Portal de Periódicos da CAPES

The First Nucleotide Binding Domain of Cystic Fibrosis Transmembrane Conductance Regulator Is a Site of Stable Nucleotide Interaction, whereas the Second Is a Site of Rapid Turnover

2002; Elsevier BV; Volume: 277; Issue: 18 Linguagem: Inglês

10.1074/jbc.m111713200

1083-351X

Luba A. Aleksandrov, Andrei A. Aleksandrov, Xiu-Bao Chang, John R. Riordan,

Advanced biosensing and bioanalysis techniques

As in other adenine nucleotide binding cassette (ABC) proteins the nucleotide binding domains of the cystic fibrosis transmembrane conductance regulator (CFTR) bind and hydrolyze ATP and in some manner regulate CFTR ion channel gating. Unlike some other ABC proteins, however, there are preliminary indications that the two domains of CFTR are nonequivalent in their nucleotide interactions (Szabo, K., Szakacs, G., Hegeds, T., and Sarkadi, B. (1999)J. Biol. Chem. 274, 12209–12212; Aleksandrov, L., Mengos, A., Chang, X., Aleksandrov, A., and Riordan, J. R. (2001)J. Biol. Chem. 276, 12918–12923). We have now characterized the interactions of the 8-azido-photoactive analogues of ATP, ADP, and 5′-adenyl-β,γ-imidodiphosphate (AMP-PNP) with the two domains of functional membrane-bound CFTR. The results show that the two domains appear to act independently in the binding and hydrolysis of 8-azido-ATP. At NBD1 binding does not require a divalent cation. This binding is followed by minimal Mg2+-dependent hydrolysis and retention of the hydrolysis product, 8-azido-ADP, but not as a vanadate stabilized post-hydrolysis transition state complex. In contrast, at NBD2, MgN3ATP is hydrolyzed as rapidly as it is bound and the nucleoside diphosphate hydrolysis product dissociates immediately. Confirming this characterization of NBD1 as a site of more stable nucleotide interaction and NBD2 as a site of fast turnover, the non-hydrolyzable N3AMP-PNP bound preferentially to NBD1. This demonstration of NBD2 as the rapid nucleotide turnover site is consistent with the strong effect on channel gating kinetics of inactivation of this domain by mutagenesis. As in other adenine nucleotide binding cassette (ABC) proteins the nucleotide binding domains of the cystic fibrosis transmembrane conductance regulator (CFTR) bind and hydrolyze ATP and in some manner regulate CFTR ion channel gating. Unlike some other ABC proteins, however, there are preliminary indications that the two domains of CFTR are nonequivalent in their nucleotide interactions (Szabo, K., Szakacs, G., Hegeds, T., and Sarkadi, B. (1999)J. Biol. Chem. 274, 12209–12212; Aleksandrov, L., Mengos, A., Chang, X., Aleksandrov, A., and Riordan, J. R. (2001)J. Biol. Chem. 276, 12918–12923). We have now characterized the interactions of the 8-azido-photoactive analogues of ATP, ADP, and 5′-adenyl-β,γ-imidodiphosphate (AMP-PNP) with the two domains of functional membrane-bound CFTR. The results show that the two domains appear to act independently in the binding and hydrolysis of 8-azido-ATP. At NBD1 binding does not require a divalent cation. This binding is followed by minimal Mg2+-dependent hydrolysis and retention of the hydrolysis product, 8-azido-ADP, but not as a vanadate stabilized post-hydrolysis transition state complex. In contrast, at NBD2, MgN3ATP is hydrolyzed as rapidly as it is bound and the nucleoside diphosphate hydrolysis product dissociates immediately. Confirming this characterization of NBD1 as a site of more stable nucleotide interaction and NBD2 as a site of fast turnover, the non-hydrolyzable N3AMP-PNP bound preferentially to NBD1. This demonstration of NBD2 as the rapid nucleotide turnover site is consistent with the strong effect on channel gating kinetics of inactivation of this domain by mutagenesis. adenine nucleotide