1 Alfred I. duPont Hospital for Children, Wilmington, Delaware 19803; 2 Wistar Institute and 3 Institute for Human Gene Therapy, University of Pennsylvania, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR is a chloride channel whose activity requires protein kinase A-dependent phosphorylation of an intracellular regulatory domain (R-domain) and ATP hydrolysis at the nucleotide-binding domains (NBDs). To identify potential sites of domain-domain interaction within CFTR, we expressed, purified, and refolded histidine (His)- and glutathione-S-transferase (GST)-tagged cytoplasmic domains of CFTR. ATP-binding to his-NBD1 and his-NBD2 was demonstrated by measuring tryptophan fluorescence quenching. Tryptic digestion of in vitro phosphorylated his-NBD1-R and in situ phosphorylated CFTR generated the same phosphopeptides. An interaction between NBD1-R and NBD2 was assayed by tryptophan fluorescence quenching. Binding among all pairwise combinations of R-domain, NBD1, and NBD2 was demonstrated with an overlay assay. To identify specific sites of interaction between domains of CFTR, an overlay assay was used to probe an overlapping peptide library spanning all intracellular regions of CFTR with his-NBD1, his-NBD2, and GST-R-domain. By mapping peptides from NBD1 and NBD2 that bound to other intracellular domains onto crystal structures for HisP, MalK, and Rad50, probable sites of interaction between NBD1 and NBD2 were identified. Our data support a model where NBDs form dimers with the ATP-binding sites at the domain-domain interface.
tryptophan fluorescence; peptide array; molecular structure; peptide library; phosphorylation
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INTRODUCTION |
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CYSTIC FIBROSIS (CF)
is a genetic disease caused by mutations in the gene encoding the
cystic fibrosis transmembrane conductance regulator (CFTR), an
epithelial chloride channel (35, 37). CFTR is a member of
the ATP-binding cassette (ABC) transporter gene superfamily
(14). More than 1,000 ABC transporters are known, and they
are found in both prokaryotic and eukaryotic cells where they carry out
the unidirectional ATP-dependent transport of a wide variety of
molecules. All known ABC transporters have two membrane-spanning
domains (MSDs), usually composed of six transmembrane helices, and two
nucleotide-binding domains (NBDs) (14). These domains
can be expressed in a single subunit, as in CFTR and
multidrug resistance protein (MDR), or in multiple subunits, as
in the bacterial maltose and histidine transporters. ABC transporters
like CFTR and MDR have intracellular NH2- and COOH-terminals and four intracellular loops, L1 to L4, between transmembrane helices 2 and 3, 4 and 5, 8 and 9, and 10 and 11, respectively. CFTR is unique among ABC family members because it is the
only family member to contain an additional intracellular domain, the
regulatory or R-domain. CFTR is also the only ABC family member that is
known to function as an ion channel (37). The R-domain
contains multiple protein kinase A (PKA)-dependent phosphorylation
sites that are involved in the regulation of CFTR channel activity
(35). The regulation of CFTR channel activity involves a
two-step process; phosphorylation of the R-domain, most likely on
multiple sites, is required so that ATP hydrolysis by the NBDs can
regulate CFTR gating (1, 11, 34, 36, 46). Mutations in the
R-domain, most notably at phosphorylation sites, alter ATP-dependent
CFTR gating (46). In addition, the CF-causing F508
mutation, located in NBD1, has been shown to alter in situ
phosphorylation (18). The fact that alterations in one
domain can modulate the function of a second domain indicates that
functionally important domain-domain interactions must occur (11). The primary goal of the study was to identify the
sites of domain-domain interaction.
A number of studies have suggested that specific domain-domain associations are required for optimal CFTR activity. An in vitro association between NBD1-R and NBD2 constructs has been demonstrated by fluorescence quenching and exclusion chromatography (26). Interactions between the NH2-and COOH-terminals with other domains of CFTR have been postulated on the basis of CFTR channel activity (29, 32, 42). However, there is no information on the specific sites of interaction among CFTR domains. In addition, recent electrophysiological and cytochemical studies have suggested that native CFTR may exist as a homodimer (9, 32, 42, 47), but any dimeric form would appear to be relatively unstable because biochemical studies have failed to detect multimeric forms of CFTR (6, 28). Despite the fact that CFTR has been shown by both immunological and yeast two-hybrid studies to interact with a number of membrane-associated proteins (12, 13, 29, 39).
