for the isolated NBD1 is available
(9), there is only a low resolution structure of the whole protein
(15). High-resolution structures of several bacterial ABC trans-
porters have recently been determined by x-ray crystallography
(16–19). These structures provided important new insights into
the mechanisms by which these proteins accomplish transmem-
brane translocation of their substrates (20). Although function-
ing as a channel rather than either an importer or an exporter,
CFTR is a member of the exporter class of ABC proteins.
Sequence and biochemical similarities between several of these
exporters, and those for which atomic structures have been
determined, Sav1866 (16) and MsbA (21), suggest that there are
likely to be strong structural similarities as well.
Molecular Modeling. We constructed a 3D structure of CFTR by
molecular modeling [for detailed methods, see supporting in-
formation (SI) Text]. CFTR consists of nucleotide-binding do-
mains NBD1 and NBD2, membrane-spanning domains MSD1
and MSD2, and a regulatory region called the R domain (Fig. 1).
There are existing 3D structures of NBD1 and also a homology
model of NBD2 derived from structures of NBDs of other ABC
transporters (22). To arrive at a structural model of the complete
CFTR, we built models of the membrane-spanning domains
from the known structure of the full-length ABC exporter,
Sav1866 (SI Fig. 5). Full-length ABC proteins can be grouped
into two classes according to the number and conformation of
Author contributions: A.W.R.S., T.H., N.V.D., and J.R.R. designed research; A.W.R.S., T.H.,
A.A.A., L.H., and L.C. performed research; N.V.D. contributed new reagents/analytic tools;
A.A.A. and L.H. analyzed data; and J.R.R. wrote the paper.
The authors declare no conflict of interest.
?To whom correspondence may be addressed. E-mail: email@example.com or jack?riordan@
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
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their transmembrane helices. Bacterial importers have variable
numbers of helices that are short, positioning their NBDs close
to the membrane plane. The exporters such as Sav1866 possess
12 transmembrane helices that are longer than those of the
importers, thus their NBDs are farther from the membrane
plane. CFTR contains 12 transmembrane helices, and its intra-
cellular loops are of a length similar to those of Sav1866 (16),
which suggested that CFTR MSDs can be modeled from those
of Sav1866 (See SI Text). To organize the different domains of
CFTR, we followed the tertiary organization of the Sav1866
domains. The structural model is consistent with available
experimental data on the orientation and packing of CFTR
transmembrane helices (see SI Text and SI Fig. 6). At least some
of these data derive from measurements of open-channel cur-
rents, whereas the Sav1866 structure is believed to represent the
closed state of the translocation pathway of that transporter (16).
Nevertheless, the interdomain contacts in the modeled CFTR
structure, which are our primary focus here, are strongly con-
firmed by biochemical experiments (see below). Significantly,
analogous contacts have been shown to be maintained in both
open and closed conformations of the MsbA exporter (21).
The R domain unique to CFTR is largely unstructured (23).
Secondary structure prediction algorithms as well as our ab initio
folding simulations (see SI Text) suggested the formation of
persistent secondary structural elements and tertiary arrange-
ment of the chain. We approximated the R domain by con-
structing an ensemble of observed conformations and repre-
senting it by the centroid of the most dominant structure in this
ensemble (Fig. 1B). The placement of the R domain in the
structure is consistent with preliminary EM observations of
purified CFTR, with nanogold labeling of polyhistidine se-
quences inserted into the R domain and localized dynamic
changes on phosphorylation by protein kinase A (PKA) (R. C.
Ford and J.R.R., unpublished observations).
Cytoplasmic–Membrane Domain Interfaces. Most of the electro-
physiological and biochemical constraints accommodated by the
structure relate to the interactions of the two NBDs or MSDs (SI
Fig. 6) with each other but not those between NBDs and MSDs.
The NBD/MSD interfaces are likely to be crucial in both
assembly during biogenesis and in mediating conformational
signals that influence channel activity. Therefore, we focused on
template for building the structural model, these interfaces
comprise contacts between the NBD in one half of the molecule
and the MSD in the other. Earlier studies had shown many
disease-associated missense mutations in CL4, including several
coupling helix in Sav1866 (24, 25). The positions of three of these
mutated residues in proximity to NBD1 are shown in Fig. 2A.
