Deletion of Phe508 in the first nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator increases its affinity for the heat shock cognate 70 chaperone Toby S Scott-Ward1,2 and Margarida D Amaral1,2 ˆ Universidade de Lisboa, Faculdade de Ciencias de Lisboa, BioFIG, Centre for Biodiversity, Functional and integrative Genomics, Portugal ´ ´ Centro de Genetica Humana, Instituto Nacional de Saude Dr Ricardo Jorge, Lisboa, Portugal Keywords CFTR-interacting proteins; correctors; mechanism of disease; small molecules; surface plasmon resonance Correspondence M D Amaral, EMBL – European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany Fax: +49 6221 387 8306 Tel: +49 6221 387 8199 E-mail: mdamaral@fc.ul.pt (Received August 2009, revised 29 September 2009, accepted October 2009) doi:10.1111/j.1742-4658.2009.07421.x The primary cause of cystic fibrosis (CF), the most frequent fatal genetic disease in Caucasians, is deletion of phenylalanine at position 508 (F508del), located in the first nucleotide-binding domain (NBD1) of the CF transmembrane conductance regulator (CFTR) protein F508del-CFTR is recognized by the endoplasmic reticulum quality control (ERQC), which targets it for proteasomal degradation, preventing this misfolded but partially functional Cl) channel from reaching the cell membrane We recently proposed that the ERQC proceeds along several checkpoints, the first of which, utilizing the chaperone heat shock cognate 70 (Hsc70), is the major one directing F508del-CFTR for proteolysis Therefore, a detailed characterization of the interaction occurring between F508del-CFTR and Hsc70 is critical to clarify the mechanism that senses misfolded F508del-CFTR in vivo Here, we determined by surface plasmon resonance that: (a) F508del-murine (m)NBD1 binds Hsc70 with higher affinity (KD, 2.6 nm) than wild-type (wt) mNBD1 (13.9 nm); (b) ATP and ADP dramatically reduce NBD1–Hsc70 binding; (c) the F508del mutation increases by approximately six-fold the ATP concentration required to inhibit the NBD1–Hsc70 interaction (IC50; wt-mNBD1, 19.7 lm ATP); and (d) the small molecule CFTR corrector 4a (C4a), but not VRT-325 (V325; both rescuing F508del-CFTR traffic), significantly reduces F508del-mNBD1 binding to Hsc70, by $ 30% Altogether, these results provide a novel, robust quantitative characterization of Hsc70–NBD1 binding, bringing detailed insights into the molecular basis of CF Moreover, we show how this surface plasmon resonance assay helps to elucidate the mechanism of action of small corrective molecules, demonstrating its potential to validate additional therapeutic compounds for CF Structured digital abstract l MINT-7265886: mNBD1 (uniprotkb:P26361) binds (MI:0407) to Hsc70 (uniprotkb:P19120) by anti bait coimmunoprecipitation (MI:0006) Abbreviations Ab, antibody; C4a, corrector 4a; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; ER, endoplasmic reticulum; ERQC, endoplasmic reticulum quality control; F508del, deletion of phenylalanine residue at position 508; h, human; Hsc70, heat shock cognate 70; Hsp70, heat shock protein 70; I172, inhibitor CFTRinh-172; LA, apo-a-lactalbumin; m, murine; NBD1, first nucleotide-binding domain; Red-LA, reduced apo-a-lactalbumin; SEM, standard error of the mean; SPR, surface plasmon resonance; V325, corrector VRT-325; wt, wild-type FEBS Journal 276 (2009) 7097–7109 ª 2009 The Authors Journal compilation ª 2009 FEBS 7097 Interaction of Hsc70 with F508del-NBD1 T S Scott-Ward and M D Amaral l l l MINT-7265906, MINT-7265964, MINT-7265981, MINT-7265951: mNBD1 (uniprotkb:P26361) binds (MI:0407) to Hsc70 (uniprotkb:P19120) by surface plasmon resonance (MI:0107) MINT-7265924, MINT-7265939: hNBD1 (uniprotkb:P13569) binds (MI:0407) to Hsc70 (uniprotkb:P19120) by surface plasmon resonance (MI:0107) MINT-7265996: Hsc70 (uniprotkb:P19120) binds (MI:0407) to Apo-alpha-lactalbumin (uniprotkb:P00711) by surface plasmon resonance (MI:0107) Introduction Cystic fibrosis (CF) is a life-threatening genetic disease caused by malfunction of CF transmembrane conductance regulator (CFTR) [1], a Cl) channel that plays a central role in transepithelial ion transport [2] A single amino acid deletion, of phenylalanine 508 (F508del), in the first nucleotide-binding domain (NBD1) of CFTR accounts for approximately 70% of CF chromosomes worldwide [2] This mutation prevents the correct folding of CFTR, thus causing its retention by the endoplasmic reticulum (ER) quality control (ERQC) as an immature intermediate that is rapidly degraded by the ubiquitin–proteasome pathway [3] Like that of other secretory proteins, the proper folding of CFTR is highly dependent on molecular chaperones, such as heat shock protein 70 (Hsp70) and its constitutively expressed homologue, heat shock cognate 70 (Hsc70) [4,5] Hsc70 and Hsp70 are single-polypeptide cytoplasmic proteins composed of an N-terminal ATPase domain, a substrate-binding region, and a C-terminal 15 kDa ‘lid’ regulating binding affinity for ‘client’ proteins These chaperones bind short (approximately seven residues) hydrophobic regions exposed in either mutant or immature wild-type (wt) client proteins [6,7] The binding of Hsc70 and Hsp70 to substrates is tightly coupled to cycles of ATP binding and hydrolysis followed by ADP release [8] In our previously proposed model of the ERQC, the folding states of CFTR are assessed within the ER at multiple checkpoints [9,10], the first of these involving the Hsc70 ⁄ Hsp70 machinery, the most critical one for F508del-CFTR degradation Biochemical data suggest that both immature wt-CFTR and F508del-CFTR have Hsc70-binding sites, but the Hsc70 association with F508del-CFTR is prolonged [11] However, there is evidence that critical interaction sites for Hsc70 reside within the first half of CFTR, in