NMR evidence for differential phosphorylation-dependent interactions in WT and ΔF508 CFTR
2009; Springer Nature; Volume: 29; Issue: 1 Linguagem: Inglês
10.1038/emboj.2009.329
ISSN1460-2075
AutoresVoula Kanelis, Rhea P. Hudson, Patrick H. Thibodeau, Philip Thomas, Julie D. Forman‐Kay,
Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoArticle19 November 2009free access NMR evidence for differential phosphorylation-dependent interactions in WT and ΔF508 CFTR Voula Kanelis Voula Kanelis Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario, CanadaPresent address: Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road N., Mississauga, Ontario, Canada L5L 1C6 Search for more papers by this author Rhea P Hudson Rhea P Hudson Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario, Canada Search for more papers by this author Patrick H Thibodeau Patrick H Thibodeau Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX, USAPresent address: Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, 3500 Terrace Street, Pittsburgh, PA 15261, USA Search for more papers by this author Philip J Thomas Philip J Thomas Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Julie D Forman-Kay Corresponding Author Julie D Forman-Kay Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario, Canada Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Voula Kanelis Voula Kanelis Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario, CanadaPresent address: Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road N., Mississauga, Ontario, Canada L5L 1C6 Search for more papers by this author Rhea P Hudson Rhea P Hudson Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario, Canada Search for more papers by this author Patrick H Thibodeau Patrick H Thibodeau Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX, USAPresent address: Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, 3500 Terrace Street, Pittsburgh, PA 15261, USA Search for more papers by this author Philip J Thomas Philip J Thomas Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Julie D Forman-Kay Corresponding Author Julie D Forman-Kay Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario, Canada Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Author Information Voula Kanelis1, Rhea P Hudson1, Patrick H Thibodeau2, Philip J Thomas2 and Julie D Forman-Kay 1,3 1Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario, Canada 2Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX, USA 3Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada *Corresponding author. Structural Biology & Biochemistry, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G1X8. Tel.: +1 416 813 5358; Fax: +1 416 813 5022; E-mail: [email protected] The EMBO Journal (2010)29:263-277https://doi.org/10.1038/emboj.2009.329 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The most common cystic fibrosis (CF)-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of Phe508 (ΔF508) in the first of two nucleotide-binding domains (NBDs). Nucleotide binding and hydrolysis at the NBDs and phosphorylation of the regulatory (R) region are required for gating of CFTR chloride channel activity. We report NMR studies of wild-type and ΔF508 murine CFTR NBD1 with the C-terminal regulatory extension (RE), which contains residues of the R region. Interactions of the wild-type NBD1 core with the phosphoregulatory regions, the regulatory insertion (RI) and RE, are disrupted upon phosphorylation, exposing a potential binding site for the first coupling helix of the N-terminal intracellular domain (ICD). Phosphorylation of ΔF508 NBD1 does not as effectively disrupt interactions with the phosphoregulatory regions, which, along with other structural differences, leads to decreased binding of the first coupling helix. These results provide a structural basis by which phosphorylation of CFTR may affect the channel gating of full-length CFTR and expand our understanding of the molecular basis of the ΔF508 defect. Introduction Cystic fibrosis (CF) is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) (Rommens et al, 1989), a 1480-residue multi-domain, integral