No crystal structures have been generated for any domain of CFTR; however, structures for bacterial NBDs have been reported. These include HisP from the histidine transporter of Salmonella typhimurium and MalK from the maltose transporter of Thermococcus litoralis (7, 17). In addition, a crystal structure for Rad50, a DNA-binding protein with a similar motif to HisP and MalK, has been reported (15). Comparison of the crystal structures for the NBD monomers suggests that the basic fold is conserved. Recently, two additional crystal structures for NBD monomers have been reported; they have the same structure as the NBDs of HisP and MalK (21, 44). However, crystallographic structures for HisP, MalK, and Rad50 dimers vary considerably. Because the relative orientation of the two NBDs should be conserved among all ABC family members (19), we suggest that no more than one, and possibly none, of the observed dimeric structures are related to the structure in native ABC transporters. Although additional crystal structures may resolve this issue, we have taken a biochemical approach. To this end, individual cytoplasmic domains of CFTR were expressed, purified, and refolded. Sites of domain-domain association were determined by in vitro binding to an overlapping peptide library generated from the intracellular regions of CFTR. Peptides within the NBDs that bound cytoplasmic domains were mapped onto the known NBD structures. Predicted sites of domain-domain interaction were compared with published structures for NBD dimers.
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MATERIALS AND METHODS |
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Generation of expression constructs. Polymerase chain reaction (PCR) was employed to amplify cDNA fragments of CFTR from a pcDNA vector (Invitrogen) containing CFTR (obtained from Dr. W. Skach, Oregon Health Sciences University). DNA fragments of NBD1 (nucleotides 1249-1899; amino acids 373-589), NBD2 (nucleotides 3583-4560; amino acids 1151-1476), and NBD1-R (nucleotides 1249-2709; amino acids 373-859) were cloned into pProEx HT (Qiagen) with an NH2-terminal his-x6 tag. Because of its poor expression as a His-tagged protein, R-domain (nucleotides 1891-2709; amino acids 589-830) was cloned into pGEX-5X (Amersham Pharmacia Biotech) with glutathione-S-transferase (GST) fused to its NH2 terminus. The correct recombinants were identified by restriction mapping and sequencing.
Expression, purification, and refolding. CFTR recombinants were expressed in Escherichia coli BL21codon-plus (Strategene). His-tagged CFTR domains were purified from inclusion bodies in 8 M urea by Ni-affinity chromatography according to the manufacturer's instruction (Qiagen). GST-R protein was purified on glutathione-Sepharose 4B (10). Protein purity was analyzed by SDS-PAGE, and protein concentrations were determined with bicinchoninic acid (40). GST-R domain was renatured by dialyzing overnight against phosphate-buffered saline (PBS, pH 7.2). His-tagged proteins were renatured by diluting 10-fold into binding buffer (50 mM Tris, pH 7.5, 0.15 M NaCl, 1 mM EDTA, 0.5% NP-40, 0.5 mM dithiothreitol, and 0.1 mg/ml BSA) for peptide binding assays or PBS for fluorescence studies.
Fluorescence studies. Fluorescence measurements were performed at room temperature using a Perkin-Elmer LS50B spectrofluorometer. Samples were excited at 295 nm and emission spectra were recorded at 345 nm. Excitation and emission bandwidths were 5.0 nm, and spectra were corrected for background fluorescence. For measurements of ATP-dependent fluorescence quenching, ATP was added to domains in binding buffer and the percent change in fluorescence was calculated at each ATP concentration. For the assay of domain-domain association, separate samples of a single domain and two domains were generated for each experimental condition.
Phosphorylation and two-dimensional peptide mapping.
In situ CFTR labeling was performed as previously described
(33). Briefly, NIH-3T3 cells stably expressing human CFTR
(NIH-CFTR) were incubated with 32Pi for 2 h before stimulation with 10 µM forskolin for 2 min. Cells were lysed
in 4°C lysis buffer (100 mM NaCl, 50 mM NaF, 0.1% SDS, 1%
Na-deoxycholate, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 0.1 mM
phenylmethylsulfonyl fluoride, 0.1 mg/mL aprotinin, 1 mM orthovanadate,
and 50 mM Tris · HCl, pH 7.5) and lysate-cleared by
centrifugation. CFTR was immunoprecipitated with a COOH-terminal CFTR
antibody (R&D) and protein A beads (Calbiochem) and was then purified
by SDS-PAGE. For in vitro labeling, CFTR was immunoprecipitated from
NIH-CFTR cells as described, with the exception that cells were not
exposed to 32Pi or forskolin.
Immunoprecipitated CFTR was suspended in 50 µl of kinase buffer
(50 mM Tris, 10 mM MgCl2, and 100 µg/ml BSA, pH
7.5) and phosphorylated with 2 units of PKA catalytic subunit (Sigma) and 10 µCi [-32P]ATP. CFTR was resolved
by SDS-PAGE. For in vitro labeling of NBD1-R, purified protein
(1-2 µg) was placed in kinase buffer plus 0.8 M urea and
phosphorylated with 2 units of PKA and 10 µCi
[
-32P]ATP. Phosphorylated NBD1-R was separated from
the reaction mixture by SDS-PAGE. In all cases, phosphorylated products
were visualized with a Storm 860 PhosphorImager.