The abilities of synthetic 18-mer peptides containing the wild
type or each of the mutated CL4 sequences to bind NBD1 were
tested in pull-down experiments. The wild-type peptide bound
strongly, whereas the two mutations (L1065P and R1066C) that
prevent conformational maturation (24) reduced this binding to
control levels (Fig. 2B). The third mutation (G1069R), which
does not prevent maturation but impairs channel function (25),
causes a lesser reduction in binding. Surface plasmon resonance
analysis (Fig. 2C) of the binding yielded essentially the same
result in which interaction of purified NBD1 with the wild-type
membrane-spanning domains (MSD1 and MSD2), and a regulatory region (R domain). Each MSD contains two cytoplasmic loops (CL) that form interfaces with
R domain, which is largely unstructured (23), was approximated by constructing an ensemble of dynamically accessible conformations derived from ab initio
folding (see SI Text). R domain backbone size is rendered in proportion to variations of C?atoms. (C) Close-up view of the interfaces formed between NBD1/CL4
and NBD2/CL2. Cross-linking of Cys pairs F508C/L1065C, F508C/F1068C, F508C/G1069C, and F508C/F1074C confirms that Phe-508 in NBD1 associates with CL4 in
MSD2 (Fig. 3 and SI Fig. 7). Cross-linking of C276/Q1280C and C276/K1284C confirms interaction of CL2 and NBD2.
Theoretical model of CFTR structure. (A) Schema of CFTR primary structure containing two nucleotide-binding domains (NBD1 and NBD2), two
Serohijos et al.
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Cysteine Cross-Linking. As a second means of assessing the NBD1/
CL4 interface and to locate specifically the contact sites between
CL4 and NBD1, pairs of cysteine residues were introduced into a
functional Cys-less CFTR construct (26) at positions in CL4 and
NBD1 that are in close proximity according to our modeled
structure (Fig. 3A, Top Left). As seen in the Left column of Fig. 3A,
several such Cys pairs could be cross-linked by bifunctional meth-
ane-thiosulfonate (MTS) reagents. In control experiments where
separate constructs containing individual rather than both mem-
Fig. 7). Strikingly, Phe-508 apparently plays a central role in this
interface because it can be cross-linked to cysteines introduced at
many positions in CL4. These positions include Leu-1065, Phe-
1068, Gly-1069, and Phe-1074 (Fig. 3A and SI Fig. 8). Cross-linking
also occurs between cysteines substituted for another hydrophobic
residue (Val-510) near Phe-508 on the NBD1 surface and G1069C
in CL4 (SI Fig. 8). In addition to the Phe-508-containing NBD1
surface patch, residues in other regions of the domain also interact
with CL4 residues as evidenced by cross-linking of Cys pairs
involving amino acids closer to the Q loop (Gln-493), including
W496C/T1064C and M498C/L1065C as well as nearer the Walker
B motif (Asp-572) such as K564C/G1069C (Fig. 3A and SI Fig. 8).
These data confirm the extensive NBD1/CL4 contact apparent in
Most of the specific Cys pair cross-links were mediated by
MTS reagents with spacer arms ranging from 3.9 to 24.7 Å in
length, which initially seemed somewhat surprising but must
indicate considerable flexibility in the positioning of each mem-
ber of the pair with respect to each other (Fig. 3 A and B, Right
and SI Fig. 8). Nevertheless, the proximity or relative orientation
of the F508C/F1068C, F508C/G1069C, and V510C/G1069C
pairs permitted very little disulfide bond formation on oxidation
catalyzed by copper phenanthroline, i.e., only a very small
proportion of mature band was converted to cross-linked band
tions (L1065P, R1066C, and G1069R) at the NBD1/CL4 interface. (B) Disease-
causing mutations in CL4 abolish or diminish the CL4 and NBD1 interaction.
were eluted with sample buffer and detected by Western blotting with CFTR
antibody 660. NeutrAvidin beads without bound peptide were used as control.
(C) CL4 binds to NBD1 as detected with surface plasmon resonance. Biotinylated
peptides were immobilized on a BIAcore streptavidin sensor chip to 200 reso-
resonance and BIAcore 2000. The binding of NBD1 to the chip without peptide
was subtracted from NBD1 binding to the peptides.