membrane protein that functions as a chloride channel. CFTR belongs to the ABC transporter superfamily of proteins and consists of two repeats, each composed of a membrane-spanning domain (MSD) followed by a cytosolic nucleotide-binding domain (NBD) (Supplementary Figure 1). A large intrinsically disordered regulatory (R) region, unique to CFTR, is located between the first NBD and the second MSD. Intracellular domains (ICDs) extend beyond the transmembrane helices to link the MSDs with the NBDs. The ICDs are composed of residues between the transmembrane segments and residues N-terminal to the NBDs that extend the transmembrane helices into the cytoplasm. Connecting these long α-helical segments of the ICDs are short helical elements or coupling helices (Supplementary Figure 1a). Coupling helices 1 and 3 are located between the second and third transmembrane helices in MSD1 and MSD2, respectively. Coupling helices 2 and 4 are between the fourth and fifth transmembrane segments in MSD1 and MSD2, respectively. CFTR channel gating is regulated by ATP binding and hydrolysis at the NBDs, and phosphorylation by protein kinase A (PKA) and protein kinase C (PKC) at multiple Ser residues within the R region (Anderson et al, 1991; Cheng et al, 1991; Kartner et al, 1991; Tabcharani et al, 1991; Picciotto et al, 1992; Ma et al, 1996). During the gating cycle, two ATP molecules bind at the interface of a proposed NBD1/NBD2 heterodimer, as shown by crystal structures of homologous bacterial NBD homodimers (Smith et al, 2002) and crosslinking studies in CFTR (Mense et al, 2006). Conformational changes associated with the formation and disruption of the NBD dimer are suggested to be transmitted to the MSDs through the ICDs, allowing the channel to open and close (Wang et al, 2004; Ward et al, 2007). Currently, over 1500 mutations in the CFTR gene (http://www.genet.sickkids.on.ca/cftr) have been associated with CF with different symptoms and varying severities. The most common, and a severe CF-causing mutation, is deletion of Phe508 (ΔF508) located in the first nucleotide-binding domain (NBD1), which affects the folding (Du et al, 2005; Thibodeau et al, 2005), maturation (Cheng et al, 1990; Denning et al, 1992), gating (Hwang et al, 1994; Schultz et al, 1999; Cui et al, 2006), and cell-surface stability (Lukacs et al, 1993) of CFTR. Crystal structures of wild-type (WT) and mutant CFTR NBD1, including variants of ΔF508, have been solved (Lewis et al, 2004, 2005; Thibodeau et al, 2005). The structure of the NBD core, which consists of the α/β subdomain (that contains the ATP-binding site) and the ABC-specific α-helical and β-sheet subdomains, is similar to that of NBDs from other ABC transporters. NBD1 from CFTR also contains a unique regulatory insertion (RI) that has one site for PKA phosphorylation (Dahan et al, 2001). The RI links the first two strands of the β-sheet subdomain and is composed of two short α-helices separated by a disordered linker, most of which is not observed in the various crystal structures of CFTR NBD1 (Supplementary Figure 1b). The constructs that have been used for most of the crystallization studies (residues 389–673) include a C-terminal regulatory extension (RE) (Lewis et al, 2004) that extends beyond the C-terminus of NBD1, which is defined as residue 650 by sequence alignment with NBDs from bacterial and other eukaryotic ABC transporters (Gibson et al, 1991). By this definition, the RE comprises the first ∼25 residues of the R region (Supplementary Figure 1a). A recent crystal structure of the NBD1 that lacks the RI and extends only to Gly646 contains the entire canonical NBD fold (PDB code 2PZE). Recent NMR data from our laboratory indicate significant conformational flexibility of the C-terminus of this construct (Chong and Forman-Kay, unpublished results). Therefore, the R region in CFTR likely begins before Gly646, possibly as far back as Leu633, which was defined as the functional C-terminus of NBD1 (Chan et al, 2000). Thus, our construct of NBD1, which extends to 653, likely still contains some of the R region, although it lacks all