Peptide binding assay.
Peptide walking was used to identify sites of domain-domain interaction
(20). One hundred fifty-seven overlapping 20-mer peptides,
spanning all cytoplasmic regions of CFTR, were synthesized by the
multipin synthesis method (41) and were purity-analyzed on
HPLC (Chiron). Peptides overlapped by 13 residues. All peptides were
acetylated at the NH2 terminus and amidated at the COOH
terminus. Peptides were dissolved in 100% DMSO to a concentration of 2 mM and stored frozen at 70 °C. For binding assays, peptides in DMSO were diluted with distilled water (1:10), and then 10 µg of each peptide were blotted onto polyvinylide difluoride membrane (Bio-Rad). The membranes were air-dried, blocked with 5% milk in PBST (PBS-0.5% Tween), and then incubated with 10 µg/ml of a purified CFTR domain overnight in binding buffer. Bound proteins were detected with monoclonal antibodies against GST or His tags (Upstate Biotechnology) or with a polyclonal antibody against NBD2 (generated against the
expressed domain by Covance Research Products). Signals were visualized
by enhanced chemiluminescence (Amersham Pharmacia Biotech).
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RESULTS |
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Expression and characterization of CFTR domains.
His-tagged expression vectors for NBD1, NBD2, and the NBD1-R-domain of
CFTR were constructed. In addition, we expressed a GST-tagged R-domain
because of poor expression of His-tagged R-domain. These proteins,
his-NBD1 (amino acids 373-589), his-NBD2 (amino acids
1151-1476), his-NBD1-R (amino acids 373-859), and GST-R (amino acids 589-830), were expressed in E. coli and
purified by affinity chromatography. As shown in Fig.
1, the purified domains contained >95%
of the intended product. His-NBD1, his-NBD2, and GST-R migrated at the
expected molecular masses of 24, 36, and 52 kDa, respectively, whereas
his-NBD1-R migrated at 60 kDa as opposed to the predicted molecular
mass of 53 kDa. In all cases, the identity of the expressed domain was
confirmed by Western blotting with the antibodies against individual
domains (data not shown).
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Binding of expressed domains to peptides from CFTR.
To identify epitopes that are involved in domain-domain interactions,
an overlapping peptide library of 20-mers spanning all intracellular
amino acids of CFTR was synthesized. Peptides 1-10, 11-17,
18-25, 97-103, and 105-111 spanned the NH2
terminus and intracellular loops L1-L4, respectively. Peptides
26-96 spanned NBD1 and the R-domain, whereas peptides 112-157
spanned NBD2 and the COOH-terminus. The identity of each peptide is
indicated in Fig.
6A. As
shown in Fig. 6B, each peptide was assayed for the ability
to bind the expressed domains with an overlay assay. Peptides were
spotted onto nitrocellulose membranes and incubated with a domain, and
bound domain was probed with antibodies to the domain or the fused
tag. To identify false positives, the binding of antibodies, or
GST plus anti-GST, in the absence of expressed domains was determined.
Our anti-His tag antibody bound to none of our peptides, whereas the
anti-NBD2 antibody bound to epitopes on three overlapping peptides
(135-137) from NBD2 (data not shown). As shown in
Fig. 6A, GST was bound by peptides 11, 12, 48, 63, 96, and
106, and more protein was bound to peptide 13. In addition, all three
expressed domains bound to peptides 13, 59, 60, 96, and 106, indicated
in gray in Fig. 6A; these five interactions were considered
false positives and not included in the following analysis. In
addition, there are undoubtedly peptides that form associations with a
domain in the native structure but do not bind the domain in our assay.
These false negatives could occur if the association between peptide
and domain were to weak, if the peptide were not retained on the
membrane, or if spatial restrictions prevented peptide-domain
interaction on the membrane. It is likely that some of the more
hydrophilic peptides were lost from the membrane because the percentage
of bound peptides and Kyte-Dolittle hydrophobicity (24)
were correlated (R2 = 0.91; data not
shown). Therefore, it is unlikely that we have identified all
sites of domain-domain interaction. Because some peptides were likely
to be lost from the membrane, comparisons were made only on the basis
of the amount of bound domain and not of the relative strength of
association. Despite the uncertainty caused by the potential loss of
peptides, the specificity of domain binding can be assessed for any
peptide that shows differential binding to the tested domains. For
example, data with his-NBD2 and GST-R indicate that peptides
135-137 and 144-148 are retained on the membrane.
Because there is no evidence for the binding of his-NBD1 to these
peptides, these peptides establish a background for nonspecific binding
by NBD1.
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Mapping of binding data to known NBD structures.