binding cassette cystic fibrosis transmembrane conductance regulator nucleotide binding domain 5′-adenylyl-β,γ-imidodiphosphate baby hamster kidney 8-azidoadenosine 5′-triphosphate tosylphenylalanyl chloromethyl ketone n-dodecyl-β-d-maltoside The ubiquity of adenine nucleotide binding cassette (ABC)1 proteins in all kingdoms of life and their involvement in many human disease processes (1Saurin W. Hofnung M. Dassa E. J. Mol. Evol. 1999; 48: 22-41Crossref PubMed Scopus (266) Google Scholar, 2Dean M. Rzhetsky A. Allikmets R. Genome Res. 2001; 11: 1156-1166Crossref PubMed Scopus (1530) Google Scholar, 3Holland I.B. Blight M.A. J. Mol. Biol. 1999; 293: 381-399Crossref PubMed Scopus (498) Google Scholar) emphasizes the need to understand their structure and function. The presence of two homologous nucleotide binding domains where ATP binding and hydrolysis can occur is the defining feature of these molecules. It is assumed that the energy of ATP binding is used to do the mechanical work of the molecule, in most cases the vectorial translocation of a solute across a biological membrane (4Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3422) Google Scholar, 5Rosenberg M.F. Velarde G. Ford R.C. Martin C. Berridge G. Kerr I.D. Callaghan R. Schmidlin A. Wooding C. Linton K.J. Higgins C.F. EMBO J. 2001; 20: 5615-5625Crossref PubMed Scopus (272) Google Scholar). There are currently two prominent models for the utilization of the two NBDs in this process. In the first, ATP hydrolysis occurs alternatively and in a mutually exclusive fashion at each NBD, to drive solute export (6Senior A.E. al-Shawi M.K. Urbatsch I.L. FEBS Lett. 1995; 377: 285-289Crossref PubMed Scopus (436) Google Scholar). In a second model, hydrolysis at one NBD drives transport whereas that at the second "resets" the protein for the subsequent step (7Ambudkar S.V. Dey S. Hrycyna C.A. Ramachandra M. Pastan I. Gottesman M.M. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 361-398Crossref PubMed Scopus (1948) Google Scholar). In both of these models, the NBD at which the initial hydrolysis occurs is chosen randomly; the two domains are distinct only temporally. Indeed, in the P-glycoprotein multidrug transporter in which studies supporting each of these models have been performed, the amino acid sequences of the two domains are very similar and only minor functional asymmetry has been observed (8Hrycyna C.A. Ramachandra M. Germann U.A. Cheng P.W. Pastan I. Gottesman M.M. Biochemistry. 1999; 38: 13887-13899Crossref PubMed Scopus (131) Google Scholar). However, in some other members of the large family, including the ABCC subfamily, this similarity is far less and distinctive properties have been observed in the case of SUR1 (9Matsuo M. Kioka N. Amachi T. Ueda K. J. Biol. Chem. 1999; 274: 37479-37482Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 10Matsuo M. Tanabe K. Kioka N. Amachi T. Ueda K. J. Biol. Chem. 2000; 275: 28757-28763Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), MRP1 (11Gao M. Cui H.R. Loe D.W. Grant C.E. Almquist K.C. Cole S.P. Deeley R.G. J. Biol. Chem. 2000; 275: 13098-13108Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 12Hou Y.-X. Cui L. Riordan J.R. Chang X.-B. J. Biol. Chem. 2000; 275: 20280-20287Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 13Nagata K. Nishitani M. Matsuo M. Kioka N. Amachi T. Ueda K. J. Biol. Chem. 2000; 275: 17626-17630Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 14Hou Y.