phosphorylation sites of the R region of CFTR. Many of the crystallization studies to date have used murine CFTR NBD1 because of its increased solubility relative to the human protein (Lewis et al, 2004; Thibodeau et al, 2005). Solubilizing mutants of human NBD1 used for crystallization lead to partial correction of ΔF508 (Pissarra et al, 2008), making it difficult to elucidate the structural differences between true human WT and ΔF508 NBD1. Murine and human NBD1 share nearly 80% sequence identity. Further, there is strong conservation of PKA phosphorylation sites among CFTR proteins from mammalian species (Dahan et al, 2001) and conservation of domain–domain interactions in related ABC proteins even from bacteria (Dawson and Locher, 2006, 2007; Hollenstein et al, 2007; Ward et al, 2007; Aller et al, 2009). Although human NBD1 constructs can now be obtained with reasonable solubility and yield, they require deletions of the phosphoregulatory regions, notably the RI, thus precluding analysis of the structural basis of phosphorylation-dependent interactions. We have chosen to perform NMR studies on the natural murine WT and ΔF508 NBD1 sequences that do not contain any other substitutions or deletions of the phosphoregulatory regions. Notably, a recent study indicates that a reduction in open probability in human, mouse, and pig ΔF508 CFTR occurs by the same mechanism (Ostedgaard et al, 2007). Thus, the natural murine WT and ΔF508 NBD1 sequences can be used to elucidate the molecular basis of some regulatory features of CFTR. Our previous NMR work on the isolated human R region (Baker et al, 2007) showed a dynamic interaction between murine NBD1 and multiple R region segments with significant helical propensity. Phosphorylation by PKA at multiple Ser residues alters the conformational ensemble of the R region to reduce the helical propensity and disrupt interactions with NBD1. In this study, we describe NMR studies of regions of murine WT and ΔF508 CFTR that contain NBD1 (residues 389–653) and the RE (NBD1–RE; residues 389–673). We have made resonance assignments for WT NBD1–RE and probed changes in the conformation of the protein and its interactions with coupling helix 1 upon phosphorylation and deletion of Phe508, shedding light on the mechanisms underlying regulation of CFTR and its dysfunction in CF. Results NMR spectra of WT NBD1–RE The NMR spectrum of WT NBD1–RE that is bound to ATP is presented in Figure 1A and B, showing dispersion characteristics of a folded protein. Identical spectra of WT NBD1–RE recorded at different concentrations (0.10, 0.25, and 0.42 mM) or different times (over 7 days) indicate that there is no concentration-dependent aggregation, change in oligomeric state, or increase in disorder occurring under these conditions (data not shown). NMR resonances of WT NBD1–RE have different intensities, varying from sharp to broad, weak signals (Figure 1C). The sharp resonances, which are centred at ∼8.2 p.p.m. (Figure 1B), are due to the disordered segments of NBD1–RE, and the number of these peaks (∼20) suggests that significant segments of the protein are disordered in solution, including the RI and RE, with only transient sampling of ordered conformations. The significant broadening observed for some resonances is indicative of motion on the μs–ms timescale, as shown by elevated R2 relaxation rates (Supplementary Table 1) and different peak intensities at different magnetic field strengths, as broadening due to μs–ms dynamic processes is field dependent (Supplementary Figure 2). Spectra of WT NBD1–RE in the ATP-bound state recorded with varying concentrations of glycerol and at different temperatures and buffer conditions were very similar to the spectrum shown in Figure 1A (data not shown). Therefore, the variable peak intensity that is observed likely reflects the general motional properties of NBD1–RE rather than specific solution conditions. Figure 1.Conformational and dynamic changes in NBD1–RE with the ΔF508 mutation. (A) Comparison of 15N–1H correlation spectra for WT (400 μM) and ΔF508 (175 μM) NBD1–RE with 5 mM Mg2+ and 5 mM ATP in 20 mM