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• W1990508717 abstract "Mutations in the chloride channel cystic fibrosis transmembrane regulator (CFTR) cause cystic fibrosis, a genetic disorder characterized by defects in CFTR biosynthesis, localization to the cell surface, or activation by regulatory factors. It was discovered recently that surface localization of CFTR is stabilized by an interaction between the CFTR N terminus and the multidomain cytoskeletal protein filamin. The details of the CFTR-filamin interaction, however, are unclear. Using x-ray crystallography, we show how the CFTR N terminus binds to immunoglobulin-like repeat 21 of filamin A (FlnA-Ig21). CFTR binds to β-strands C and D of FlnA-Ig21 using backbone-backbone hydrogen bonds, a linchpin serine residue, and hydrophobic side-chain packing. We use NMR to determine that the CFTR N terminus also binds to several other immunoglobulin-like repeats from filamin A in vitro. Our structural data explain why the cystic fibrosis-causing S13F mutation disrupts CFTR-filamin interaction. We show that FlnA-Ig repeats transfected into cultured Calu-3 cells disrupt CFTR-filamin interaction and reduce surface levels of CFTR. Our findings suggest that filamin A stabilizes surface CFTR by anchoring it to the actin cytoskeleton through interactions with multiple filamin Ig repeats. Such an interaction mode may allow filamins to cluster multiple CFTR molecules and to promote colocalization of CFTR and other filamin-binding proteins in the apical plasma membrane of epithelial cells. Mutations in the chloride channel cystic fibrosis transmembrane regulator (CFTR) cause cystic fibrosis, a genetic disorder characterized by defects in CFTR biosynthesis, localization to the cell surface, or activation by regulatory factors. It was discovered recently that surface localization of CFTR is stabilized by an interaction between the CFTR N terminus and the multidomain cytoskeletal protein filamin. The details of the CFTR-filamin interaction, however, are unclear. Using x-ray crystallography, we show how the CFTR N terminus binds to immunoglobulin-like repeat 21 of filamin A (FlnA-Ig21). CFTR binds to β-strands C and D of FlnA-Ig21 using backbone-backbone hydrogen bonds, a linchpin serine residue, and hydrophobic side-chain packing. We use NMR to determine that the CFTR N terminus also binds to several other immunoglobulin-like repeats from filamin A in vitro. Our structural data explain why the cystic fibrosis-causing S13F mutation disrupts CFTR-filamin interaction. We show that FlnA-Ig repeats transfected into cultured Calu-3 cells disrupt CFTR-filamin interaction and reduce surface levels of CFTR. Our findings suggest that filamin A stabilizes surface CFTR by anchoring it to the actin cytoskeleton through interactions with multiple filamin Ig repeats. Such an interaction mode may allow filamins to cluster multiple CFTR molecules and to promote colocalization of CFTR and other filamin-binding proteins in the apical plasma membrane of epithelial cells. Cystic fibrosis (CF) 4The abbreviations used are: CFcystic fibrosisCFTRcystic fibrosis transmembrane conductance regulatorFlnA and FlnBfilamin A and B, respectivelyFlnA-Ig21immunoglobulin-like repeat 21 of filamin AGSTglutathione S-transferasePDBProtein Data BankHSQCheteronuclear single quantum coherenceCSPchemical shift perturbationPBSphosphate-buffered saline. is a genetic disorder caused by mutations in an apical chloride channel, cystic fibrosis transmembrane regulator (CFTR). This disorder is characterized by high sweat chloride concentration, pulmonary disease with high production of dehydrated viscous secretions, and pancreatic insufficiency (1Gadsby D.C. Vergani P. Csanády L. Nature. 