-X. Cui L. Riordan J.R. Chang X. J. Biol. Chem. 2001; 277: 5110-5119Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), and CFTR (15Szabo K. Szakacs G. Hegeds T. Sarkadi B. J. Biol. Chem. 1999; 274: 12209-12212Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 16Aleksandrov L. Mengos A. Chang X. Aleksandrov A. Riordan J.R. J. Biol. Chem. 2001; 276: 12918-12923Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Utilizing [α-32P]8-N3ATP, both NBDs of CFTR could be photolabeled (15Szabo K. Szakacs G. Hegeds T. Sarkadi B. J. Biol. Chem. 1999; 274: 12209-12212Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 16Aleksandrov L. Mengos A. Chang X. Aleksandrov A. Riordan J.R. J. Biol. Chem. 2001; 276: 12918-12923Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). However, a non-hydrolyzable analogue, 8-N3AMP-PNP, competed much more strongly for N3ATP binding and capture at NBD1 than at NBD2 (16Aleksandrov L. Mengos A. Chang X. Aleksandrov A. Riordan J.R. J. Biol. Chem. 2001; 276: 12918-12923Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). We have now further explored this apparent asymmetry of the domains in photolabeling experiments employing [γ-32P]- as well as [α-32P]8-N3ATP, [α-32P]8-N3ADP, and [α-32P]8-N3AMP-PNP. The latter compound selectively labeled NBD1. NBD1 could be labeled by each of these compounds in the absence of a divalent cation such as Mg2+. Labeling of NBD2 that occurred only when azido-nucleotides were not washed out prior to photoactivation was Mg2+-dependent. When [γ-32P]8-N3ATP was employed at temperatures and divalent cation concentrations where hydrolysis could occur, only NBD1 was labeled, indicating that the intact nucleoside triphosphate was bound and retained there but not at NBD2. This revealed that the labeling of NBD2 detected with [α-32P]8-N3ATP must have reflected bound [α-32P]8-N3ADP formed by hydrolysis. This hydrolysis must have been rapid and complete because no [γ-32P]8-N3ATP radioactivity could be detected at NBD2. The [α-32P]8-N3ADP bound at NBD2 after presentation of the protein with [α-32P]8-N3ATP could have arisen from hydrolysis at either NBD1 or NBD2. To distinguish between these possibilities, the Walker A lysine mutants K464A and K1250A were used; K464A ablated labeling of NBD1 without influencing that at NBD2, and hence the N3ADP that labeled NBD2 could not have arisen from hydrolysis at NBD1. This implicates NBD2 as a site of rapid hydrolysis, whereas hydrolysis is very limited at NBD1 where there is labeling to a similar extent with [α-32P]- and [γ-32P]8-N3ATP. Not only is hydrolysis at NBD2 rapid but release of the product, N3ADP, is also as it is completely removed by washing away of the free compound prior to photo-activation. In contrast, after incubation with [α-32P]- or [γ-32P]8-N3ATP or [α-32P]8-N3ADP, bound nucleotide is retained at NBD1 to the same extent with or without washout of free azido-nucleotide. [α-32P]8-N3ATP, [γ-32P]8-N3ATP, [α-32P]8-N3ADP, and [α-32P]8-N3AMP-PNP were obtained from Affinity Labeling Technologies, Inc. Non-radioactively labeled 8-N3AMP-PNP was from the same source; other nucleotides were purchased from Sigma as were other reagents employed, including TPCK-treated trypsin. Stable BHK-21 cell lines expressing wild-type and K464A and K1250A variants of CFTR were established and cultured as described previously (16Aleksandrov L. Mengos A. Chang X. Aleksandrov A. Riordan J.R. J. Biol. Chem. 2001; 276: 12918-12923Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 17Chang X.-B. Tabcharani J.A. Hou Y.