Na+ phosphate, pH 7, 150 mM NaCl, 2% glycerol, 5 mM DTT, 10% (v/v) D2O at 20 °C at 600 MHz, with selected residues indicated. The spectrum of WT NBD1–RE is shown in the foreground with resonances of backbone nuclei, as well as those from side chain nuclei from Trp, Asn, and Gln residues, in black. The blue resonances are of opposite sign, caused by spectral aliasing, and are from side chain Arg NεHε correlations and one backbone NH correlation. The spectrum of ΔF508 NBD1-RE is shown in the background. Resonances coloured red and green in the ΔF508 NBD1–RE spectrum correspond to those coloured black and blue in the WT NBD1–RE spectrum, respectively. (B) Selected region of the spectrum in (A) with assigned residues in the RI and RE highlighted. The asterisks indicate sharp resonances that are overlapped from residues in the RI or RE and residues in the NBD core. (C) Selected regions of the spectra with peaks of different intensities. A trace through the approximate centre of the peak is shown at the bottom of each spectrum, illustrating the lineshape. (D) The combined chemical shift difference, Δδ(tot), from deletion of F508 is plotted as a function of residue number. The combined chemical shift difference is calculated with the equation ((ΔHp.p.m.)2+(ΔNp.p.m./5.3)2)1/2. The ΔNp.p.m. value was divided by 5.3 to account for the difference in digital resolution between proton and nitrogen dimensions in our spectra. Significant chemical shift changes defined as values higher than the average of all Δδ(tot) values plus 1 s.d. are shown by the red dashed line. Δδ(tot) values greater than the maximum value shown on the y axis reflect very large chemical shift changes that limit our ability to assign that residue in ΔF508 NBD1–RE. For situations in which there are multiple possibilities for the ΔF508 assignment, the closest peak was used to calculate the Δδ(tot) value. (E) A schematic ribbon diagram of the crystal structure of the WT NBD1–RE from murine CFTR (PDB code 1R0X) is shown. The ribbon is coloured blue for residues for which we have resonance assignments, light grey for those not assigned, and dark grey for those assigned in the G550E/R553M/R555K mutant but not transferable to WT NBD1–RE. The Cα atoms for residues that show chemical shift changes in ΔF508 NBD1–RE are shown as spheres coloured with a linear gradient from light pink (Δδ(tot)=0.04) to magenta (Δδ(tot) ⩾0.1), as indicated by the bar in the top right. The Cα atom of Phe508 is coloured yellow. All structure figures were created using MOLMOL (Koradi et al, 1996). Download figure Download PowerPoint Resonance assignment of G550E/R553M/R555K NBD1–RE The weak intensity of many of the resonances and the limited stability of the WT NBD1–RE NMR samples precluded resonance assignment. Therefore, we used a variant of NBD1–RE containing the revertant mutations, G550E (DeCarvalho et al, 2002), R553M (Teem et al, 1993), and R555K (Teem et al, 1996). These mutations increase the amount of soluble recombinantly expressed NBD1–RE and the solubility of the purified protein. The G550E/R553M/R555K mutant NBD1–RE could be concentrated to ∼600 μM and was stable for >20 days, allowing NMR data for backbone resonance assignment to be recorded. More resonances are present in the spectra of the G550E/R553M/R555K mutant compared with WT NBD1–RE (Supplementary Figure 3), pointing to less severe broadening than in the spectra of WT protein because of differences in motion on the μs–ms timescale. Although not as extensive as observed for the WT NBD1–RE, spectra of the G550E/R553M/R555K mutant also show broadening, with some of the weak resonances having elevated R2 rates from μs–ms timescale motion (Supplementary Table 1). Relaxation data recorded on 360 and 550 μM samples of the G550E/R553M/R555K mutant were very similar for most residues, indicating that the elevated R2 rates are not caused by sample aggregation at high concentrations (Supplementary Table 1). Many resonances are weak, especially in the spectra of the lower concentrated sample of the G550E/R553M/R555K mutant (i.e., Val562, Asp572, and Ser573), precluding reliable