2006; 440: 477-483Crossref PubMed Scopus (542) Google Scholar). CF affects all exocrine epithelia, with morbidity and mortality primarily caused by bacterial infection and inflammation in the lung. CF affects ∼30,000 individuals in North America, of whom about 70% carry one copy of the mutation ΔF508, the most common of >1,000 CF-associated mutations. ΔF508 is a folding mutation that leads to rapid degradation at the endoplasmic reticulum. The small fraction of ΔF508-CFTR that is not degraded is characterized by inefficient trafficking to the apical plasma membrane and reduced residency in the plasma membrane (2Sharma M. Pampinella F. Nemes C. Benharouga M. So J. Du K. Bache K.G. Papsin B. Zerangue N. Stenmark H. Lukacs G.L. J. Cell Biol. 2004; 164: 923-933Crossref PubMed Scopus (279) Google Scholar, 3Swiatecka-Urban A. Brown A. Moreau-Marquis S. Renuka J. Coutermarsh B. Barnaby R. Karlson K.H. Flotte T.R. Fukuda M. Langford G.M. Stanton B.A. J. Biol. Chem. 2005; 280: 36762-36772Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). cystic fibrosis cystic fibrosis transmembrane conductance regulator filamin A and B, respectively immunoglobulin-like repeat 21 of filamin A glutathione S-transferase Protein Data Bank heteronuclear single quantum coherence chemical shift perturbation phosphate-buffered saline. Although the levels of ΔF508-CFTR in the apical plasma membrane are low, ΔF508-CFTR retains partial function as a cAMP-activated chloride channel (4Li C. Ramjeesingh M. Reyes E. Jensen T. Chang X. Rommens J.M. Bear C.E. Nat. Genet. 1993; 3: 311-316Crossref PubMed Scopus (155) Google Scholar, 5Nguyen T.D. Kim U.S. Perrine S.P. Biochem. Biophys. Res. Commun. 2006; 342: 245-252Crossref PubMed Scopus (9) Google Scholar). This justifies therapeutic approaches to promote delivery of ΔF508-CFTR and other functionally impaired CFTR mutants to the plasma membrane. A detailed understanding of factors that stabilize and regulate CFTR at the plasma membrane is important for the development of new therapies to correct CF-causing defects in vivo. CFTR is regulated by intracellular cAMP levels and phosphorylated at multiple sites by cAMP-activated protein kinase, which modulates CFTR trafficking (6Chang S.Y. Di A. Naren A.P. Palfrey H.C. Kirk K.L. Nelson D.J. J. Cell Sci. 2002; 115: 783-791Crossref PubMed Google Scholar) and activity (7Chappe V. Irvine T. Liao J. Evagelidis A. Hanrahan J.W. EMBO J. 2005; 24: 2730-2740Crossref PubMed Scopus (67) Google Scholar, 8Mense M. Vergani P. White D.M. Altberg G. Nairn A.C. Gadsby D.C. EMBO J. 2006; 25: 4728-4739Crossref PubMed Scopus (154) Google Scholar). We and others have identified and characterized additional regulatory proteins that interact with cytoplasmic domains of CFTR. For example, the PDZ-containing adaptor molecule NHERF1/EBP50 binds to the C terminus of CFTR, where it interacts with microtubules and with receptor for activated C kinase-1 (RACK1) (9Liedtke C.M. Yun C.H. Kyle N. Wang D. J. Biol. Chem. 2002; 277: 22925-22933Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). By interacting with ERM (ezrin-radixin-moesin) domain actin-binding proteins such as ezrin, NHERF1 also connects CFTR to the cortical actin cytoskeleton (10Short D.B. Trotter K.W. Reczek D. Kreda S.M. Bretscher A. Boucher R.C. Stutts M.J. Milgram S.L. J. Biol. Chem. 1998; 273: 19797-19801Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 11Sun F. Hug M.J. Lewarchik C.M. Yun C.H. Bradbury N.A. Frizzell R.A. J. Biol. Chem. 2000; 275: 29539-29546Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). CFTR associates with SNARE proteins (syntaxin 1A, SNAP23) and endocytic adaptors such as AP2, undergoing clathrin-mediated endocytosis (6Chang S.Y. Di A. Naren A.P. Palfrey H.C. Kirk K.L. Nelson D.J. J. Cell Sci. 2002; 115: 783-791Crossref PubMed Google Scholar, 12Cormet-Boyaka E. Di A. Chang S.Y. Naren A.P. Tousson A. Nelson D.J. Kirk K.L. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 12477-12482Crossref PubMed Scopus (69) Google Scholar, 13Li C. Roy K. Dandridge K. Naren A.P. J. Biol. Chem. 2004; 279: 24673-24684Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 14Ameen N. Silvis M. Bradbury N.A. J. Cyst. Fibros. 2007; 6: 1-14Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). A balance between cytoskeletal tethering and capture by the endocytic machinery may be crucial to maintain a sufficient population of CFTR at the cell surface. Recently, Thelin et al. (15Thelin W.R. Chen Y. Gentzsch M. Kreda S.M. Sallee J.L. Scarlett C.O. Borchers C.H. Jacobson K. Stutts M.J. Milgram S.L. J. Clin. Invest. 2007; 117: 364-374Crossref PubMed Scopus (75) Google Scholar) identified the dimeric cytoskeletal adaptors filamin A and filamin B (FlnA and FlnB) as new and important binding partners of CFTR. FlnA and FlnB, which have high sequence similarity to each other, are homodimeric rod-like proteins that cross-link actin filaments at high-angle orientations (16Hartwig J.H. Shevlin P. J. Cell Biol. 1986; 103: 1007-1020Crossref PubMed Scopus (138) Google Scholar). The filamins confer mechanical strength as well as flexibility and reversible deformability to cellular actin networks under mechanical stress (17Gardel M.L. Nakamura F. Hartwig J.H. Crocker J.C. Stossel T.P. Weitz D.A. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 1762-1767Crossref PubMed Scopus (322) Google Scholar). Filamins, however, also bind to cytosolic effectors and to membrane proteins such as integrin β-subunits, the platelet adhesion receptor GPIBα, HCN1 pacemaker channels, calcitonin receptor, glutamate receptor type 7, D2/D3 dopamine receptors, CD4 receptor, Ror2 receptor tyrosine kinase, μ-opioid receptor, and others (for reviews, see Refs. 18Feng Y. Walsh C.A. Nat. Cell Biol. 2004; 6: 1034-1038Crossref PubMed Scopus (417) Google Scholar, 19Popowicz G.M. Schleicher M. Noegel A.A. Holak T.A. Trends Biochem. Sci. 2006; 31: 411-419Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Filamins tether such proteins to the membrane-proximal actin cytoskeleton and regulate their surface localization and dynamics. Filamins may also mediate direct signaling between these proteins and the cytoskeleton (20Stossel T.P. Condeelis J. Cooley L. Hartwig J.H. Noegel A. Schleicher M. Shapiro S.S. Nat. Rev. Mol. Cell Biol. 2001; 2: 138-145Crossref PubMed Scopus (826) Google Scholar). The filamins exhibit common structural and functional properties (for review, see Ref. 19Popowicz G.M. Schleicher M. Noegel A.A. Holak T.A. Trends Biochem. Sci. 2006; 31: 411-419Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Filamins contain N-terminal globular actin-binding domains consisting of two calponin homology domains. These are followed by two extended rod domains connected by a hinge. The rod domains, respectively, consist of 15 and 8 immunoglobulin-like repeats, termed Ig1–Ig23. At the C terminus, a second hinge connects the second rod domain to a final repeat, Ig24, which is the dimerization element of the protein (21Pudas R. Kiema T.R. Butler P.J. Stewart M. Ylänne J. Structure. 2005; 13: 111-1119Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Those Ig repeats that have been structurally characterized have 7-stranded β-sandwich folds (21Pudas R. Kiema T.R. Butler P.J. Stewart M. Ylänne J. Structure. 