particular NBD1 [12,13] Indeed, analysis of the human (h) and murine (m) CFTR-NBD1 sequences with the online program limbo (http://switpc7.vub.ac.be/) performed here predicts that they both contain at least three hepapeptide Hsp70-binding sites, with a high degree of certainty (99%) Biochemical analyses of purified 7098 NBD1 indicate that F508del reduces domain stability, and hence promotes aggregation [14,15] Molecular dynamics modelling studies suggest that F508delNBD1 has more conformational freedom than the wild-type (wt), thus exposing its hydrophobic interior to the solution and impairing its interdomain contacts [16,17] Collectively, these data implicate NBD1 as the most probable site for the interaction of CFTR with Hsc70 Several small molecules that improve CFTR folding, biogenesis and function were recently identified in high-throughput screens [18], such as corrector 4a (C4a) and corrector VRT-325 (V325), which promote trafficking of F508del-CFTR to the plasma membrane [19,20] However, the exact mechanism of action of these compounds and their putative binding sites on CFTR remain undefined Here, we used a novel approach, surface plasmon resonance (SPR) [21], to quantify the interaction occurring between F508del-CFTR and Hsc70, versus that of the chaperone with wt-NBD1 Our data show that F508del-mNBD1 binds Hsc70 with approximately fivefold higher affinity than wt-mNBD1, and that both ATP and ADP dramatically reduce NBD1–Hsc70 binding Moreover, we also show that, in the presence of a small molecule known to rescue the traffic of fulllength F508del-mCFTR to the plasma membrane, the strength of the F508del-mNBD1–Hsc70 interaction is reduced by $ 30% Results Interaction of purified NBD1 and Hsc70 under the SPR conditions To confirm whether NBD1 binds specifically to Hsc70 under conditions that would subsequently be used in SPR interaction studies, we immunoprecipitated Hsc70–NBD1 complexes in vitro from a mixture of the two purified proteins Electrophoresis of the purified F508del-mNBD1 and bovine Hsc70 prior to immunoprecipitation (Fig 1A) resolved single 30 and 73 kDa FEBS Journal 276 (2009) 7097–7109 ª 2009 The Authors Journal compilation ª 2009 FEBS T S Scott-Ward and M D Amaral Interaction of Hsc70 with F508del-NBD1 Analysis of stability of NBD1 under the SPR interaction conditions 50 25 16 37 25 B – – 1 – – 1 – 1 – 1 1 – – IB: Hsc70 – – – – IP: NBD1 NBD1 Hsc70 (µM) NBD1 (µM) MgATP (mM) BSA (µM) Fig In vitro interaction of NBD1 with Hsc70 confirmed by immunoprecipitation (IP) under conditions used in SPR studies (A) SDS ⁄ PAGE (P) and western blot (WB) analyses of purified F508del-mNBD1 (F508del-NBD1; lg) and bovine Hsc70 (1 lg) with L12B4 and SPA-815, respectively, to confirm their specificity (see Experimental procedures) (B) In vitro analysis of the interaction of Hsc70 with NBD1 by immunoprecipitation under conditions equivalent to those used for SPR Protein G beads coated with L12B4–NBD1 complexes were incubated with purified Hsc70 (1 lM; 10 at 25 °C) and analysed by western blot using either SPA-815 or L12B4 (see Experimental procedures) Lanes: 1, BSA (3 lM; without NBD1); 2, wt-hNBD1 (1 lM); 3, wt-hNBD1 (5 lM); 4, F508del-mNBD1 (1 lM); 5, wt-mNBD1; 6, wt-mNBD1 with MgATP (2 mM); 7, wt-hNBD1 with beads (without L12B4); 8, L12B4-coated beads only (without NBD1 and Hsc70) Similar results were obtained in three experiments (n = 3) protein bands (left lane, both panels), which were confirmed to be specific by the parallel western blot analysis with L12B4 [antibody (Ab) against NBD1 of CFTR] and SPA-815 (Ab against Hsc70), respectively (right lane, both panels) The data in Fig 1B show that purified Hsc70 is present in immunoprecipitated complexes of either wt-mNBD1 (lanes 2, and 5) or F508del-mNBD1 (lane 4) incubated in SPR flow buffer Moreover, the absence of Hsc70 in immunoprecipitates when BSA (Fig 1B, lane 1) was used instead of NBD1, or in the absence of L12B4 (lane 7), demonstrates that the binding of Hsc70 does not occur with all protein substrates Increasing the concentration of wt-hNBD1 by five-fold (Fig 1B, lane 3) did not noticeably increase the amount of NBD1 or Hsc70 recovered (compare with lane 2), suggesting that the L12B4-coated beads were already saturated with NBD1 Addition of MgATP (2 mm) markedly reduced the binding of Hsc70 to wt-mNBD1 (Fig 1B, lane 6), consistent with the results of previous studies with other Hsc70 substrates [8] Assessment of optimal conditions for studying the Hsc70–NBD1 interaction by SPR Before determining the effect of the F508del mutation on the NBD1–Hsc70 interaction by SPR, we performed A B F508del-m 10 Buffer 300 340 380 420 Wavelength (nm) Fluorescence (units) 47 We used intrinsic tryptophan fluorescence spectroscopy to assess the structure and stability of NBD1 under assay conditions that would subsequently be used in SPR interaction studies A comparison of emission spectra obtained before (0 min) and after (10 min) incubation (Fig 2A) reveals that there was only a small decrease in the overall intensity of the fluorescence emitted from F508del-mNBD1 and no shift in the peak wavelength Furthermore, the intensity of fluorescence emitted at 328 nm and 343 nm from diluted F508del-mNBD1 (Fig 2B) displayed a minor initial decrease, but was essentially stable during the 10 incubation period (similar data were obtained with wt-mNBD1; not shown; n = 2) Previous studies indicate that, as compared with their folded forms, denatured wt-hNBD1 and F508del-hNBD1 display a dramatically reduced overall intensity of emitted fluorescence, and peak values are red-shifted to higher wavelengths [15] Hence, our data suggest that: (a) the wt-mNBD1 and F508del-mNBD1 used in this study have a structure consistent with a native fold [15,22]; and (b) the domain is stable under the conditions used in SPR experiments 343 75 328 Hsc70 kDa P WB Fluorescence (units) A NBD1 kDa P WB 83 F508del-m 343 nm 328 nm 0 200 400 600 800 Time (s) Fig Spectroscopic analyses show that NBD1 is stable under the conditions used in SPR studies (A) Fluorescence emission spectra (300–400 nm) of F508del-mNBD1 (1 lM) before (0 min) and after (10 min) incubation in buffer at 25 °C (excitation wavelength of 295 nm) (B) Change in intrinsic tryptophan fluorescence emitted at 328 and 343 nm from F508del-mNBD1 (1 lM) during incubation in buffer (25 °C) Data are corrected for buffer fluorescence Similar results were obtained with other NBD1 preparations (F508delmNBD1, n = 3; wt-mNBD1, not shown, n = 2) FEBS Journal 276 (2009) 7097–7109 ª 2009 The Authors Journal compilation ª 2009 FEBS 7099 Interaction of Hsc70 with F508del-NBD1 T S Scott-Ward and M D Amaral a series of control experiments to determine the binding specificity and optimal assay conditions To this end, wt-hNBD1 and BSA were immobilized on sensor chip surfaces, and the binding of L12B4 was investigated The data in Fig 3A show that there was a measurable and time-dependent interaction of Ab (15 nm) with wt-hNBD1, but not with BSA-coated chip surfaces (n = 3–5) These data indicate that even at a low nanomolar concentration, there was potent binding of L12B4 to immobilized NBD1 (23.7 ± 0.9 pmolỈnmol)1; n = 3), which is characteristic of Ab–antigen interactions [23] The data also indicate that we can use SPR to assay the binding of interacting proteins to NBD1 To further optimize the conditions for investigating the NBD1–Hsc70 interaction, we tested sensor chips coated with NBD1 (on-chip NBD1) Hsc70 (0.3 lm) bound specifically to sensor surfaces coated with wt-mNBD1, F508del-mNBD1, and wt-hNBD1 (Fig 3B; F508del-mNBD1, 2.5 ± 0.2 pmolỈnmol)1; wt-mNBD1, 2.9 ± 0.1 pmolỈnmol)1; wt-hNBD1, 3.8 ± 0.2 pmolỈnmol)1; n = 3), with minimal adsorption of Hsc70 onto BSA-coated surfaces (< 0.2 pmolỈnmol)1) Hence, under these conditions, the magnitude of Hsc70 binding to wt-mNBD1 was substantially (approximately six-fold) lower than that observed for L12B4 Applied: L12B4 Ab On-chip: wt-h 20 Binding (pmol·nmol–1) 500 Time (s) On-Chip: Hsc70 Applied: Red-LA Red-LA +ATP 20 LA BSA 0 7100 500 Time (s) 1000 Applied: Hsc70 On-chip: wt-h wt-m F508del-m BSA 1000 D 40 BSA C Binding (pmol·nmol–1) B 40 Binding (pmol·nmol–1) Binding (pmol·nmol–1) A Then, we tested the reverse situation by immobilizing Hsc70 on CM5 sensor chips (on-chip Hsc70), to investigate its ability to bind folded or unfolded proteins The immobilized chaperone was found to potently bind reduced apo-a-lactalbumin (Red-LA) (Fig 3C; 10 lm; 29.1 ± 3.8 pmolỈnmol)1; n = 3), an Hsc70 substrate [13], whereas minimal binding was detected with the nonreduced, folded form of the protein (LA) (10 lm; 1.1 ± 0.7 pmolỈnmol)1; n = 3) As expected, virtually no binding of BSA (15 lm) to Hsc70-coated surfaces could be detected (< 0.1 pmolỈnmol)1; n = 20) under these conditions These data confirm that Hsc70 binding detected by SPR was only significantly detected for unfolded protein substrates Interestingly, when Red-LA was applied in the presence of ATP (100 lm), the level of binding was reduced to $ 80% of that under nucleotide-free conditions (23.9 ± 1.9 pmolỈnmol)1; n = 3) Next, we determined Hsc70 binding of all three NBD1 variants (0.5 lm; Fig 3D), and found that all bound potently to immobilized Hsc70 (F508del-mNBD1, 20.9 ± 2.0 pmolỈnmol)1; wt-mNBD1, 17.8 ± 2.3 pmolỈnmol)1; wt-hNBD1, 27.3 ± 3.8 pmolỈnmol)1; n = 4) Under these conditions, wt-hNBD1 displayed the highest level of Hsc70 binding, followed by F508del-mNBD1 Moreover, the addition of MgATP 40 500 Time (s) 1000 On-Chip: Hsc70 Applied: wt-h F508del-m 20 wt-m wt-m + ATP BSA 0 500 Time (s) 1000 Fig Hsc70 binds specifically to NBD1 and control proteins in SPR studies Characterization of the interaction of: (A) applied L12B4 (15 nM) with covalently immobilized (‘on-chip’) wt-hNBD1; and (B) applied Hsc70 (0.3 lM) with on-chip wt-hNBD1, wtmNBD1, or F508del-mNBD1 (see Experimental procedures) Hsc70 and L12B4 displayed minimal interaction with on-chip BSA [ , (A) and (B)] (C, D) The interaction of applied Red-LA or LA (10 lM) (C) and wt-hNBD1, wt-mMBD1 or F508del-mNBD1 (0.5 lM) (D) with on-chip Hsc70 (On-Chip Hsc70; see Experimental procedures) BSA (15 lM) showed minimal interaction with on-chip Hsc70 [–, (C) and (D), n = 15] Periods of protein application (and association) are indicated by the solid bars After protein application, dissociation was measured by injecting flow buffer over the protein-coated surface Similar results were obtained in additional experiments (n = or n = 4) FEBS Journal 276 (2009) 7097–7109 ª 2009 The Authors Journal compilation ª 2009 FEBS T S Scott-Ward and M D Amaral Interaction of Hsc70 with F508del-NBD1 (100 lm) dramatically reduced the binding of wtmNBD1 to Hsc70 (Fig 1A; 3.7 ± 1.3 pmolỈnmol)1; n = 3) These data show that we can use SPR to accurately measure the specific binding of NBD1 to immobilized Hsc70 The substantially higher binding of NBD1 to immobilized Hsc70 over that of Hsc70 chaperone binding to immobilised NBD1 can be attributed to the ability of Hsc70 to better survive the acidic, low-salt SPR immobilization conditions than NBD1 [24] Hence, in subsequent experiments, we decided to immobilize Hsc70 and quantify the binding of wt- and F508del-mNBD1 Assessment of the impact of F508del on the NBD1–Hsc70 interaction To determine the strength of the NBD1–Hsc70 interaction, we thus immobilized the chaperone and quantified the binding of increasing concentrations of wt-mNBD1 and F508del-mNBD1 (Fig 4A,B) Using kinetic analyses, we determined that rates of association (ka) and dissociation (Fig 