To better understand the relationship of the binding studies to the
structure of the NBD1 and NBD2, we have mapped the binding sites in
NBD1 and NBD2 onto crystallographic structures of the HisP and MalK
monomers, two bacterial NBDs (7, 17). These proteins are
overlaid in Fig. 7A. They have
sequence identities of only 26%, but comparison of the crystal
structures by combinatorial extension (38) gives a root
mean square deviation (RMSD) for the -carbons of 2.7 Å with a
Z-score (measure of spatial significance of the fit relative to the
alignment of random structures) of 6.7 and a gap size of 18 amino
acids. The structure of Rad50, a DNA-binding protein where a structure
analogous to that of a HisP monomer is formed from two domains, was
also examined (15). An overlay of HisP and Rad50 is shown
in Fig. 7B. The sequence identity between Rad50 and HisP is
20.2%, but the RMSD for
-carbon atoms is 2.7 Å, with a Z-score of
5.3 and a gap size of 29 amino acids. A sequence alignment of MalK,
HisP, NBD1, NBD2, and Rad50 is shown in Fig.
8, which also shows the alignment of
secondary structural elements for MalK, HisP, and Rad50. These results
suggest that NBD1 and NBD2, with sequence identities to HisP of 18%
and 17% and gap sizes of 32 and 17, respectively, may have similar three-dimensional structures to those of HisP, MalK, and Rad50. Figure
8 also indicates epitopes in NBD1 that bind NBD2 (blue), epitopes in
NBD2 that bind NBD1 (red), and epitopes in either NBD that bind to the
R-domain (green). Whereas the monomeric structures of these three
proteins are very similar, the crystallographic dimers (Fig.
9) show no similarity. In addition to the
crystallographic dimers for HisP, MalK, and Rad50, the comparison of
NBD sequences from a large number of ABC transporters has led to the
development of an alternative model for the HisP dimer, aHisP
(19). In Fig. 9, A-D, the aqua monomer
(NBD1) is always in the same orientation, and the arrow indicates the
orientation of the helix in NBD2 that is projecting toward the viewer
in Fig. 9A. Epitopes from one NBD of CFTR that bind to the
other NBD are indicated with a darker color. Because they are found in
the interfacial region of the crystal structures for MalK
(9C), Rad50 (9D), and aHisP (9B), our
data are more consistent with these models than with the HisP crystal
structure (9A).
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DISCUSSION |
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Since the discovery of the gene for cystic fibrosis and the identification of transmembrane helices with hydropathy plots (35), there has been little success in efforts to further refine the structure of CFTR. At present, there is consensus on the domain boundaries (3). Yeast two-hybrid studies suggest the presence of interactions between intracellular domains and intracellular loops in the transmembrane domains (22), and binding of the NH2 and COOH termini to the intracellular domains has been proposed on the basis of functional studies (29, 32, 42). Whereas the NBDs of CFTR have been expressed and shown by various groups to bind or hydrolyze ATP (16, 23, 31) and the conformation of expressed R-domain has been shown to be altered by phosphorylation (8), there is presently no structural information for any of these domains. In a recent study, the CD spectrum of an R-domain construct (amino acids 708-831) that lacks the first 119 amino acids of our construct was analyzed and found to have little defined secondary structure (30). This construct was shown to restore kinase-dependent channel activity to a CFTR construct that lacked amino acids 708-831, but the presence of the NH2-terminus region of the R-domain in the CFTR construct may induce bound R-domain to assume a native structure. A similar effect could occur in solution. If this were the case, results should be more consistent with earlier studies of an expressed R-domain construct from amino acids 595-831, in which considerably more secondary structure was observed and in which PKA-dependent phosphorylation caused an appreciable change in the CD spectrum (8). It has been suggested that expressed R-domain is largely unstructured in solution. Whereas an unstructured R-domain might bind to the NBDs and specific peptides, the binding data in Figs. 5 and 6 and the phosphorylation data in Fig. 3 would appear to be more consistent with an R-domain with a defined tertiary structure. We therefore suggest that the absence of amino acids from the NH2 terminus of the R-domain peptide may in large part cause the reported absence of R-domain structure.
At present, the best structural information for CFTR comes from studies of other ABC transporters. Crystallographic structures have been reported for NBDs from two bacterial ABC transporters, the HisP subunit of the S. typhimurium histidine permease (17) and the MalK subunit of the T. litoralis maltose transporter (7). In addition, a crystal structure for Rad50 from Pyroccus furiosus has also been solved (15). The structures of two additional NBDs, MJ1267 and MJ0796, have recently been reported (21, 44). The monomeric structures are very similar to those of MalK and HisP, but they have not been considered in this analysis because the authors chose not to infer dimeric structures from their crystallographic data. In addition, the recent structure of the ABC transporter homolog MsbA was not considered because much of the NBD is unresolved in the crystal (4). Also, the authors' conclusion that the NBDs may not interact appears to be dependent on a series of interactions between extracellular loops in the transmembrane domains. Because these loops are considerably shorter in CFTR (see Fig. 6A), it is highly unlikely that CFTR could form a structure similar to that described from MsbA.