2005; 13: 111-1119Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 22Sjekloæa L. Pudas R. Sjöblom B. Konarev P. Carugo O. Rybin V. Kiema T.R. Svergun D. Ylänne J. Djinoviæ Carugo K. J. Mol. Biol. 2007; 368: 1011-1023Crossref PubMed Scopus (23) Google Scholar). Selected Ig-like repeats, primarily the odd-numbered repeats in the second rod domain, bind a diverse array of linear motifs from the cytoplasmic portions of the integral membrane protein-binding partners of filamins (23Kiema T. Lad Y. Jiang P. Oxley C.L. Baldassarre M. Wevener K.L. Campbell I.D. Ylänne J. Calderwood D.A. Mol. Cell. 2006; 21: 337-347Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 24Nakamura F. Pudas R. Heikkinen O. Permi P. Kilpeläinen I. Munday A.D. Hartwig J.H. Stossel T.P. Ylänne J. Blood. 2006; 107: 1925-1932Crossref PubMed Scopus (136) Google Scholar, 25Lad Y. Jiang P. Ruskamo S. Harburger D.S. Ylänne J. Campbell I.D. Calderwood D.A. J. Biol. Chem. 2008; 283: 35154-35163Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 26Ithychanda S.S. Das M. Ma Y.Q. Ding K. Wang X. Gupta S. Wu C. Plow E.F. Qin J. J. Biol. Chem. 2009; 284: 4713-4722Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Interestingly, some binding partners such as integrin β7 can bind to more than one of the Ig-like repeats in vitro (23Kiema T. Lad Y. Jiang P. Oxley C.L. Baldassarre M. Wevener K.L. Campbell I.D. Ylänne J. Calderwood D.A. Mol. Cell. 2006; 21: 337-347Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 27Nakamura F. Osborn T.M. Hartemink C.A. Hartwig J.H. Stossel T.P. J. Cell Biol. 2007; 179: 1011-1025Crossref PubMed Scopus (212) Google Scholar). A CF-associated mutation, S13F, disrupts the interaction between the N terminus of CFTR and FlnA or FlnB (15Thelin W.R. Chen Y. Gentzsch M. Kreda S.M. Sallee J.L. Scarlett C.O. Borchers C.H. Jacobson K. Stutts M.J. Milgram S.L. J. Clin. Invest. 2007; 117: 364-374Crossref PubMed Scopus (75) Google Scholar). Disruption of the CFTR-filamin interaction results in greatly reduced CFTR surface levels due to rapid endocytosis. Unlike wild-type CFTR, the internalized S13F CFTR is targeted preferentially to lysosomes rather than being recycled to the plasma membrane (15Thelin W.R. Chen Y. Gentzsch M. Kreda S.M. Sallee J.L. Scarlett C.O. Borchers C.H. Jacobson K. Stutts M.J. Milgram S.L. J. Clin. Invest. 2007; 117: 364-374Crossref PubMed Scopus (75) Google Scholar). The CFTR-filamin interaction is thus crucial to maintain sufficient plasma membrane levels of CFTR, but the details of the interaction are unclear. It is also unclear how filamins fit into the diverse network of proteins that associate with CFTR and regulate its trafficking and activity at the plasma membrane. In this article, we present the crystal structure of the CFTR N terminus with immunoglobulin-like repeat 21 of filamin A (FlnA-Ig21). We also characterize the binding of the CFTR N terminus to other repeats in the C-terminal rod domain of filamin A using NMR. Our results explain why the S13F mutation disrupts the interaction between CFTR and filamins. In addition, we show that FlnA-Ig21 acts in a dominant negative fashion in cultured epithelial cells, disrupting the CFTR-filamin interaction and resulting in loss of surface CFTR. Our studies present the molecular details of the CFTR-filamin interaction and emphasize that coupling of CFTR to the actin cytoskeleton through filamin is crucial for the regulation of surface CFTR levels in epithelial tissues. Anti-human CFTR (C terminus-specific) monoclonal antibody was obtained from R&D Systems. Anti-FlnA antibody was from Millipore. Anti-NHERF1 was from ABCAM, and anti-RACK1 monoclonal antibody was from Transduction Laboratories. Horseradish peroxidase-coupled secondary antibodies were purchased from Santa Cruz Biotechnology. BioPORTER protein delivery system was obtained from Gene Therapy Systems, Inc. An enhanced chemiluminescence reagent was purchased from Denville. All other chemicals were reagent grade. Human FlnA-Ig17 (residues 1861–1950), FlnA-Ig19 (residues 2045–2140), FlnA-Ig21 (residues 2236–2329), FlnA-Ig23 (residues 2424–2516), and FlnA-Ig10 (residues 1158–1252) were cloned into pGST‖1 (28Sheffield P. Garrard S. Derewenda Z. Protein Expr. Purif. 