4C, kd) of wt-mNBD1 and Hsc70 are extremely slow [ka, 3660 ± 596 m)1Ỉs)1; kd, (5.2 ± 1.4) · 10)5 s)1; n = 3] In contrast, F508del-mNBD1 bound Hsc70 with a comparable association rate, but had a five-fold lower dissociation rate [ka, 4030 ± 655 m)1Ỉs)1; kd, (1.0 ± 0.2) · 10)5 s)1; n = 3) Using these constants, we calculated that Hsc70 bound F508del-mNBD1 with five-fold higher affinity than wt-mNBD1 [dissociation constant (KD): F508del-mNBD1, 2.6 ± 0.5 nm; wt-mNBD1, 13.9 ± 0.8 nm; n = 3] These data indicate that when Phe508 is deleted, there is a significant increase in the real-time affinity of mNBD1 for Hsc70 (P < 0.01) Analysis of the dose–response data (Fig 4D) revealed that the maximum amount of F508del-mNBD1 bound to Hsc70 at saturating concentration appears to be lower than that of wt-mNBD1 (Bmaxapp: F508del-mNBD1, 73.1 ± 3.9 pmolỈnmol)1; wt-mNBD1, 106.6 ± 6.6 pmolỈ nmol)1; n = 3) Moreover, apparent dissociation constants determined directly from the dose–response data (Eqn 1) indicate that F508del-mNBD1 bound Hsc70 with at least three-fold higher affinity than wt-mNBD1 (KDapp: F508del-mNBD1, 23.2 ± 1.7 nm; wt-mNBD1, 70.2 ± 4.7 nm; n = 3) Analysis of the effect of adenine nucleotides on the binding of NBD1 to Hsc70 To further explore the effect of adenine nucleotides on the NBD1–Hsc70 interaction, we quantified the effect of ATP and ADP on the binding of wt-mNBD1 and F508del-mNBD1 to immobilized Hsc70 As shown by the solid lines in Fig 5A,B, increasing concentrations of ATP caused a dramatic reduction in the binding of A B 0.5 50 0.2 0.1 0.05 Binding (pmol·nmol–1) NBD1 (µM) 0.02 0 1000 Time (s) F508del-m NBD1 (µM) 0.5 50 0.2 0.1 0.05 0.02 2000 1000 Time (s) 2000 D C 0.1 µM 0.2 µM F508del-m 102 Normalised binding Fig F508del increases the affinity of NBD1 for on-chip Hsc70 (A, B) The interaction of increasing concentrations of (A) wt-mNBD1 and (B) F508del-mNBD1 with on-chip Hsc70 (C) Dissociation of Hsc70-bound wt-mNBD1 and F508 del-mNBD1 (0.1 and 0.2 lM) shown on a highly expanded scale Binding was normalized independently for each dissociation curve to an initial maximum value (100) at the start of each dissociation phase (920 s) First-order fits ( ) to the data denote relative dissociation rates (D) The change in amount of NBD1 bound by on-chip Hsc70 at equilibrium with increasing concentrations of wt-mNBD1 (d) and F508del-mNBD1 (s; mean ± SEM; n = 3) Other details as in legend to Fig 100 wt-m F508del-m 100 98 wt-m wt-m 96 1000 1500 1000 1500 Time (s) FEBS Journal 276 (2009) 7097–7109 ª 2009 The Authors Journal compilation ª 2009 FEBS wt-m 100 Binding (pmol·nmol–1) Binding (pmol·nmol–1) 100 75 50 F508del-m 25 0.01 0.1 [NBD1] (µM) 1.0 7101 Interaction of Hsc70 with F508del-NBD1 wt-m ATP (µM) 20 23 53 103 203 10 0 500 Time (s) 30 C F508del-m ATP (µM) 20 23 53 103 203 503 10 1000 Binding (pmol·nmol–1) B 30 Binding (pmol·nmol–1) Binding (pmol·nmol–1) A T S Scott-Ward and M D Amaral 500 Time (s) 30 F508del-m 20 NADP wt-m 10 ADP ATP 10 100 1000 [Adenine nucleotide] (µM) 1000 Fig Adenine nucleotides reduce the binding of NBD1 to on-chip Hsc70 The interaction of (A) wt-mNBD1 (0.5 lM) and (B) F508delmNBD1 (0.5 lM) with on-chip Hsc70 in the presence of NADP (– –, 500 lM), MgADP ( , 500 lM) or increasing concentrations of MgATP (–) (C) The change in binding of wt-mNBD1 (d) and F508del-mNBD1 (s) to on-chip Hsc70 in the presence of NADP (D, ; 500 lM), MgADP (j,h; 500 lM), or increasing concentrations of MgATP (d,s) (mean ± SEM; n = 3) Other details as in the legends to Figs and wt-mNBD1 and F508del-mNBD1 (0.5 lm) to Hsc70 In contrast, the introduction of a control molecule, NADP (500 lm), did not significantly reduce the binding of either wt-mNBD1 or F508del-mNBD1 (0.5 lm) to immobilized Hsc70 (Fig 5C; wt-mNBD1, P = 0.45; F508del-mNBD1, P = 0.46; n = 3) Interestingly, the binding of wt-mNBD1 and F508del-mNBD1 to Hsc70 was also reduced by the addition of ADP (500 lm), although, at this concentration, the degree of inhibition was less than for ATP (P = 0.01; n = 3) Figure 5C, displaying a comparison of the summarized data for wt-mNBD1 and F508del-mNBD1, shows that the interaction of wt-mNBD1 with Hsc70 was most potently inhibited by increasing ATP concentration (IC50, 19.7 ± 2.1 lm; n = 3) For F508del-mNBD1, the ability of ATP to inhibit the Hsc70 interaction was 25 F508del-m (1.0 µM) I172 25 * V325 C4a 0 500 Time (s) 1000 50 Incubation Incubation + ATP * 25 0 40 [Compound] (µM) 80 n C4 V3 a 25 I172 Con V325 C4a C 50 Co F508del-m (1.0 µM) Small molecules recently identified in high-throughput screens have been proposed to affect CFTR-NBD1 conformation [25,26] Here, we tested by SPR whether and how these molecules affect the NBD1–Hsc70 interaction The data in Fig 6A show there was a significant reduction in the Hsc70 binding of F508del-mNBD1 (1 lm) upon acute application of C4a, which was not Co n C4 V3 a 25 B (IC50, 111 ± 4.8 lm; P = 0.001; our data suggest that the NBD1– inversely dependent on the ATP that F508del stabilizes this The effect of small molecule compounds on the NBD1–Hsc70 interaction Binding (pmol·nmol–1) 50 Binding (pmol·nmol–1) Binding (pmol·nmol–1) A significantly reduced n = 3) Collectively, Hsc70 interaction is concentration and interaction Fig C4a alters the binding of F508del-NBD1 to on-chip Hsc70 (A) Interaction of F508del-mNBD1 (1 lM) with on-chip Hsc70 in the absence ( , Con) and presence (50 lM) of: C4a (–), V325 ( ), or I172 (gray ) (B) Change in amount of F508del-mNBD1 bound by on-chip Hsc70 in the presence of I172 ( ; 50 lM ) or increasing concentrations of V325 (s) and C4a (d; mean ± SEM; n = 3) (C) Binding of F508del-mNBD1 to Hsc70 ( h; Con ) following incubation of NBD1 (1 lM) with C4a ( ) or V325 ( ; 50 lM ) for 30 at 16 °C in SPR buffer alone (Incubation) or in