R2 values from being obtained for these residues. Importantly, for resonances observed for both the WT and G550E/R553M/R555K mutant forms of the protein, backbone chemical shifts are very similar (Supplementary Figure 3), allowing the straightforward transfer of assignments for most resonances. Using triple resonance experiments and specific labelling on Leu, the combination of Gly, Ser, Asp, and Asn residues, or aromatic residues, we have assigned 70% of the 1HN and 15N resonances in the G550E/R553M/R555K mutant and 60% of the 1HN and 15N resonances in WT NBD1–RE (Supplementary Figure 4a). Fewer assignments for the WT are due to chemical shift changes primarily for residues Glu543–Ala559 near the sites of mutations. Similarly, transfer of many assignments was possible to the ΔF508 NBD1–RE, to the WT NBD1 (lacking the RE), and to the phosphorylated states of WT and ΔF508 proteins. The more the changes in spectral positions, however, the fewer the assignments that could be transferred. The secondary structures of the G550E/R553M/R555K mutant, WT, and ΔF508 NBD1–RE were determined using 1HN and 15N chemical shifts, as well as 13Cα, 13Cβ, and 13CO chemical shifts where available (Supplementary Figure 5). As expected from the similarity of the NMR spectra, secondary structures of the G550E/R553M/R555K mutant, WT, and ΔF508 NBD1–RE proteins are very similar and largely agree with that of the crystal structures. The resonance assignments and different peak intensities give insights into the motional properties of the CFTR NBD1–RE. Consistent with the crystal structures of CFTR NBD1–RE (Lewis et al, 2004, 2005; Thibodeau et al, 2005), many of the sharp resonances located near 8.2 p.p.m. in the 1H dimension are from residues in the RE and the disordered linker of the RI (Figure 1A–C and Supplementary Table 1). Additional peaks in this region, which are not as intense as those from the disordered linker but are more intense than the average, are from residues in the H1c helix of the RI, implying increased mobility of this fragment (Supplementary Table 1). This observation is in contrast with the predominantly ordered structure of the H1b and H1c helices of the RI when bound to the NBD core that is observed in crystal structures of CFTR (Lewis et al, 2004, 2005; Thibodeau et al, 2005). Interestingly, the unassigned residues in the G550E/R553M/R555K mutant map to distinct regions on NBD1–RE (Supplementary Figure 4b). One set of residues involved includes those in the N-terminus of helix H1, the N-terminus of helix H6, the N-terminus of helix H7, the C-terminus of helix H8, and the N-terminus of helix H9. The N-termini of helices H1, H6, and H7 form part of the NBD1/NBD2 dimerization interface (Smith et al, 2002; Mense et al, 2006). The H1b helix of the RI and the H9b helix of the RE are also not assigned and, when bound to the NBD core, these elements form a nearly contiguous surface with helices H1 and H6. This surface changes when the RE adopts an alternate conformation and contacts only helices H6 and H7, as observed in the crystal structure of human NBD1–RE F508A (Lewis et al, 2004, 2005). Helix H4, which is located in the α-helical subdomain and is adjacent to helix H6, is also not assigned. The few broadened resonances that could be assigned and for which R2 rates could be extracted (Ser478, Ser490, Cys524, Val591, and Ser641) map to sites around the unassigned residues, implicating dynamic interactions of the RI and RE with multiple surfaces of the NBD core on the μs–ms timescale. In addition, the broadening of residues near the Walker A sequence in the triple resonance assignment experiments (data not shown) implicates motion at the ATP-binding site, possibly caused by exchange of ATP on and off of the binding site and/or the effects of dynamic interactions of the RI and RE at this site. Effect of the ΔF508 mutation on NBD1–RE The spectra of NBD1–RE for WT and ΔF508, containing no additional mutations, are very similar (Figure 1A and D), showing that WT and ΔF508 NBD1–RE adopt similar structures, as previously observed (Lewis et al, 2004, 2005). Exceptions include