-X. Jensen T.J. Kartner N. Alon N. Hanrahan J.W. Riordan J.R. J. Biol. Chem. 1993; 268: 11304-11311Abstract Full Text PDF PubMed Google Scholar). Membranes for photolabeling experiments were isolated from these cells by methods described previously (16Aleksandrov L. Mengos A. Chang X. Aleksandrov A. Riordan J.R. J. Biol. Chem. 2001; 276: 12918-12923Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) and outlined below for purification of CFTR from them. Photoaffinity labeling of CFTR in BHK membranes with the different 8-azido-nucleotides was carried out exactly as we have described previously for 8-N3[α-32P] ATP (16Aleksandrov L. Mengos A. Chang X. Aleksandrov A. Riordan J.R. J. Biol. Chem. 2001; 276: 12918-12923Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Membrane suspensions (50–200 μg of protein) were incubated for 10 min with the concentration of 8-azido-nucleotide indicated in the figure legends under the conditions also specified there. The membranes were irradiated directly or pelleted and washed with and resuspended in 40 mm Tris-HCl, pH 7.4, 0.1 mm EGTA and then irradiated for 2 min on ice in a Stratalinker UV cross-linker (λ = 254 nm). The labeled membranes were incubated with TPCK-treated trypsin for 15 min on ice (enzyme to membrane protein mass ratio of 1:180). After termination of digestion with soybean trypsin inhibitor, the entire mix was solubilized in radioimmune precipitation buffer (50 mm Tris-HCl, pH 7.4, 1% deoxycholate, 1% Triton X-100, 0.1 SDS, 150 mm NaCl) and immunoprecipitated with monoclonal antibody 596, which recognizes an epitope between residues 1204 and 1211. The immunoprecipitates were fractionated by SDS-PAGE and transferred to nitrocellulose sheets for conventional (x-ray film) and electronic (Packard Instant Imager) autoradiography. Bands corresponding to tryptic fragments containing NBD1 and NBD2 were assigned by Western blots of the same nitrocellulose probed with monoclonal antibodies L12B4 and M3A7 (18Kartner N. Augustinas O. Jensen T.J. Naismith A.L. Riordan J.R. Nat. Genet. 1992; 1: 321-327Crossref PubMed Scopus (329) Google Scholar), as described in detail elsewhere (16Aleksandrov L. Mengos A. Chang X. Aleksandrov A. Riordan J.R. J. Biol. Chem. 2001; 276: 12918-12923Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). A protocol very similar to that we developed for the purification of the MRP1 protein from membranes of BHK cells was employed (19Chang X.B. Hou Y.X. Riordan J.R. J. Biol. Chem. 1997; 272: 30962-30968Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). CFTR with 10 C-terminal histidine residues was stably expressed in BHK cells as described (19Chang X.B. Hou Y.X. Riordan J.R. J. Biol. Chem. 1997; 272: 30962-30968Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Membranes were isolated following homogenization of these cells in ice-cold hypotonic buffer (10 mm HEPES, pH 7.2, containing 1 mmEDTA and a protease inhibitor mixture (Ref. 19Chang X.B. Hou Y.X. Riordan J.R. J. Biol. Chem. 1997; 272: 30962-30968Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar)) by sedimentation of the post-nuclear supernatant at 100,000 × g for 30 min. Stripping of peripheral membrane proteins was achieved by incubation of resuspended membranes at pH 10.8 for 20 min at 4 °C. The alkali-stripped membranes were resuspended in 20 mmTris-HCl, pH 7.4, containing 500 mm NaCl, 2 mmMgCl2, 20% glycerol containing 0.2% lipids (Escherichia coli crude lipids/phosphatidylcholine, 3:1) and 1% n-dodecyl-β-d-maltoside (DDM) and incubated with agitation for 40 min at 4 °C. Following centrifugation at 100,000 × g for 30 min, the soluble supernatant was made 5 mm in imidazole and incubated overnight at 4 °C with nickel-nitrilotriacetic acid resin. The mixture was poured into a column and washed with 10 volumes of the same buffer solution first containing 40 mm imidazole and 0.5% DDM and then an additional 10 volumes with 60 mm imidazole and 0.1% DDM. For elution imidazole was increased to 400 mm and DDM kept at 0.1%. The eluate was diluted 2-fold and mixed with wheat germ lectin-Sepharose 6 MB for incubation overnight at 4 °C with gentle agitation. After washing extensively with the buffer containing 0.1% DDM, 1 mm dithiothreitol, 500 mm