SPR buffer plus 100 lM MgATP (Incubation + ATP; mean ± SEM; n = 3) Other details as in the legend to Fig 7102 FEBS Journal 276 (2009) 7097–7109 ª 2009 The Authors Journal compilation ª 2009 FEBS T S Scott-Ward and M D Amaral observed for V325 or inhibitor CFTRinh-172 (I172; n = 3) We quantified the Hsc70 binding of F508del-mNBD1 in the presence of increasing concentrations of C4a and V325 (Fig 6B) The data show that only at the higher concentration tested (50 lm) could C4a, but not V325 or I172, significantly reduce the Hsc70 binding of F508del-mNBD1, by $ 30% (control, 32.2 ± 2.2 pmolỈnmol)1; C4a, 23.4 ± 1.9 pmolỈnmol)1; P = 0.04; n = 3) To better understand the mechanism of action of these correctors, we also preincubated F508del-NBD1 with either C4a or V325 (50 lm, 30 min, 16 °C) prior to application, and then quantified NBD1 binding to Hsc70 As shown in Fig 6C, again only C4a, and not V325, significantly reduced the binding of F508del-mNBD1 to Hsc70 (C4a, P = 0.04; V325, P = 0.58; n = 3) Comparison of the data with those in Fig 6B indicates that the magnitude of inhibition ($ 30%) was comparable to that observed with acute compound application However, following preincubation of NBD1 with C4a or V325 at increased MgATP concentration (106 lm), we observed no effect of these corrector compounds on the NBD1–Hsc70 interaction (Fig 6C; P = 0.74–0.95; n = 3) Discussion Various components of the ERQC recognize aberrant conformations of secretory proteins and target them for proteasomal degradation so as to avoid clogging of the secretory pathway In the case of the CFTR protein bearing F508del, the major CF-causing mutation, it has been shown that the molecular chaperone Hsc70 plays a major role in this disposal mechanism [11,12] In the present study, we have used an SPR approach to investigate how deleting F508 from NBD1 of mCFTR alters the interaction of the domain with the molecular chaperone Hsc70, and whether small molecule correctors of CFTR folding can affect this critical interaction Both wt-NBD1 and F508del-NBD1 bind Hsc70 Our data indicate that Hsc70 can interact with both wt-NBD1 and F508del-NBD1, in agreement with previous studies [12] showing that both wt-CFTR and F508del-CFTR can associate with this chaperone via NBD1 The strength of NBD1–Hsc70 binding determined here by SPR is substantially higher (low nanomolar KD) than the majority of previous non-SPR assessments of Hsc70 ⁄ Hsp70–substrate interactions (nanomolar to micromolar KD values [27–29]) This is likely to reflect the fact that we have used a potent Hsc70 substrate (isolated NBD1 of CFTR) Moreover, the conditions under which the experiments were performed favour Interaction of Hsc70 with F508del-NBD1 high-affinity interaction of Hsc70 with a client protein Nonetheless, although we could detect interaction of Hsc70 with Red-LA, this was not the case for LA In addition, previous measurements of the affinity of Hsc70–substrate binding have almost exclusively been the result of peptide, and not protein, binding by Hsp70 ⁄ Hsc70 [6–8,30] Previous studies have used SPR to quantify the binding of nonchaperone proteins (the cytoskeletal, PDZ-anchoring protein NHERF ⁄ EBP50 and the related, PDZ-based scaffold protein Shank2) to C-terminal CFTR peptides and, in one case (the Ca2+ ⁄ lipid-dependent annexin A5), isolated NBD1 Although the proteins studied are unrelated to Hsc70 ⁄ Hsp70, they were also found to bind CFTR fragments with high affinities (KD; C-terminus–EBP50, 22 nm; C-terminus–Shank2, 56 nm [31]; NBD1–annexin A5, nm [32]) However, in contrast to our findings, Trouve et al [32] found that ATP increased the binding of NBD1 to annexin A5, and that deleting Phe508 had no effect on the annexin A5–NBD1 interaction Interestingly, they demonstrated that CPX, a potential NBD1 small molecule ligand [33], inhibited binding Overall, the data suggest that Hsc70 interacts with NBD1 at alternative sites to annexin A5 and with different characteristics The enhanced binding of Hsc70 to wt-hNBD1 relative to wt-mNBD1 that we find here may be due to variations in amino acids between these forms of NBD1 that help to stabilize this domain against interaction with Hsc70 Interestingly, when some of the variant residues in NBD1 of F508del-mCFTR are substituted for residues at corresponding positions in F508del-hCFTR, they act as revertants of the folding and trafficking defects [14] This is the case for Thr539 (Ile539 in humans), a so-called revertant of F508delhCFTR [34], and also Ser429 (Phe429 in humans), recently shown to contribute to rescue of the trafficking defect of F508del-hCFTR [35] The presence of these ‘profolding’ residues in mNBD1 is a probable explanation for the recently reported attenuated processing ⁄ trafficking defect of F508del-mCFTR in comparison with F508del-hCFTR [36] F508del increases the affinity of CFTR NBD1 for Hsc70 Our data demonstrate that deleting Phe508 from mNBD1 increases five-fold the affinity of the domain for the Hsc70 chaperone However, our data also indicate that Hsc70 binds $ 30% more wt-mNBD1 than F508del-mNBD1 at saturation One explanation for this reduction is that the deletion of Phe508 increases FEBS Journal 276 (2009) 7097–7109 ª 2009 The Authors Journal compilation ª 2009 FEBS 7103 Interaction of Hsc70 with F508del-NBD1 T S Scott-Ward and M D Amaral the affinity of NBD1 for not only Hsc70 but also itself Indeed, as F508del-NBD1 is more prone to aggregation than wt-NBD1 [14,15], the aggregated, mutant form would thus be incapable of binding to Hsc70 This would effectively reduce the average concentration of the free monomeric form of F508del-mNBD1 available to bind Hsc70 and hence the maximum binding In a recent study, it was found that, in vivo, both wt-CFTR and F508del-CFTR bind equal amounts