As described in RESULTS, these proteins show little sequence similarity but a great deal of structural homology. The regions with the most highly conserved sequences are those indicated in Fig. 8: the Walker A, B, and C regions and the Q-, D-, and H-loops. These regions have been linked to the binding or hydrolysis of ATP (7, 15, 17). With regard to domain-domain interactions between the NBDs, the crystallographic studies have generated vastly different dimeric structures. This is perhaps to be expected as the conditions for NBD crystallization are quite different from those found within the cell. One difference is that the MSDs are not present during crystallization, and, therefore, any effect that these domains have on interactions between the NBDs will not be reflected in the crystal structure. A better model for the structure of the NBD dimer in CFTR may be the soluble Rad50 dimer, formed in the presence of ATP, in which alterations in structure due to the absence of MSDs should not occur. In general, there are two different models for NBD-NBD interaction. The HisP dimer places the ATP binding sites on opposite sides of the dimer (17), whereas the Rad50 and MalK structures place the ATP binding sites in a cleft formed by the NBD-NBD interface (7, 15). For both of these models, ATP binding sites are likely to be composed of residues from both NBDs. Whereas this issue may be resolved only with the crystal structure of a complete ABC transporter, cysteine mutagenesis of amino acids in the ATP binding sites of MDR allows disulfide cross-links to be generated between the NBDs (25). This result is consistent only with an interfacial model. An ATP binding site at the interface of an NBD-NBD dimer may also explain why it has been difficult to observe ATP hydrolysis by NBD constructs.
In this study, we expressed, purified, and refolded tagged NBD1, NBD2, NBD1-R, and R-domain. These expressed CFTR domains had properties that were consistent with a native-like structure. All NBD-containing proteins bound ATP. The affinities for ATP binding to his-NBD1 and his-NBD2 were similar to values reported previously. However, it is somewhat surprising that the affinity of NBD2 is greater than that of NBD1, because previous studies have suggested the opposite (23, 31). The only explanation we can offer is that, in the previous studies, NBD1 and NBD2 constructs from amino acids 433-589 and 1208-1399 were fused to the maltose binding protein, whereas our NBD1 and NBD2 constructs were His-tagged and spanned amino acids 373-589 and 1151-1476 (23, 31). Recent studies have indicated that NBD1 may extend as far as amino acid 640 (3). However, the observation that, when fused to the maltose-binding protein, an NBD1 construct that terminates at amino acid 589 can hydrolyze ATP (26) suggests that amino acids after 589 are not essential for the formation of a native-like structure. In vitro phosphorylation of NBD1-R by PKA occurred at the same sites as in situ phosphorylation of full-length CFTR. In addition, the sites of R-domain phosphorylation differed from those observed when immunoprecipitated CFTR was in vitro phosphorylated with PKA. Direct associations between the expressed domains were observed with overlay assays and by tryptophan fluorescence quenching. The dose dependence of tryptophan fluorescence quenching allowed binding to be quantified. However, since we have no data regarding homodimerization, caution must be used with regard to quantification of the association between NBD1-R and NBD2. Recently, it has been reported that dimers of NBDs are not formed in solution (21). This has been used as an argument in favor of the possibility that crystallographic NBD dimers may not reflect the structure within native ABC transporters. Although we are in agreement with this conclusion, our fluorescence quenching data indicate that NBDs can associate in solution. Perhaps the presence of the R-domain stabilizes the interaction, because an NBD1-R/NBD2 complex has been observed previously (26). We also suspect that conditions designed to precipitate NBDs may disfavor solution dimer formation, whereas ours, designed to maintain NBDs in solution, favored dimer formation. Differences in the size of the expressed domains may also affect domain-domain interactions. Lastly, we have shown that an association between NBD1-R and NBD2 alters the effects of ATP binding on tryptophan fluorescence.
The principle finding of our study is that specific epitopes in a peptide library spanning all cytoplasmic regions of CFTR bind to the expressed domains of CFTR. Epitopes from one domain that bind to another domain are likely to define sites of interaction between the two domains. Because the epitopes in one NBD that bind to the other NBD are at the interface in models that place the ATP binding sites at the NBD-NBD interface, our data are most consistent with the crystal structures of MalK, Rad50, and the alternative structure for HisP. Our data are in agreement with crystallographic (7, 15) and cross-linking (25) studies of other ABC superfamily members. However, because there are complications with each approach, the confluence of data with three different techniques and with three different ABC superfamily members is comforting.