the loss of resonance for Phe508 in the mutant, as well as chemical shift changes for the backbone amides of Ile506, Ile507, and Val562–Ala566, all located in the α-helical subdomain and within 6 Å of Phe508 (Figure 1D and E). Resonances of Asn505 and Gly509–Ser511 are overlapped, masking any chemical shift changes. The sharp resonances observed in the spectrum of ΔF508 indicate that the RI and RE are also significantly disordered in ΔF508 NBD1–RE. Chemical shift changes are also observed for some residues that are not close to Phe508 (Figure 1D and E). These include residues at the interface between the α/β and α-helical subdomains (Val488, Cys491, and Gln493), residues located on the other side of the α-helical subdomain (Cys524, Asp529, Thr531, Lys532, Ala534, Glu535, Asp537 Asn538, Val540, and Val591), as well as the RI residue Gly437 and α/β subdomain residues Ile616, Gly622, and Arg647, which are 30–40 Å away from Phe508 in the various crystal structures (Lewis et al, 2004, 2005; Thibodeau et al, 2005). These chemical shift differences may result from conformational changes in ΔF508 compared with WT, including, but not limited to, changes in the relative orientation of the α-helical and α/β subdomains and/or different interactions of the RI and/or RE with the NBD core. Significant conformational changes, apart from differences in the local surface properties at the mutation site, were not observed in the crystal structures of ΔF508 NBD1–RE (also containing F429S, F494N, and Q637A mutations required for protein solubility and crystallization) (Lewis et al, 2004, 2005) and of ΔF508 NBD1 lacking the RI and the RE (PDB code 2PZF). Differential effect of phosphorylation of WT and ΔF508 NBD1–RE Our previous work on the isolated R region indicates that PKA phosphorylation decreases the helical propensity of many residues in the R region and disrupts binding of NBD1 to most segments of the full-length phospho-R region (Baker et al, 2007). To probe the structural basis of these differences from the perspective of NBD1, we have compared the spectra of phosphorylated (phospho)-WT NBD1–RE, phospho-WT NBD1 lacking the RE, and phospho-ΔF508 NBD1–RE with their non-phospho counterparts (Figures 2 and 3). Constructs of CFTR NBD1 lacking the RE (residues 389–653) are referred to as NBD1 to distinguish them from NBD1–RE. The murine WT and ΔF508 NBD1–RE contain four PKA phosphorylation sites, with three located in the RE at Ser659, Ser660, and Ser670 and one in the RI at Ser422, while NBD1 lacking the RE has only the single RI phosphorylation site. Human NBD1–RE does not have a phosphorylation site at position 659. Analysis of tryptic fragments using mass spectrometry indicated complete phosphorylation of Ser422 in the RI and Ser659 and Ser670 in the RE, with almost complete phosphorylation of Ser660 in the RE (Supplementary Figure 6). The NMR spectra of two different samples of phospho-WT NBD1–RE are very similar (data not shown), illustrating the reproducibility of our protocol. Figure 2.Phosphorylation of the RI and RE disrupts interactions with the NBD core. 15N–1H correlation spectra of (A) non-phospho-WT NBD1–RE (400 μM) and phospho-WT NBD1–RE (150 μM), (B) non-phospho-WT NBD1–RE (400 μM) and non-phospho-WT NBD1 (85 μM), and (C) non-phospho WT NBD1 (85 μM) and phospho-WT NBD1 (25 μM) are overlayed. The solution conditions for each sample are identical to those described in the legend to Figure 1. The spectrum of non-phospho-WT NBD1–RE is in the foreground in (A, B), with resonances coloured in black or blue as in Figure 1. The spectra of phospho-WT NBD1–RE and non-phospho-WT NBD1 are in the background in (A, B), respectively, with resonances coloured red and green as in Figure 1. Blue circles in (A, B) highlight chemical shift changes in non-phospho-WT NBD1–RE upon both phosphorylation and removal of the RE, respectively. The spectrum of non-phospho-WT NBD1 is shown in the foreground in black and blue in (C) whereas that of phospho-WT NBD1 is shown in the background in red and green. Light blue circles in (C) highlight the subset of resonances in non-phospho-WT NBD1 with