of the Hsp70 chaperone [37] Hence, the differences in binding caused by the F508del mutation observed here might be due to the in vitro nature of the SPR approach versus in vivo complexity Within the cell, additional factors influence Hsc70–CFTR binding, e.g cobinding and competition of Hsc70 cochaperones, such as the E3-ubiquitin ligase CHIP, which promotes the fast dissociation of the Hsc70–CFTR complex, targeting CFTR for proteasomal degradation [38–40] Moreover, some Hsc70 ⁄ Hsp70 interaction sites are likely to occur only in the context of the native conformation of the full-length protein [5], as proposed in a recent structural model of CFTR, where the surface of NBD1 containing Phe508 mediates an interdomain contact with intracellular cytoplasmic loop of membrane-spanning domain [16] Nevertheless, our findings are consistent with the reported prolonged association of Hsc70 with F508del-CFTR relative to that with wt-CFTR [11], which appears to constitute the first ERQC checkpoint occurring in vivo [9,12] Our data reported here thus suggest that the absence of Phe508 increases the accessibility of Hsc70 to one or more binding sites on NBD1 Binding sites on NBD1 promoting Hsc70 interaction A critical issue in this field is the location of the functionally important Hsc70-binding site(s) on NBD1; in particular, whether removal of Phe508 creates a novel Hsc70-binding site in NBD1 Although it is known that Hsc70 and Hsp70 bind short, hydrophobic peptide pockets exposed on substrate proteins, the exact primary sequence of these peptides is variable [6,30] Accordingly, the NBD1 proteins employed in this study (Thr389–Gly673) contain many of these short, hydrophobic sequences, including the region around Phe508, constituting potential Hsc70-binding sites Analysis of hNBD1 and mNBD1 sequences with the limbo program, which predicts likely binding sites of the Hsp70 ⁄ Hsc70 homologue, DnaK, identified three novel regions (Ser466–Leu472, Leu568–Pro574, and Asp614–Gln621), all three of which are distant from Phe508 Qu and Thomas [14] localized a putative 7104 Hsc70-binding site to Gly545–Ala561 [13], a hydrophobic pocket that is partially exposed in the crystal structure of NBD1 and that is also distant from Phe508 However, the residue limits of the NBD1 used in this study (Gly404–Ser589) were different from those used in the present study Nevertheless, even if no additional binding sites occur in F508del-mNDB1, they may be more accessible to Hsc70, in comparison with wt-mCFTR Supporting this notion, biochemical analyses of purified NBD1 indicate that F508del promotes aggregation, a property known to result from exposure of hydrophobic residues [14,15] Moreover, molecular dynamics modelling studies suggest that F508del-NBD1 exposes its hydrophobic interior to the solution more often than wt-NBD1 [16,17] Effect of ATP on the Hsc70–NBD1 interaction Adenine nucleotides are of critical importance to the binding of Hsc70 to CFTR [8] Consistently, our data show that ATP dramatically reduces the ability of mNBD1 to bind Hsc70 However, ATP can mediate this effect in two distinct ways First, the binding and hydrolysis of ATP at the nucleotide-binding domain of Hsc70 is known to accelerate its binding and release of substrates, reducing the affinity of their interaction Once ADP occupies the nucleotide-binding site of Hsc70, it converts the chaperone to a high-affinity binding form [8] Second, ATP, a native ligand of CFTR-NBD1, may bind to this domain, stabilizing its structure [14,41] and possibly hindering one (or more) Hsc70-binding site(s) Several lines of evidence argue that the effect of ATP on the NBD1–Hsc70 interaction observed here is mediated predominantly via NBD1 and not by Hsc70 First, although ATP reduced NBD1 binding to Hsc70, it did not alter the association–dissociation profile, suggesting that it did not alter the kinetics of the interaction Second, ADP, rather than enhancing NBD1–Hsc70 binding, as predicted for an Hsc70-mediated effect, substantially reduced binding Third, the IC50 values for ATP inhibition of wt-mNBD1 and F508del-mNBD1 binding to Hsc70 ($ 20 and $ 110 lm, respectively, this study) are comparable to the previously reported apparent dissociation constants for ATP and wt-hNBD1 and F508del-hNBD1 ($ 90 lm [42]) but substantially higher than the dissociation constant for ATP and Hsc70, which is in the order of $ 0.7 lm [43] Finally, ATP caused a reduction of only $ 20% in the Hsc70 binding of denatured lactalbumin, itself not predicted to interact with ATP, whereas Hsc70 binding to wt-mNBD1 was reduced by $ 80% Our data also FEBS Journal 276 (2009) 7097–7109 ª 2009 The Authors Journal compilation ª 2009 FEBS T S Scott-Ward and M D Amaral show that the ATP concentration required to inhibit the mNBD1–Hsc70 interaction is dramatically increased by deleting Phe508 Less optimal ATP binding to F508del-NBD1 would be consistent with the gating defect of F508del-CFTR, characterized by long interburst intervals [44], according to the current model of CFTR channel gating [45] However, regarding the isolated domain, ATP binds wt-NBD1 and F508del-NBD1 with equivalent affinity, arguing against such an effect However, it remains plausible that the F508del-induced exposure of Hsc70-binding sites on NBD1 diminishes the ability of ATP binding to stabilize this domain against its Hsc70 interaction Altogether, interpretation of these data would be consistent with ATP promoting ordered homodimerization of NBD1, a process that conceivably occludes the chaperone-binding site to reduce Hsc70 binding It has, in fact, been reported that wt-NBD1 can form homodimers under certain conditions [46,47] Thus, a reduced dimerization ability of F508del-NBD1 relative to wt-NBD1 would explain why higher ATP