In the present study, care has been taken to exclude false positive results. Our ad hoc procedures for identifying false positives are described in RESULTS, but an additional test for specific binding made use of the fact that each six-amino acid sequence is expressed on three different peptides (see Fig. 6A). As a consequence, a series of sequential peptides that bound a domain was considered to be a stronger indication of a specific interaction than binding by a single peptide. However, since peptides may form structures that obscure the relevant epitope, strong binding by an isolated peptide cannot be ignored. Binding to sequential peptides also defines the site of interaction more precisely. In addition, the peptides that bound NBD1 and NBD2 were not the same. These observations strongly suggest that, in general, peptide binding to our expressed domains was specific.
In addition to interactions between NBD1 and NBD2, interactions between R-domain and both NBDs were observed. Previous functional studies of CFTR channel activity have suggested that the activities of the NBDs and R-domain are linked (18). In an NBD1-R-domain construct, R-domain phosphorylation inhibits nucleotide binding, and the presence of R-domain alters the kinetics of ATP hydrolysis by NBD1 (27, 43). Our results provide direct physical evidence for these interactions. The analysis of peptide binding to the R-domain in terms of NBD crystal structure is more speculative than that for NBD-NBD interaction because the model proteins do not have an R-domain. However, because we have based our analysis on the assumption that arrangement of NBDs and MSDs in ABC superfamily members are the same, regions of NBD1 and NBD2 that associate with R-domain should form a contiguous surface. Although none of the structures in Fig. 9 are entirely consistent with our assumption, the peptides that interact with the R-domain are most contiguous in the Rad50 and aHisP structures. The MalK and HisP crystals show binding of the R-domain at two distant locations. On the basis of these observations, we conclude that the Rad50 structure and the alternative aHisP (20) structures represent the most likely structures for the NBDs in CFTR.
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ACKNOWLEDGEMENTS |
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The technical assistance of Mu-Young Kim is acknowledged.
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FOOTNOTES |
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This work was supported by grants from the Cystic Fibrosis Foundation, Cystic Fibrosis Research, and the Nemours Foundation.
Address for reprint requests and other correspondence: W. W. Reenstra, Institute for Human Gene Therapy, Univ. of Pennsylvania, Philadelphia, PA 19104 (E-mail: Reenstra{at}mail.med.upenn.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 2, 2002;10.1152/ajpcell.00337.2001
Received 20 July 2001; accepted in final form 27 November 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Anderson, MP,
Berger HA,
Rich DR,
Gregory RJ,
Smith AE,
and
Welsh MJ.
Nucleoside triphosphates are required to open the CFTR chloride channel.
Cell
67:
775-784,
1991[ISI][Medline].
2.
Boyle, WJ,
van der Geer P,
and
Hunter T.
Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer plates.
Methods Enzymol
210:
110-148,
1991.
3.
Chan, KW,
Csanady L,
Seto-Young D,
Nairn AC,
and
Gadsby DC.
Severed molecules functionally define the boundaries of the cystic fibrosis transmembrane conductance regulator's NH2-terminal nucleotide binding domain.
J Gen Physiol
116:
163-180,
2000
4.
Chang, G,
and
Roth CB.
Structure of MsbA from E. coli: a homolog of the multidrug resistance ATP binding cassette (ABC) transporters.
Science
293:
1793-1800,
2001
5.
Cheng, SH,
Rich DP,
Marshall J,
Gregory RJ,
Welsh MJ,
and
Smith AE.
Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel.
Cell
66:
1027-1036,
1991[ISI][Medline].
6.
Chen, JH,
Chang XB,
Aleksandrov AA,
Hammerk MM,
Shanmugam K,
and
Riordan JR.
Biochemical and electrophysiological evaluation of CFTR quaternary structure (Abstract).
Pediatr Pulmonol Suppl
20:
174,
2000.
7.
Diederichs, K,
Diez J,
Greller G,
Muller C,
Breed J,
Schnell C,
Vonrhein C,
Boos W,
and
Welte W.
Crystal structure of MalK, the ATPase subunit of the trehalose/maltose ABC transporter of the archaeon Thermococcus litoralis.
EMBO J
19:
5951-5961,
2000
8.
Dulhanty, AM,
and
Riordan JR.
Phosphorylation by cAMP-dependent protein kinase causes a conformational change in the R-domain of the cystic fibrosis conductance transmembrane regulator.
Biochemistry
33:
4072-4079,
1994[ISI][Medline].
9.
Eskandari, S,
Wright EM,
Kreman M,
Starace DM,
and
Zampighi GA.
Structural analysis of cloned plasma membrane proteins by freeze-fracture electron microscopy.
Proc Natl Sci USA
95:
11235-11240,
1998
10.
Frangioni, JV,
and
Neel BG.
Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins.
Anal Biochem
210:
179-187,
1993[ISI][Medline].
11.
Gadsby, DC,
and
Nairn AC.
Control of CFTR channel gating by phosphorylation and nucleotide hydrolysis.
Physiol Rev
79:
S77-S107,
1999[Medline].
12.