1999; 15: 34-39Crossref PubMed Scopus (530) Google Scholar) for expression as GST fusions. GST-Ig repeats were expressed in Rosetta2(DE3) cells (EMD Biosciences) and purified by glutathione-affinity column chromatography in buffers containing 150 mm NaCl, 50 mm sodium phosphate, pH 8.0. The GST tag was removed by overnight cleavage with tobacco etch virus protease followed by additional glutathione-Sepharose and gel filtration chromatography. 15N-Labeled FlnA-Ig repeats were expressed as GST fusions in minimal medium supplemented with 1 g/liter [15N]NH4Cl (Isotec), cleaved, and purified in the same manner as the unlabeled repeats. CFTR N-terminal peptides were synthesized in the Molecular Biotechnology Core, Lerner Research Institute (Cleveland Clinic). The peptides are acetylated at their N termini and amidated at their C termini and were purified by high performance liquid chromatography. Crystallization was carried out using 1 mm FlnA-Ig21 in 20 mm Tris, pH 7.4, 50 mm NaCl, mixed with 3 mm CFTR4–22 peptide (splekasvvsklffswtrp) in water. The complex crystallized at 20 °C in hanging drops against 60% Tacsimate, 0.1 m Bistris-propane, pH 7.0. Crystals were cryoprotected directly in mother liquor and frozen in liquid nitrogen before data collection. Diffraction data were collected at Advanced Light Source beamline 4.2.2., Lawrence Berkeley National Laboratory. Diffraction from these crystals was typically very anisotropic. Data to 2.8 Å were indexed, integrated, and scaled, using D*TREK (29Pflugrath J.W. Acta Crystallogr. Sect. D. 1999; 55: 1718-1725Crossref PubMed Scopus (1417) Google Scholar). Crystals belonged to space group P6522 with unit cell dimensions a = 74.15 Å, b = 74.15 Å, c = 289.06 Å, as confirmed by molecular replacement in Phaser (30McCoy A.J. Grosse-Kunstleve R.W. Adams P.D. Winn M.D. Storoni L.C. Read R.J. J. Appl. Crystallogr. 2007; 40: 658-674Crossref PubMed Scopus (14528) Google Scholar). The FlnA-Ig21 monomer structure and a polyalanine model of the integrin β7 C terminus (Protein Data Bank (PDB) code 2BRQ) (Ref. 23Kiema T. Lad Y. Jiang P. Oxley C.L. Baldassarre M. Wevener K.L. Campbell I.D. Ylänne J. Calderwood D.A. Mol. Cell. 2006; 21: 337-347Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar) were used as search models. Refmac (31Murshudov G.N. Vagin A.A. Lebedev A. Wilson K.S. Dodson E.J. Acta Crystallogr. Sect. D. 1999; 55: 247-255Crossref PubMed Scopus (1010) Google Scholar) and CNS (32Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D. 1998; 54: 905-921Crossref PubMed Scopus (16965) Google Scholar) were used for refinement, and model rebuilding was carried out in COOT (33Emsley P. Cowtan K. Acta Crystallogr. Sect. D. 2004; 60: 2126-21232Crossref PubMed Scopus (23344) Google Scholar) to generate a final model with Rwork and Rfree of 26.3% and 29.8%, respectively. The anisotropy of the diffraction data contributed to a high average B factor. More than 97% of residues are in the most favored or additionally allowed regions of Ramachandran space, whereas no residues are in the disallowed region. The asymmetric unit of the crystal contains two copies of FlnA-Ig21 and one CFTR4–22 peptide. Coordinates and structure factors have been deposited with the RCSB Protein Data Bank (PDB code 3ISW). Crystallographic data