concentrations are required to inhibit its binding to Hsc70 Interaction of Hsc70 with F508del-NBD1 association In particular, our data show that ATP dramatically reduces the ability of mNBD1 to bind Hsc70, and indicate that it is mostly this ability of ATP to displace Hsc70 from NBD1 binding that is impaired by F508del C4a significantly inhibits the F508del-mNBD1 interaction with Hsp70, suggesting direct binding As this effect is abolished at high ATP concentrations, ATP and C4a may compete for the same F508del-mNBD1 binding site ⁄ surface We conclude that this SPR approach constitutes a useful assay for the determination of whether and how correctors affect the Hsc70– F508del-NBD1 interaction, which will improve our understanding of the mechanism of action of small molecules with therapeutic potential for CF, a critical step in bringing them to the clinical setting Future studies should focus on the quantification of the effect of these correctors on the NBD1–Hsc70 interaction in the presence of other relevant CFTR domains (e.g intracellular cytoplasmic loop and ⁄ or nucleotide-binding domain 2) at controlled MgATP concentrations Experimental procedures Effect of small molecules on the interaction of Hsc70 with F508del-NBD1 Here, we also used SPR to determine whether C4a and V325 have an effect on the NBD1–Hsc70 interaction Our data demonstrate that C4a did indeed reduce F508del-mNBD1 binding to Hsc70 This suggests that C4a binds directly to F508del-mNBD1, either at the same binding site, or by allosteric stabilization of NBD1, to influence its folding and thus decrease the affinity of the domain for the Hsc70 chaperone This finding is consistent with the results of recent in vivo studies on full-length F508del-hCFTR [20,26] However, it should be noted that substantially higher concentrations of C4a were required to affect the NBD1–Hsc70 interaction than those needed to correct cell surface expression of CFTR, although, in vivo, a more complex mechanism of action may occur than the simple disruption of an NBD1–Hsc70 complex A plausible explanation, nevertheless, is that C4a binds directly to a site on monomeric NBD1 that stabilizes homodimerization, similarly to the ATP effect (see above) This could likewise occlude the Hsc70-binding site(s) and thus reduce Hsc70 binding Strikingly, we observed a significantly reduced effect of C4a at high ATP concentrations Our data demonstrate that SPR provides a powerful approach to quantifying the Hsc70–NBD1 interaction and the impact of F508del (or other NBD1 mutations) and corrector compounds on this folding-sensitive Reagents All three forms of NBD1 used in this study (wt-hNBD1, wt-mNBD1, and F508del-mNBD1; Thr389–Gly673) were prepared using the same protocol as previously described [22] Briefly, the proteins were expressed in BL21 (DE3 strain) Escherichia coli from pSmt3 vectors, purified by nickel-affinity and size-exclusion chromatography, and stored at )80 °C [10 mgỈmL)1 NBD1, 20 mm Tris, 150 mm NaCl, mm MgCl2, mm ATP, mm b-mercaptoethanol, 12.5% (v ⁄ v) glycerol, pH 7.6] Postpurification, each preparation of NBD1 protein (wt-hNBD1, wt-mNBD1, and F508del-mNBD1) was analysed by intrinsic tryptophan fluorescence and CD spectroscopy to confirm that it carried a native fold (personal communication: data at http://www cftrfolding.org/reagentRequestshWT.asp) Because F508delhNBD1 CFTR is highly insoluble and prone to aggregation during purification [14], it was not available for use in this study Hence, wt-mNBD1 and F508del-mNBD1 were used to characterize the impact of the F508del mutation on CFTR–Hsc70 interactions Purified bovine Hsc70 protein (SPA-751) and monoclonal rat Ab against Hsc70 (SPA815) were obtained from Assay Designs (Ann Arbor, MI, USA) Monoclonal mouse Ab against NBD1 (L12B4) was obtained from Chemicon (Temecula, CA, USA) Biacore materials were obtained from GE Healthcare (Milwaukee, WI, USA) All other chemicals, proteins (BSA and bovine apo-a-lactalbumin) and reagents were purchased from Sigma Aldrich (St Louis, MO, USA) or BDH (Poole, UK), and were of research grade or higher (‡ 99% purity) FEBS Journal 276 (2009) 7097–7109 ª 2009 The Authors Journal compilation ª 2009 FEBS 7105 Interaction of Hsc70 with F508del-NBD1 T S Scott-Ward and M D Amaral Biochemical analysis of purified NBD1 and Hsc70 Data analysis Coimmunoprecipitation of purified NBD1 (1 lm, unless otherwise stated) and Hsc70 (1 lm) in SPR flow buffer [150 mm KCl, mm MgCl2, 0.1% (v ⁄ v) Triton X-100, 0.1% (v ⁄ v) dimethylsulfoxide, mm b-mercaptoethanol, 20 mm Hepes, pH 7.0] was performed using L12B4, as previously described [39] SDS ⁄ PAGE and western blot using SPA-815 (Hsc70) and L12B4 (NBD1) were also performed as previously described [39] Protein quantification was performed with a modified Lowry method All SPR sensograms were corrected for buffer-induced refractive index changes at an uncoated reference surface, analysed using biaevaluation software (biaeval; v 3.2; GE Healthcare), and displayed in sigmaplot (v 10; Systat, San Jose, CA, USA) as pmoles of interacting protein (e.g NBD1) bound per nmole of immobilized protein (e.g Hsc70) Molar concentrations of the proteins were calculated from their measured concentration (mgỈmL)1), using their molecular masses as determined from amino acid composition (wt-NBD1, 31 976 Da; F508del-NBD1, 31 829 Da; wt-hNBD1, 31 969 Da; bovine Hsc70, 71 241 Da) The kinetics of interaction (association, ka, and dissociation, kd, rates) were determined from each set of dose– response data by global fitting of the association and dissociation phases of all binding curves in that dataset (biaeval) The dissociation constant (KD) for each dose– response set was then determined (kd ⁄ ka), and the values were averaged (mean KD) To determine the