Hall, RA,
Ostedgaard LS,
Premont RT,
Blitzer JT,
Rahman N,
Welch MJ,
and
Lefkowitz RJ.
A C-terminal motif found in the beta2-adrenergic receptor, P2Y1 receptor, and cystic fibrosis transmembrane conductance regulator determines binding to the Na+/H+ exchanger regulatory factor family of PDZ proteins.
Proc Natl Acad Sci USA
95:
8496-8501,
1998
13.
Hallows, KR,
Raghuram V,
Kemp BE,
Witters LA,
and
Foskett JK.
Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase.
J Clin Invest
105:
1711-1721,
2000
14.
Higgins, CF.
ABC transporters: from microorganisms to man.
Annu Rev Cell Biol
8:
67-113,
1992[ISI].
15.
Hopfner, KP,
Karcher A,
Shin DS,
Craig L,
Arthur LM,
Carney JP,
and
Tainer JA.
Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily.
Cell
101:
789-800,
2000[ISI][Medline].
16.
Howell, LD,
Borchardt R,
and
Cohn JA.
ATP hydrolysis by a CFTR domain: pharmacology and effects of G551D mutation.
Biochem Biophys Res Commun
271:
518-525,
2000[ISI][Medline].
17.
Huang, LW,
Wang IX,
Nikaido K,
Liu PQ,
Ames GFL,
and
Kim SH.
Crystal structure of the ATP-binding subunit of an ABC transporter.
Nature
396:
703-707,
1998[ISI][Medline].
18.
Hwang, TC,
Wang F,
Yang CH,
and
Reenstra WW.
Potentiation of F508 channel function by genistein binding to CFTR.
Am J Physiol Cell Physiol
273:
C988-C998,
1997
19.
Jones, PM,
and
George AM.
Subunit interactions in ABC transporters: towards a functional architecture.
FEMS Microbiol Lett
179:
187-200,
1999[ISI][Medline].
20.
Joseph, G,
and
Pick E.
"Peptide walking" is a novel method for mapping functional domains in proteins. Its application to the Rac1-dependent activation of NADPH oxidase.
J Biol Chem
270:
29079-29082,
1995
21.
Karpowich, N,
Martsinkevich O, L,
Millen Yuan YR,
Dei PL,
MacVey K,
Thomas P,
and
Hunt JF.
Crystal structures of the MJ1267 ATP-binding cassette reveal an induced-fit effect at the ATPase active site of an ABC transporter.
Structure
9:
571-586,
2001[ISI][Medline].
22.
Kiser, GL,
Chang XB,
and
Riordan JR.
Two-hybrid analysis of CFTR domain interactions (Abstract).
Pediatr Pulmonol Suppl
13:
213,
1996.
23.
Ko, YH,
and
Pedersen PL.
The first nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator can function as an active ATPase.
J Biol Chem
270:
22093-22096,
1995
24.
Kyte, J,
and
Doolittle RF.
A simple method for displaying the hydropathic character of a protein.
J Mol Biol
157:
105-132,
1982[ISI][Medline].
25.
Loo, TW,
and
Clarke DM.
Covalent modification of human P-glycoprotein mutants containing a single cysteine in either nucleotide-binding fold abolishes drug-stimulated ATPase activity.
J Biol Chem
270:
22957-22961,
1995
26.
Lu, NT,
and
Pedersen PL.
Cystic fibrosis transmembrane conductance regulator: the purified NBF1+R protein interacts with the purified NBF2 domain to form a stable NBF+R/NBF2 complex while inducing a conformational change transmitted to the C-terminal region.
Arch Biochem Biophys
375:
7-20,
2000[ISI][Medline].
27.
Ma, J,
Zhao J,
Drumm ML,
Xie J,
and
Davis PB.
Function of the R-domain in the cystic fibrosis transmembrane conductance regulator chloride channel.
J Biol Chem
272:
28133-28141,
1997
28.
Marshall, J,
Fang S,
Ostedgaard LS,
O'Riordan CR,
Ferrara D,
Amara JF,
Hoppe H,
Scheule RK,
Welsh MJ,
and
Smith AE.
Stoichiometry of recombinant cystic fibrosis transmembrane conductance regulator in epithelial cells and its functional reconstitution into cells in vitro J.
Biol Chem
269:
2987-2995,
1994
29.
Naren, AP,
Quick MW,
Collawn JF,
Nelson DJ,
and
Kirk KL.
Syntaxin 1A inhibits CFTR chloride channels by means of domain-specific protein-protein interactions.
Proc Natl Acad Sci USA
95:
10972-10977,
1998
30.
Ostedgaard, LS,
Baldursson O,
Vermeer DW,
Welsh MJ,
and
Robertson AD.
A functional R-domain from cystic fibrosis transmembrane conductance regulator is predominantly unstructured in solution.
Proc Natl Acad Sci USA
97:
5657-5662,
2000
31.