NaCl, and a lipid mixture (phosphatidylethanolamine/phosphatidylcholine/phosphatidylserine/cholesterol, 5:3:1:1) to achieve a final protein:lipid ratio of ∼1:100, elution was with the same solution also containing 50% ethylene glycol and 0.5mN-acetylglucosamine. The eluate was dialyzed against 20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 2 mm MgCl2, concentrated ∼10-fold, and made 10% in glycerol. ATPase activity was assayed employing [α-32P]ATP as substrate, separation of nucleotides by thin layer chromatography, and quantitation by electronic autoradiography. Previous experiments had shown that, when incubated with [α-32P]8-N3ATP under hydrolysis conditions, i.e. at 37 °C in the presence of Mg2+ ions, CFTR was photolabeled more strongly at NBD1 than at NBD2 (15Szabo K. Szakacs G. Hegeds T. Sarkadi B. J. Biol. Chem. 1999; 274: 12209-12212Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 16Aleksandrov L. Mengos A. Chang X. Aleksandrov A. Riordan J.R. J. Biol. Chem. 2001; 276: 12918-12923Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). However, because this labeling was only slightly enhanced by orthovanadate, known to trap the N3ADP product of hydrolysis at the active site of other ATPases (20Eckstein F. Annu. Rev. Biochem. 1985; 54: 367-402Crossref PubMed Google Scholar), it was unclear whether labeling was with N3ATP or N3ADP or both. To resolve this issue and further characterize nucleotide interactions of the two NBDs of CFTR, we have tested conditions that influence hydrolysis and trapping. Fig.1A shows the results of labeling with [α-32P]8-N3ATP after incubation at different temperatures and in the presence or absence of Mg2+ ions and orthovanadate, i.e. conditions expected to influence hydrolysis and N3ADP trapping, respectively. From lanes 1 and 2 with incubation at 0 °C where hydrolysis should be minimal, Mg2+ is seen to slightly stimulate but not be essential for labeling of NBD1, whereas labeling of NBD2 is more dependent on the divalent cation. With incubation at 37 °C (lanes 3–5) where hydrolysis is enabled, there is much stronger labeling of NBD1 than at 0 °C. This labeling is increased by Mg2+ but less so by orthovanadate. Strikingly, labeling of NBD2 is almost completely dependent on Mg2+ ions and is also increased by vanadate. Additional clarification of whether labeling was with N3ATP or N3ADP was obtained by using [γ-32P]8-N3ATP (Fig. 1B). In that case after γ-phosphate is removed by hydrolysis, labeling by N3ADP would not be visible. At 0 °C (lanes 1and 2), there was labeling at NBD1 that was not strongly influenced by Mg2+ ions. Hence, consistent with the result with [α-32P]N3ATP at 0 °C, the first domain was clearly labeled by the intact unhydrolyzed nucleoside triphosphate. There was also weak labeling of NBD2 by the γ-32P-labeled nucleotide at 0 °C. It also labeled NBD1 more strongly at 37 °C than at 0 °C, again indicating strong labeling of that domain with the unhydrolyzed nucleoside triphosphate. However, the incremental amount of labeling of NBD1 with the α-32P-reagent on shifting from 0 °C to 37 °C (Fig.1A, lanes 1 and 2 compared withlanes 3 and 4) is even greater than with the γ-32P-reagent (Fig. 1B, lanes 1 and 2 compared with lanes 3 and4). This is not surprising because hydrolysis, which is reflected as well as binding in the former case, is expected to have a large temperature coefficient typical of enzymatic reactions. These observations are consistent with hydrolysis occurring at both domains but more completely at NBD2 than NBD1 during the 10-min preincubation prior to photoactivation because labeling of NBD2 is detected only using the nucleotide with 32P in the α-phosphate position and not in the γ-phosphate position. The most striking revelation