and statistics are summarized in Table 1.TABLE 1Crystallographic data collection statisticsData collectionBeamlineALS 4.2.2Wavelength (Å)1.0Space groupP6522Cell dimensionsa, b, c (Å)74.15, 74.15, 289.06α, β, γ (degree)90, 90, 120Resolution range (Å)42.97–2.80 (2.90–2.80)aValues in parentheses correspond to the highest resolution shell of reflections.Rsym (%)7.1 (47.2)I/σI13.8 (3.3)Completeness (%)96.6 (98.5)Redundancy8.53 (10.41)RefinementResolution range (Å)42.97–2.80 (2.90–2.80)No. of reflectionsRefinement11427 (829)Test set580 (46)Rwork/Rfree (%)26.3/29.8 (37.9/40.8)Correlation coefficients Fo − Fc/Fo − Fc free0.919/0.907No. of atoms1,518Protein1,504Solvent14Root mean square differencesBond lengths (Å)0.015Bond angles (degree)1.644Average B factor (Å2)69.19Protein69.92Peptide62.96Solvent61.51B factor from Wilson plot75.7Amino acids in Ramachandran diagram (%)In most favored regions88.8In additional allowed regions8.8In generously allowed regions2.4a Values in parentheses correspond to the highest resolution shell of reflections. Open table in a new tab NMR spectra for 15N-labeled FlnA repeats 10, 17, 19, 21, and 23 were collected at 25 °C on a Bruker 600 MHz Avance ICE spectrometer equipped with a cryoprobe. Two-dimensional 1H/15N HSQC spectra were acquired utilizing a spin-state selective gradient-enhanced HSQC pulse sequence (34Schleucher J. Schwendinger M. Sattler M. Schmidt P. Schedletzky O. Glaser S.J. Sørensen O.W. Griesinger C. J. Biomol. NMR. 1994; 4: 301-306Crossref PubMed Scopus (714) Google Scholar). All NMR datasets were processed with in-house scripts using NMRPipe (35Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11554) Google Scholar) and visualized with Sparky (36Goddard T.D. Kneller D.G. SPARKY3. University of California, San Francisco2006Google Scholar). NMR samples contained 25 μm15N-labeled protein in 25 mm HEPES, pH 7.0, 5 mm sodium chloride, 1 mm dithiothreitol, and 10% D2O. Backbone chemical shift assignments for FlnA-Ig21 were determined using 1H/13C/15N HNCA, HNCO, and CBCA(CO)NH experiments. The assignments will be published separately. 5J. Liu, S. Ithychanda, and J. Qin, unpublished results. Backbone chemical shift assignments were obtained from the Biological Magnetic Resonance Data Bank for FlnA-Ig17 (accession number 6730) (24Nakamura F. Pudas R. Heikkinen O. Permi P. Kilpeläinen I. Munday A.D. Hartwig J.H. Stossel T.P. Ylänne J. Blood. 2006; 107: 1925-1932Crossref PubMed Scopus (136) Google Scholar), FlnA-Ig19 (accession number 15925) (37Heikkinen O. Permi P. Koskela H. Ylänne J. Kilpelainen I. Biomol. NMR Assign. 2009; 3: 53-56Crossref PubMed Scopus (6) Google Scholar) and FlnA-Ig23 (accession number 15777) (38Nakamura F. Heikkinen O. Pentikäinen O.T. Osborn T.M. Kasza K.E. Weitz D.A. Kupiainen O. Permi P. Kilpeläinen I. Ylänne J. Hartwig J.H. Stossel T.P. PLoS One. 2009; 4: e4928Crossref PubMed Scopus (55) Google Scholar). Peptide binding titrations were initially pursued using CFTR4–22 or CFTR4–22/S13F. These peptides tended to aggregate or form gel-like phases, causing extensive line broadening and leading to poor spectra. Therefore, titrations were carried out using shorter peptides containing an F16E substitution to increase peptide solubility and reduce aggregation behavior. 15N-Ig repeats were titrated with CFTR7–20/F16E (EKASVVSKLEFSWT) and CFTR7–20/S13F/F16E (EKASVVFKLEFSWT) at ratios ranging from 1:0 up to 1:100 (FlnA:peptide). These spectra exhibited much less line broadening and clearly defined chemical shift perturbations. Normalized chemical shift perturbations (Δδ) (39Grzesiek S. Stahl S.J. Wingfield P.T. Bax A. Biochemistry. 