Randak, C,
Neth P,
Auerswald EA,
Eckerskorn C,
Assfalg-Machleidt I,
and
Machleidt W.
A recombinant polypeptide model of the second nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator functions as an active ATPase, GTPase, and adenylate kinase.
FEBS Lett
410:
180-186,
1997[ISI][Medline].
32.
Raghuram, V,
Mak DD,
and
Foskett JK.
Regulation of cystic fibrosis transmembrane conductance regulator single-channel gating by bivalent PDZ-domain-mediated interaction.
Proc Natl Acad Sci USA
98:
1300-1305,
2001
33.
Reenstra, WW,
Yurko-Mauro K,
Dam A,
Raman S,
and
Shorten S.
CFTR chloride channel activation by genistein: the role of serine/threonine protein phosphatases.
Am J Physiol Cell Physiol
271:
C650-C657,
1996
34.
Rich, DP,
Berger HA,
Cheng SH,
Travis SM,
Saxena M,
Smith AE,
and
Welsh MJ.
Regulation of the cystic fibrosis transmembrane conductance regulator Cl channel by negative charge in the R-domain.
J Biol Chem
268:
20259-20267,
1993
35.
Riordan, JR,
Rommens JM,
Kerem B,
Alon N,
Rozmahel R,
Grzelczak Z,
Zielenski J,
Lok S,
Plavsic N,
Chou JL,
Drumm ML,
Iannuzzi MC,
Collins FS,
and
Tsui LC.
Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA.
Science
245:
1066-1073,
1989[ISI][Medline].
36.
Seibert, FS,
Chang XB,
Aleksandrov AA,
Clarke DM,
Hanrahan JW,
and
Riordan JR.
Influence of phosphorylation by protein kinase A on CFTR at the cell surface and endoplasmic reticulum.
Biochem Biophys Acta
1461:
275-283,
1999[ISI][Medline].
37.
Sheppard, D,
and
Welsh MJ.
Structure and function of the CFTR chloride channel.
Physiol Rev
79:
S23-S45,
1999[Medline].
38.
Shindyalov, IN,
and
Bourne PE.
Protein structure alignment by incremental combinatorial extension (CE) of the optimal path.
Protein Eng
11:
739-747,
1998[Abstract].
39.
Short, DB,
Trotter KW,
Reczek D,
Kreda SM,
Bretscher A,
Boucher RC,
Stutts MJ,
and
Milgram SL.
An apical PDZ protein anchors the cystic fibrosis transmembrane conductance regulator to the cytoskeleton.
J Biol Chem
273:
19797-19801,
1998
40.
Smith, PK,
Krohn RI,
Hermanson GT,
Mallia AK,
Gartner FH,
Provenzano MD,
Fujimoto EK,
Goeke NM,
Olson BJ,
and
Klenk DC, DC
Measurement of protein using bicinchoninic acid.
Anal Biochem
150:
76-85,
1985[ISI][Medline].
41.
Valerio, RM,
Benstead M,
Bray AM,
Campbell RA,
and
Maeji NJ.
Synthesis of peptide analogs using the multipin peptide synthesis method.
Anal Biochem
197:
168-177,
1991[ISI][Medline].
42.
Wang, S,
Yue H,
Derin RB,
Guggino WB,
and
Li M.
Accessory protein facilitated CFTR-CFTR interaction, a molecular mechanism to potentiate the chloride channel activity.
Cell
103:
169-179,
2000[ISI][Medline].
43.
Winter, MC,
and
Welsh MJ.
Stimulation of CFTR activity by its phosphorylated R-domain.
Nature
389:
294-296,
1997[ISI][Medline].
44.
Yuan, YR,
Blecker S,
Martsinkevich O,
Millen L,
Thomas PL,
and
Hunt JF.
The crystal structure of the MJ0796 ATP-binding cassette: implications for the structural consequences of ATP hydrolysis in the active site of an ABC-transporter.
J Biol Chem
276:
32313-32321,
2001
45.
Yurko-Mauro, KA,
and
Reenstra WW.
Prostaglandin F2alpha stimulates CFTR activity by PKA and PKC-dependent phosphorylation.
Am J Physiol Cell Physiol
275:
C653-C660,
1998[Abstract].
46.
Zeltwanger, S,
Wang F,
Wang GT,
Gillis KD,
and
Hwang TC.
Gating of cystic fibrosis transmembrane conductance regulator chloride channels by adenosine triphosphate hydrolysis. Quantitative analysis of a cyclic gating scheme.
J Gen Physiol
113:
541-554,
1999
47.
Zerhusen, B,
Zhao J,
Xie J,
Davis PB,
and
Ma J.
A single conductance pore for chloride ions formed by two cystic fibrosis transmembrane conductance regulator molecules.
J Biol Chem
274:
7627-7630,
1999