from the use of [γ-32P]N3ATP at 37 °C is the complete lack of labeling of NBD2 (Fig. 1B, lanes 3–5). This can only mean that the compound is hydrolyzed as soon as it is bound to that domain; labeling detected with the α-32P-labeled compound (Fig. 1A, lanes 4 and 5) is by the hydrolysis product [α-32P]8-N3ADP. These results suggest that NBD2 is a site of more rapid hydrolysis than NBD1. One notable aspect of the above experiments was the observation that N3ATP binding to NBD1 was not dependent on Mg2+ ions. A similar finding was made earlier with another ABCC protein, SUR1 (21Ueda K. Inagaki N. Seino S. J. Biol. Chem. 1997; 272: 22983-22986Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). It is of particular interest for CFTR because the chloride channel can be activated by ATP in the absence of Mg2+, providing one piece of evidence that hydrolysis is not essential for activation (22Aleksandrov A.A. Chang X. Aleksandrov L. Riordan J.R. J. Physiol. 2000; 528: 259-265Crossref PubMed Scopus (66) Google Scholar, 23Ikuma M. Welsh M.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8675-8680Crossref PubMed Scopus (65) Google Scholar, 24Schultz B.D. Bridges R.J. Frizzell R.A. J. Membr. Biol. 1996; 151: 63-75Crossref PubMed Scopus (42) Google Scholar). The ion is required for photolabeling of NBD2 and an increment in labeling of NBD1 at 37 °C (Fig. 1) and for ATP hydrolysis by the whole CFTR protein (25Li C. Ramjeesingh M. Wang W. Garami E. Hewryk M. Lee D. Rommens J.M. Galley K. Bear C.E. J. Biol. Chem. 1996; 271: 28463-28468Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). This is confirmed in Fig. 2A, which shows that a maximal rate of hydrolysis occurred with approximately equimolar concentrations of ATP and Mg2+ (∼2 mm in this assay). It is also seen that Mn2+ is more potent than Mg2+ on a molar basis. Because there appears to be more rapid and complete hydrolysis at NBD2 than at NBD1, it might be anticipated that this greater effect of Mn2+ on hydrolysis by the whole protein might reflect its action on NBD2. Fig.2C indicates that this is the case, i.e.Mn2+ promotes much stronger labeling of NBD2 than Mg2+ with [α-32P]N3ATP as substrate. Because labeling of NBD2 using this substrate is entirely a result of bound [α-32P]N3ADP, this indicates that greater hydrolysis has occurred at NBD2 with Mn2+. Thus, there is a parallel between the divalent cation requirements for hydrolysis by the whole protein and that which occurs at NBD2. No divalent cation-independent nucleotide binding was detected at NBD2. That which occurs at NBD1 may account for the non-hydrolytic channel activation that occurs (22Aleksandrov A.A. Chang X. Aleksandrov L. Riordan J.R. J. Physiol. 2000; 528: 259-265Crossref PubMed Scopus (66) Google Scholar, 23Ikuma M. Welsh M.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8675-8680Crossref PubMed Scopus (65) Google Scholar, 24Schultz B.D. Bridges R.J. Frizzell R.A. J. Membr. Biol. 1996; 151: 63-75Crossref PubMed Scopus (42) Google Scholar). In the photolabeling experiments in Fig. 1, the unbound photoactive nucleotides were not removed after incubation with CFTR-containing membranes prior to UV irradiation. We had shown previously that labeling of NBD2 with [α-32P]N3ATP was greatly reduced if the reagent was washed out before photoactivation (16Aleksandrov L. Mengos A. Chang X. Aleksandrov A. Riordan J.R. J. Biol. Chem. 2001; 276: 12918-12923Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Although we had thought this might reflect just a low affinity of the domain for the NTP, the finding that only the NDP was detected at NBD2 (Fig. 1) suggested that the hydrolysis product might not be retained at NBD2 as it is at NBD1. To examine this possibility, photolabeling