1996; 35: 10256-10261Crossref PubMed Scopus (307) Google Scholar) were calculated and plotted with Octave (40Eaton J.W. Bateman D. Hauberg S. GNU Octave: A High-level Interactive Language for Numerical Computations. Network Theory Ltd., United Kingdom2007Google Scholar) according to the equation Δδ = [(Δ1HN)2 + (Δ15NH/5)2]1/2. Peptide binding constants (Kd) were determined by nonlinear regression fitting of chemical shift changes versus peptide concentration for selected resonances, using the equation Δδ/Δδmax = [L]/(Kd + [L]), where Δδmax is the maximum change in chemical shift at saturation and [L] is the CFTR peptide concentration (41Fielding L. Curr. Top. Med. Chem. 2003; 3: 39-53Crossref PubMed Scopus (160) Google Scholar, 42Van Holde K.E. Johnson W.C. Ho P.S. Principles of Physical Biochemistry. Prentice Hall, Upper Saddle River, N.J.1998Google Scholar). Binding constants are summarized in Table 2.TABLE 2Interaction affinities between CFTR N-terminal peptide and FlnA-Ig repeatsIg repeatKdaKc values represent the mean ± 1 S.D. as determined from six resonances exhibiting significant chemical shift perturbations.,bMeasured HSQC titration binding curves used to calculate each Kd value are shown in supplemental Fig. 6.Hydrogen bond donor residuecResidue donating a hydrogen bond via a backbone amide to the CFTR-Ser13 side-chain hydroxyl oxygen.Hydrogen bond lengthdHydrogen bond lengths are the serine oxygen − backbone amide proton distances, estimated as described under supplemental Methods.μmÅFlnA-Ig2190 ± 9Ala22811.81FlnA-Ig17315 ± 22Ala19082.09 ± 0.05FlnA-Ig19135 ± 8Val20901.96 ± 0.02FlnA-Ig23127 ± 9Val24721.99 ± 0.01a Kc values represent the mean ± 1 S.D. as determined from six resonances exhibiting significant chemical shift perturbations.b Measured HSQC titration binding curves used to calculate each Kd value are shown in supplemental Fig. 6.c Residue donating a hydrogen bond via a backbone amide to the CFTR-Ser13 side-chain hydroxyl oxygen.d Hydrogen bond lengths are the serine oxygen − backbone amide proton distances, estimated as described under supplemental Methods. Open table in a new tab CFTR4–22 peptide was docked to FlnA-Ig repeats 17, 19, and 23 using ambiguous intermolecular distance restraints, derived from 1H/15N chemical shift perturbations (CSPs). Restraints were input into a combined rigid body/torsion angle dynamics simulated annealing protocol in Xplor-NIH (43Clore G.M. Schwieters C.D. J. Am. Chem. Soc. 2003; 125: 2902-2912Crossref PubMed Scopus (114) Google Scholar). Docking calculations utilized molecule A from the crystal structure of FlnA-Ig17 with GPIbα peptide (PDB code 2BP3) (24Nakamura F. Pudas R. Heikkinen O. Permi P. Kilpeläinen I. Munday A.D. Hartwig J.H. Stossel T.P. Ylänne J. Blood. 2006; 107: 1925-1932Crossref PubMed Scopus (136) Google Scholar), the NMR structure of FlnA-Ig19 (PDB code 2K7Q) (37Heikkinen O. Permi P. Koskela H. Ylänne J. Kilpelainen I. Biomol. NMR Assign. 2009; 3: 53-56Crossref PubMed Scopus (6) Google Scholar), and the NMR structure of FlnA-Ig23 (PDB code 2K3T) (38Nakamura F. Heikkinen O. Pentikäinen O.T. Osborn T.M. Kasza K.E. Weitz D.A. Kupiainen O. Permi P. Kilpeläinen I. Ylänne J. Hartwig J.H. Stossel T.P. PLoS One. 2009; 4: e4928Crossref PubMed Scopus (55) Google Scholar). 1H/15N CSP maps were converted into ambiguous distance restraints. An additional distance restraint between the CFTRS13 hydroxyl oxygen and the appropriate FlnA-Ig repeat backbone amide was also introduced. For each FlnA-Ig repeat·CFTR peptide complex, 100 structures were generated; the solution with the lowest total energy and no ambiguous distance restraint violations greater than 0.5 Å was selected as the best structure. We built a homology model for FlnA-Ig repeat 10 using the I-TASSER server (44Zhang Y. BMC Bioinformatics. 