chemical shift changes upon phosphorylation common to both phosphorylation of WT NBD1–RE and removal of the RE. The green squares highlight resonances in non-phospho-WT NBD1 that show chemical shift changes that are common to phosphorylation of WT NBD1–RE but which are not observed with removal of the RE, indicating that these chemical shift changes are specific to phosphorylation of the RI. Selected regions of the spectra in (A–C) are shown in (D–F), respectively. Phosphorylation of NBD1–RE (D) and NBD1 (F) results in an increased number of resonances at approximately 8.2 p.p.m. in the 1H dimension, as shown by the asterisks (*). (G) The combined chemical shift difference, Δδ(tot), is plotted as a function of residue. Δδ(tot) is plotted for non-phospho- and phospho-WT NBD1–RE (top), non-phospho-WT NBD1–RE, and non-phospho-WT NBD1 (middle), and non-phospho-WT NBD1 and phospho-WT NBD1 (bottom). The sparseness of the data in the bottom panel is because of the lack of definitive assignments for non-phospho-WT NBD1 that can be obtained by transferring resonance assignments from non-phospho-WT NBD1–RE. Download figure Download PowerPoint Figure 3.Differential effect of phosphorylation on WT NBD1–RE and ΔF508 NBD1–RE. (A) 15N–1H correlation spectra of non-phospho-ΔF508 NBD1–RE (175 μM) and phospho-ΔF508 NBD1–RE (125 μM) are overlayed. The spectrum of non-phospho-ΔF508 NBD1–RE is shown in the foreground in black and blue, whereas that of phospho-ΔF508 NBD1–RE is shown in the background in red and green. (B) The combined chemical shift difference, Δδ(tot), for non-phospho-ΔF508 NBD1–RE and phospho-ΔF508 NBD1–RE (top) and for non-phospho-WT NBD1–RE and phospho-WT NBD1–RE (bottom; repeated from Figure 2G for comparison) is plotted as a function of residue number. (C) 15N–1H correlation spectra of phospho-WT NBD1–RE (150 μM) and phospho-ΔF508 (125 μM) NBD1–RE are overlayed. The spectrum of phospho-WT NBD1–RE is shown in the foreground in black and blue, whereas that of phospho-ΔF508 NBD1–RE is in the background in red and green. Resonances that appear upon phosphorylation of WT NBD1–RE but not ΔF508 NBD1–RE are indicated by purple boxes. (D) Schematic ribbon diagram with Cα atoms for residues that show chemical shift differences between phospho-WT NBD1–RE and phospho-ΔF508 NBD1–RE shown as spheres coloured as in Figure 1E. The backbone is coloured by resonance assignment as in Figure 1E, with additional residues coloured in dark grey that have been assigned in WT NBD1–RE but cannot be transferred to ΔF508 NBD1–RE. The Cα atom of Phe508 is coloured yellow. Download figure Download PowerPoint Large differences between the spectra of phospho- and non-phospho-WT NBD1–RE (Figure 2A and D) result from the introduction of four phosphate groups that significantly change the chemical environment around Ser422, Ser659, Ser660, and Ser670, and neighbouring residues. Chemical shift changes are observed for Ser423 and Asp424 in the RI, and Thr604 and Tyr625 in the NBD core (Figure 2G, top), which are close in space to the phosphorylation sites in the crystal structures of murine NBD1–RE (Lewis et al, 2004). Other residues close to these Ser positions have not been assigned. Phosphorylation of NBD1–RE also results in an increase in the number of sharp resonances (∼15) centred around 8.2 p.p.m. in 1H chemical shift (Figure 2D), indicating a greater number of disordered residues or population of molecules with regions that sample disordered conformations. Furthermore, phosphorylation of WT NBD1–RE induces specific chemical shift changes that are also observed upon deletion of the RE (Figure 2A, B, D and E, blue circles), indicating that phosphorylation disrupts interactions of the RE with the NBD core. Many of the residues showing phosphorylation-dependent chemical shift changes (Supplementary Table 1) are in the proposed dimer interface (Figure 4C). Our current observations are consistent with our previous data on the isolated full-length R region (Baker et al, 2007). In addition, peak intensities are more uniform for phospho-WT NBD1–RE t
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