was performed using [α-32P]8-N3ADP as well as [α-32P]8-N3ATP with and without washout of the compounds before irradiation. The results were remarkably similar with both compounds (Fig. 3),i.e. NBD1 was strongly labeled even after washout, but NBD2 was not. Hence, 8-azido-ADP is bound stably at NBD1 whether presented directly or as the product of hydrolysis of 8-azido-ATP, and this is not a result of trapping by orthovanadate as a mimic of a post-hydrolysis transition state complex. In contrast, the photoactive NDP labels NBD2 only when present in solution at the time of photoactivation; if it is removed after the 10-min period of incubation before irradiation, little or no labeling of NBD2 is detected. When combined with the results of Fig. 1, this provides strong evidence that MgN3ATP is bound and efficiently hydrolyzed by NBD2. The hydrolysis product, 8-azido-ADP, then dissociates more rapidly from NBD2 than NBD1 because none is detected at the former but is at the latter. Experiments employing both α- and γ-[32P]8-N3ATP and temperatures promoting (37 °C) or precluding (0 °C) hydrolysis have enabled an evaluation of nucleotide binding and hydrolysis at each of the NBDs. Once it is appreciated what occurs at each domain under different conditions, it is possible to estimate the proportions of N3ATP and N3ADP associated with each. As already shown in Fig. 1, when the two domains are resolved after limited trypsin digestion, there is no labeling of NBD2 with [γ-32P]8-N3ATP (Fig.4A, lanes 3 and4). Therefore, all the labeling of NBD2 not washed out prior to photoactivation (lane 1) is by the hydrolysis product [α-32P]8-N3ADP, which has dissociated, as indicated by lane 2 when reagents were washed out before irradiation. This comparison is also extremely informative for NBD1 whereby simply comparing the relative amounts of radioactivity associated with it in lanes 2 (4603 cpm in [α-32P]N3ATP and -N3ADP combined) and 4 (2049 cpm in [α-32P]N3ATP only), it can be seen that approximately equal amounts of the NTP and NDP are bound to NBD1 after 10 min of hydrolysis at 37 °C. A similar conclusion can be reached without even using limited trypsin digestion to separate fragments containing NBD1 and NBD2 if labeling is done at both 0 and 37 °C. For example, in Fig. 4B, labeling of NBD1 with radioactive N3ATP and N3ADP from [α-32P]N3ATP at 37 °C with washout before irradiation is indicated in lane 4 and the amount of NBD1 labeling by just N3ATP from [γ-32P]N3ATP of the same specific radioactivity is indicated in lane 6. Comparison of these two lanes yields the same conclusion as that obtained from the experiment in Fig. 3A, i.e. that approximately equal amounts of N3ATP and N3ADP are associated with NBD1. This interpretation is validated by the fact that binding of [γ-32P]N3ATP is similar at 0 and 37 °C (lanes 5 and 6 compared with lanes 7and 8). These experiments support the designation of NBD1 as a slow ATP turnover site and NBD2 as a rapid turnover site. We have interpreted the results of these experiments assuming that photolabeling of one NBD reflected events only at that domain. However, structural determination of NBDs of some ABC proteins has suggested that residues from both domains may participate in the binding and hydrolysis of each ATP (26Hopfner K.P. Karcher A. Shin D.S. Craig L. Arthur L.M. Carney J.P. Tainer J.A. Cell. 2000; 101: 789-800Abstract Full Text Full Text PDF PubMed Scopus (823) Google Scholar, 27Diederichs K. Diez J. Greller G. Muller C. Breed J. Schnell C. Vonrhein C. Boos W. Welte W. EMBO J. 2000; 19: 5951-5961Crossref PubMed Scopus (285) Google Scholar, 28Jones P.M. George A.M. FEMS Microbiol. Lett. 1999; 179: 187-202Crossref PubMed Google Scholar). To assess the extent to which