2008; 9: 40Crossref PubMed Scopus (3886) Google Scholar), based on the FlnB-Ig10 NMR structure (PDB code 2DIA). The I-TASSER C-score for the FlnA-Ig10 homology model was 1.15, indicating a high likelihood that the model accurately predicts the FlnA-Ig10 structure. As expected, the FlnA-Ig10 homology model is similar in structure to FlnB-Ig10 and other FlnA-Ig repeats. A more complete description of the molecular modeling procedures is provided in the supplemental Methods. Calu-3 cells were grown in cell culture on 100-mm2 tissue culture plastic or on 0.4-μm pore Transwell-Clear polyester filter inserts (Corning Costar) with a growth area of 4.4 cm2 for biotinylation experiments. For immunofluorescence, cells were seeded at a density of 0.2 × 106 cells/filter with a growth area of 1.0 cm2. Mimimum Eagle's culture medium was supplemented with 2.4 mg of l-glutamine and 10% fetal bovine serum. Cell cultures were grown at 37 °C under 5% CO2 humidified air. Culture medium was changed at 48-h intervals until the desired confluence was reached, as assessed by microscopic examination. We used BioPORTER protein delivery system to deliver FlnA repeats into Calu-3 cells, as described previously (45Liedtke C.M. Hubbard M. Wang X. Am. J. Physiol. Cell Physiol. 2003; 284: C487-C496Crossref PubMed Scopus (58) Google Scholar). BioPORTER reagent was dissolved in methanol, aliquoted in 10-μl portions, and dried under an N2 stream. Dried reagent was reconstituted in 50 μl of Hanks' balanced salt solution with 10 mm HEPES, pH 7.5 (HPSS) per filter insert containing an aliquot of protein in phosphate-buffered saline (PBS). The total volume of the peptide·BioPORTER complex per filter insert was taken to 500 μl for 24-mm filter inserts or 300 μl for 12-mm filter inserts, using HPSS. Apical surfaces of cells were incubated with the protein·BioPORTER complex or BioPORTER reagent alone for 2.5 h at 35 °C. The apical solution was replaced with HPSS and the incubation continued for 2 h at 35 °C. Polarized Calu-3 cell monolayers were grown on 24-mm-diameter Transwell permeable supports. Cell surface proteins were biotinylated using EZ-Link sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate (sulfo-NHS-SS; Pierce) (46Auerbach M. Liedtke C.M. Am. J. Physiol. Cell Physiol. 2007; 293: C294-C304Crossref PubMed Scopus (17) Google Scholar). Cells were cooled to 4 °C, washed in PBS, pH 8.2, supplemented with 0.1 mm CaCl2 and 1 mm MgCl2, and incubated twice with 1 mg sulfo-NHS-SS-biotin/ml for 30 min at 4 °C. Nonreacted sulfo-NHS-SS-biotin was quenched by washing cells with PBS, pH 8.2, with 100 mm glycine, 0.1 mm CaCl2, and 1 mm MgCl2. Cells were harvested in CFTR lysis buffer and biotinylated proteins isolated using streptavidin-agarose beads (GE Healthcare). Biotinylated CFTR was detected by immunoblot analysis using a monoclonal antibody directed to the C terminus of CFTR. Exposed bands were quantitated by densitometry. Calu-3 cell monolayers at 50% confluence were dual-labeled for proteins of interest as described previously (9Liedtke C.M. Yun C.H. Kyle N. Wang D. J. Biol. Chem. 2002; 277: 22925-22933Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Briefly, cells were washed with PBS, fixed in 4% paraformaldehyde for 15 min at room temperature, washed three times with PBS, and permeabilized with 0.2% Triton X-100 in 10% normal goat serum in PBS. Fixed cells were stained" @default.
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• W1990508717 title "Biochemical Basis of the Interaction between Cystic Fibrosis Transmembrane Conductance Regulator and Immunoglobulin-like Repeats of Filamin" @default.
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