apparent maximal binding (Bmaxapp, pmolỈnmol)1) and dissociation constant (KDapp, lm) of mNBD1 directly from the data (Eqn 1), the amount of mNBD1 bound at equilibrium (Beq, pmolỈnmol)1) was determined from kinetic analysis and plotted against [mNBD1] (sigmaplot) The [MgATP] required to inhibit binding by 50% [IC50, lm; Eqn (2)] was determined by plotting the amount of mNBD1 bound at 320 s against [MgATP] Spectroscopic analysis of NBD1 Wild-type and F508del-mNBD1 (1 lm) were diluted in NaCl ⁄ Pi (150 mm NaCl, 20 mm Na2PO4, mm b-mercaptoethanol, pH 7.4) and, following excitation at 295 nm, the emitted intrinsic tryptophan fluorescence was measured at 25 °C as previously described [15] The intensity of fluorescence emitted from NBD1 at 328 and 343 nm was corrected for background fluorescence from buffer at these two wavelengths SPR Interaction analyses were performed in SPR flow buffer at constant temperature (25 °C), using a Biacore 2000 system (GE Healthcare) as previously described [6] Ligand proteins (20 lgỈmL)1 in sodium acetate; 10 mm, pH 5.0) were covalently immobilized (‘on-chip’) on the surface of carboxymethyl-dextran (CM5) sensor chips, according to the manufacturer’s instructions (estimated final concentrations of immobilized proteins on sensor chip surface: 0.8–1.5 mm) The binding and dissociation of free analyte (‘applied’) proteins at a constant flow rate (30 lLỈmin)1, 180 lL) was then measured The surface of the chip was regenerated between sample applications with sequential injections of HCl (10 mm, 20 lL) and NaOH (10 mm, 20 lL) LA (20 mgỈmL)1) was reduced by incubating in SPR flow buffer containing dithiothreitol (20 mm, 60 min, °C) as previously described [48] RedLA was then diluted to 10 lm (145 lgỈmL)1) and applied immediately The effect of small molecules (C4a, V325, I172; 10 mm, 100% dimethylsulfoxide) was determined by diluting these compounds to different concentrations (as indicated in the figure legends) in flow buffer containing BSA (0.2 mgỈmL)1), and applying them either immediately (acute effect) or after 30 of incubation at 16 °C (incubated effect) The final dimethylsulfoxide concentration was adjusted to 0.5% Protein-coated CM5 chips were used for weeks, or until nonspecific binding increased (‡ 5%) All experiments were performed in parallel with an inactivated, or blank, flow cell not coated with protein 7106 Beq ¼ Bmax app ẵNBD1 KD app ỵ ẵNBD1 B ẳ Bmin ỵ Bmax Bmin ỵ 10IC50 ẵATPị 1ị ð2Þ where: Beq is binding at equilibrium; Bmaxapp and KDapp are the apparent maximum binding and dissociation constants, respectively; B is binding (at 320 s); Bmin and Bmax are minimum and maximum binding, respectively; and [NBD1] and [ATP] are the concentrations of NBD1 and ATP, respectively Statistical analysis Unless otherwise stated, data are presented as the mean ± standard error of the mean (SEM) (n ‡ 3) Mean data for NBD1 dose responses were calculated as follows: (a) global kinetic analysis of each dose–response set generated a kd and ka value for each [NBD1]; (b) these values were averaged; and (c) the averaged values were used to calculate a single KD (kd ⁄ ka) The mean KD was then determined by averaging KD values from repeat dose– response experiments (n = 3) For mean KDapp and Bmaxapp values, the KDapp and Bmaxapp were determined directly for each dose–response dataset as described, and the FEBS Journal 276 (2009) 7097–7109 ª 2009 The Authors Journal compilation ª 2009 FEBS T S Scott-Ward and M D Amaral values were averaged (n = 3) Owing to the small size of datasets (n = or n = 4), an unpaired, two-tailed t-test was used to determine whether two mean values were statistically different (sigmastat, v 3.5; Systat) Differences were considered to be statistically significant when the P-value (probability of the mean values being equal) was less than 0.05 (a = 0.05) In all cases, data were tested to ensure normality (Kolmogorov–Smirnov test) and equal variance (Levene median test) prior to applying the t-test Acknowledgements We are grateful to P Thomas (University of Texas South Western) and the CF Folding Consortium (http://www.cftrfolding.org) for generously providing the purified NBD1 proteins, to the Cystic Fibrosis Foundation 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Gadsby DC (2005) CFTR channel opening by ATP-driven tight dimerization of its nucleotide-binding domains Nature 433, 876–880 Interaction of Hsc70 with F508del-NBD1 46 Arispe N, Rojas E, Hartman J, Sorscher EJ & Pollard HB (1992) Intrinsic anion channel activity of the recombinant first nucleotide binding fold domain of the cystic fibrosis transmembrane regulator protein Proc Natl Acad Sci USA 89, 1539–1543 47 Howell LD, Borchardt R, Kole J, Kaz AM, Randak C & Cohn JA (2004) Protein kinase A regulates ATP hydrolysis and dimerization by a CFTR (cystic fibrosis transmembrane conductance regulator) domain Biochem J 378, 151–159 48 Dahlman JM, Margot KL, Ding L, Horwitz J & Posner M (2005) Zebrafish alpha-crystallins: protein structure and chaperone-like activity compared to their mammalian orthologs Mol Vis 11, 88–96 FEBS Journal 276 (2009) 7097–7109 ª 2009 The Authors Journal compilation ª 2009 FEBS 7109 ... that the F508del-induced exposure of Hsc70-binding sites on NBD1 diminishes the ability of ATP binding to stabilize this domain against its Hsc70 interaction Altogether, interpretation of these... to bind Hsc70 However, ATP can mediate this effect in two distinct ways First, the binding and hydrolysis of ATP at the nucleotide-binding domain of Hsc70 is known to accelerate its binding and... binding and release of substrates, reducing the affinity of their interaction Once ADP occupies the nucleotide-binding site of Hsc70, it converts the chaperone to a high -affinity binding form [8] Second,