this may occur with CFTR, we determined the influence of mutation of the Walker A lysine residues in the P-loop of each domain, which contacts the β-phosphate of the bound nucleotide (Fig.5). As expected, the mutations K464A and K1250A prevented photolabeling with either 8-azido-ATP or 8-azido-ADP of NBD1 and NBD2, respectively. However, there was no indication that the mutation in one domain had any influence on the labeling of the other, i.e. in K464A, NBD2 was labeled as in wild-type and in K1250A, NBD1 was not different from wild type. Because labeling with [α-32P]N3ATP under these conditions (Fig.5A) reflects both binding of the nucleoside triphosphate and its diphosphate hydrolysis product at NBD1 and solely the latter at NBD2, it can be concluded that neither binding nor hydrolysis at one domain is entirely dependent on those events of the other domain. Binding of the NDP at each domain detected with [α-32P]N3ATP as substrate clearly is not dependent on an intact Walker A motif in the other domain. Although these observations do not exclude cooperativity between the two domains at some level, they do indicate that the NDP, which dissociates so readily from NBD2 after NTP hydrolysis there, does not contribute significantly to the labeling of NBD1. The occlusion of the NDP at NBD1 and lack thereof at NBD2 obviously precludes any movement of the nucleotide in the opposite direction. As another means of evaluating the influence of nucleotide interaction at one domain on that of the other, the apparently selective interaction of AMP-PNP with NBD1 was employed. The concentration dependence of photolabeling with [α-32P]N3AMP-PNP is shown in Fig.6A and reveals a much higher affinity for NBD1 than for NBD2. Little interaction with NBD2 is detected at concentrations below 100 μm. Concentrations in this range were found to almost completely inhibit labeling of NBD1 with [α-32P]N3ATP (16Aleksandrov L. Mengos A. Chang X. Aleksandrov A. Riordan J.R. J. Biol. Chem. 2001; 276: 12918-12923Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). This effect is illustrated again in Fig. 6B, where 150 μmN3-AMP-PNP ablates the N3ATP labeling of the NBD1 band. This occurs, however, without any diminution of labeling of the NBD2 band. Hence, as with a mutation (K464A) that prevents labeling of NBD1 by N3ATP, occupation of the domain by AMP-PNP leaves the labeling of NBD2 with [α-32P]N3ATP entirely intact. The above experiments have shown distinct differences between the NBDs of CFTR in their interactions with nucleotides including divalent cation dependence, extent of hydrolysis, and dissociation. To focus strictly on the binding events, we utilized [α-32P]N3ADP and [γ-32P]N3ATP at 0 °C over a wide range of concentrations. Fig. 7Areveals that the NDP exhibits apparently conventional saturable binding to both domains with no marked difference in affinity for the two domains under these conditions where the nucleotide is kept present in solution until the time of photoactivation. Both sets of data are fit well by a single hyperbolic function. If the azido-nucleotide is removed from solution prior to irradiation, however, there is dissociation from NBD2 as would be expected from an equilibrium binding reaction. In contrast, NBD1 exhibits non-equilibrium behavior by occluding the NDP; hence, essentially the same curve is obtained when the nucleotide is washed out before photoactivation (data not shown). Nevertheless, the inherent binding affinities of the two domains for N3ADP do not appear to be significantly different. The binding curves for [γ-32P]N3ATP at 0 °C, however, appear quite different (Fig. 7B). Simple saturable binding occurs at NBD1, but the NBD2 curve is shifted to the right at low concentrations and i