CL4 peptide binding to NBD1. (A) Location of disease-associated muta-
by cross-linking of residues close to the Q loop (W496C/T1064C and M498C/
L1065C) and a residue near the Walker B motif (K564C/G1069C). HEK293 cells
transiently transfected with Cys-less CFTR containing Cys pairs were harvested
lengths for 15 min at room temperature. Samples with or without DTT were
subjected to SDS/PAGE and Western blotting with mAb596. Cross-linked species
of cross-linking of the Cys-less construct and individually expressed and mixed
single cysteine constructs (labeled 276C ? Q1280C) under the same conditions
(see also SI Fig. 8). (B) (Right) Cross-linking by the shortest reagent 1,1-
methanediyl bismethanethiosulfonate (M1M), in isolated membranes, indicates
close contact and probable mobility of residues across both interfaces. (Left)
There is greater propensity for disulfide bond formation at the CL2/NBD2 inter-
core-glycosylated CFTR (band B); gray arrowhead, mature complex-glycosylated
CFTR (band C); black arrowhead, cross-linked mature protein.
Cross-linking of interfacial cysteine pairs. (A) Confirmation of the
www.pnas.org?cgi?doi?10.1073?pnas.0800254105Serohijos et al.
(Fig. 3B, Left). In contrast, in all cases where strong cross-linking
occurred, only the mature form of CFTR but not the immature
one was converted to the cross-linked species of slower mobility
(Fig. 3 and SI Fig. 8). This finding provides strong evidence that
residue associations occur only in the mature native form of the
protein. There is indirect evidence that the Phe-508 side chain
Our present findings identify the site of the interdomain asso-
ciation and in so doing confirm that the so-called domain
swapping described by Dawson and Locher in the Sav1866
exporter (16) and also observed in the P-glycoprotein multidrug
exporter (27) and the MsbA lipid exporter (21) occurs in CFTR.
Unlike Sav1866, which is a homodimer containing two iden-
tical NBDs and MSDs, CFTR contains two distinct domains of
each type. The structural model predicts that domain swapping
occurs symmetrically between NBDs and MSDs in opposite
halves of the molecule. Cysteine cross-linking confirms that
domain swapping occurs between CL2 and NBD2 (Fig. 3). In
fact, the Q1280C and K1284C substitutions in NBD2 of Cys-less
CFTR containing the native Cys-276 residue in CL2 (illustrated
in Fig. 3A, Top Right) enabled cross-linking by all MTS reagents
tested. Furthermore, disulfide bond formation between cys-
teines at positions 276 and 1280 occurred spontaneously because
of oxidation during membrane isolation and was further pro-
moted by copper phenanthroline (Fig. 3B, Left). This observa-
tion indicates close contact between the native Cys-276 residue
in CL2 and Gln-1280 in NBD2. Thus, when NBD2 is synthesized
to complete the CFTR structure, it integrates with CL2 in
MSD1. However, although there are many disease-associated
mutations in both NBD1 and CL4 that compromise assembly,
few have been identified in either NBD2 or CL2 (www.genet.
sickkids.on.ca/cftr). This finding now can be understood in view
of the realization that incorporation of NBD2 is not required for
the correct assembly of the other domains into a structure that
satisfies ER quality control (13, 14).
Role of Interfaces in Channel Gating. It is likely that both of these
interfaces mediate conformational signals elicited by ATP binding
and hydrolysis to the channel gate. If this is the case, it is unclear
ylation, should influence the formation of cross-links between
exposure to 10 ?M M1M from the cis side of the bilayer followed by 10 mM DTT. For Cys-less CFTR, the last 4 min from the total 20 min of M1M treatment is
that contribute to cross-linkable pairs (SI Fig. 11). (B) NBD1 and CL4 participate early in the gating cycle. Brønsted plots for wild-type CFTR gated by 2 mM
nucleotide ligands shown by each experimental point (Left) and substitutions of CL4 residues (Right). Both graphs are linear with the slope ? values indicated.
Points on both graphs are shown as mean values ? SEM of at least four different experiments. (Inset) Hypothetical free energy landscape of CFTR gating where
troughs (stable states) are colored blue and crests (unstable states) are red. At the transition state (saddle point), NBD1 has proceeded 98% toward the open
state, and CL4 has proceeded 86%.
Role of domain–domain interactions in CFTR channel gating. (A) Inhibition of CFTR channel gating by cross-linking. Single-channel recording after
Serohijos et al.
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vol. 105 ?
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should influence channel gating. Treatment of cells with cAMP-
not alter cross-linking of cysteines between either CL4 and NBD1
cells, high concentrations of nucleotides [MgATP, adenosine 5?-
without phosphorylation by PKA also did not strongly influence
formation of the cross-links. These results would imply that if
conformational signals were being transmitted, structural elements
on either side of the interface may move in concert. If so, channel
gating might or might not be expected to be influenced by covalent
cross-linking between cysteines on either side of the interface. In
fact, we found that single-channel gating, which persists after the
introduction of a Cys pair at each interface, was completely
contact are integral elements of the structure, and covalent cou-
pling between residues on either side restricts channel activity. This
restriction is unlikely to be caused by prevention of signal trans-
interfaces, which also is suggested by cross-linking by reagents of
on the interactions between the NBDs and the CLs (SI Fig. 9) also
suggests that there may be coordinated movements of the two sides
of the interfaces.
As an independent means of comparing the stages in the gating
energy relationship (REFER) analysis (28) was applied (Fig. 4B).
The slopes (? values) of the linear log/log plots relating the rate
constants of single-channel opening and the equilibrium constants
approach 1 (? ? 0.98) for different channel-activating nucleotide
ligands and only a slightly lesser value for multiple amino acid
substitutions in CL4 (? ? 0.86). These values indicate that ligand
binding is essentially complete before the transition state for
way to the open state at the transition state. Thus, both parts of the
structure appear to participate early in the gating cycle, consistent
with a dynamic functional role of the NBD1/CL4 interface. Signif-
icantly, a ? value near 1 for NBD1 was also obtained by similar
analysis of different chimeric constructs of the domain (29).
Thus, a 3D structure of CFTR derived by molecular modeling
reveals important aspects of the interfaces between the NBDs and
MSDs of CFTR. The most critical contact in conformational
maturation during biosynthesis, which is precluded by the ?F508
mutation, is between a site containing that residue on the surface
of the N-terminal NBD and a cytoplasmic loop in the C-terminal
MSD. This finding is consistent with and helps to explain previous
observations of disruption of CFTR membrane domains by the
the first to decrease the folding yield and transitions between
intermediate states in the folding of NBD1 itself, and the second,
to prevent an interaction of the NBD1 surface elsewhere in the
protein. We have identified the site of this interaction, dependent
on the phenylalanine aromatic side chain. It appears to participate
in an aromatic cluster with residues from CL4 (Fig. 3A and SI Fig.
10), which may contribute to the stability of this vital tertiary
interaction as in other proteins (30, 31). The confirmed 3D struc-
to be restored or mimicked to counteract the defect caused by the
mutation. The corresponding crossing-over between NBD2 and
MSD1 is less crucial to maturation of the protein because NBD2 is
the last portion of the polypeptide to be translated and may be
incorporated posttranslationally (12).
In addition to establishing the precise intramolecular contact
formed by the Phe-508 side chain in the assembly of the N- and
C-terminal portions of the protein, our findings provide some
insight into the role of this interface in the regulation of the CFTR
either side of this interface arrests channel gating, indicating a
requirement for dynamic contact at these sites. This finding applies
to both the Phe-508-containing NBD1/CL4 interface and the
counterpart between NBD2 and CL2. These interactions between
NBDs and MSDs in opposite halves of the molecule, which have
been referred to as domain swapping (16) or intertwining (21)
between subunits of homodimer ABC transporters, are probably
also important in the transmission of regulatory signals. The fact
by the stimuli that activate channel gating is consistent with their
action as connecting joints between other portions of the molecule
that may undergo larger ranges of motion. This interpretation is
supported by the REFER analysis of single-channel kinetics, which
cytoplasmic loops between transmembrane helices are tightly
construct an atomic model of CFTR MSDs, The x-ray structure of NBD1 (9) and
an existing homology model of NBD2 (22) were used. The R domain was
approximated by an ensemble of conformations derived by ab initio folding.
CL4-NBD1 Binding. Biotinylated synthetic peptides corresponding to wild-type
and mutant versions of a CL4 peptide were immobilized on NeutrAvidin beads
and used to pull down purified human NBD1 protein kindly provided by P. J.
Thomas (University of Texas Southwestern Medical Center, Dallas, TX). Binding
also was assayed by surface plasmon resonance (BIAcore 2000).
Cysteine Cross-Linking. HEK293 or BHK cells or membranes expressing Cys-less
CFTR containing specific Cys pairs were incubated with MTS reagents of
different chain lengths and analyzed by SDS/PAGE with or without reduction
with 30 mM DTT and Western blotting.
to Mitra et al. (28).
Details of all of these procedures are in SI Text.
ACKNOWLEDGMENTS. This work was supported by National Institutes of
Health Grant DK051619 (to J.R.R.) and Cystic Fibrosis Foundation Grant
DOKHOL07I0 (to N.V.D.). A.W.R.S. is a Predoctoral Fellow of the American
Heart Association, Grant 0715215U.
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