NMR evidence for differential phosphorylation-dependent interactions in WT and ΔF508 CFTR

Abstract

The most common cystic fibrosis (CF)-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of Phe508 (DF508) 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 DF508 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 DF508 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 DF508 defect.

Phosphorylation-dependent Changes in Nucleotide Binding, Conformation, and Dynamics of the First Nucleotide Binding Domain (NBD1) of the Sulfonylurea Receptor 2B (SUR2B)

“…CFTR is phosphorylated by PKA and PKC at multiple sites in the disordered R region, which links NBD1 to MSD2, and by PKA at one site in the regulatory insert located within NBD1 (85)(86)(87)(88). As with SUR2B, phosphorylation of CFTR disrupts transient interactions of the regulatory insert and R region with the NBD1 core (29,31) to promote a more open CFTR NBD1 conformation (31,89,90), which then permits binding of NBD1 with coupling helix 1 (31). Furthermore, as with SUR2B, phosphorylation of CFTR leads to increased MgATP binding and hydrolysis, and subsequent channel activation (28,48).…”

“…There are differences in the conformation of phosphorylated CFTR with the severe cystic fibrosis-causing mutation ⌬F508 (31, 91), which partly explain the molecular defects of mutant CFTR compared with the wild type protein (91)(92)(93)(94). Furthermore, deletion of the regulatory insert, which mimics phosphorylation (31), increases the response of mutant CFTR to small molecule correctors (91). Phosphorylation also disrupts interactions of the R region with NBD2, but it enhances interactions of the R region with the C terminus of CFTR and the accessory proteins STAS (sulfate transporters and antisigma factor) and 14-3-3 (30).…”

“…We have obtained assignments for 58 -63% of backbone HN resonances of the NBD1-containing proteins. The combination of TROSY-based triple resonance data and specifically labeled samples has allowed for the level of resonance assignment obtained for other NBDs (31 , and N-tail-Phosphorylation reactions were carried out at 30°C on purified samples of NBD1, NBD1-⌬C, and N-tail (ϳ200 M) in the NBD1 buffer with 15 mM MgATP rather than 5 mM MgATP. The phosphorylation reaction was initiated by the addition of 750 units (or 0.6 M) of the catalytic subunit of protein kinase A (PKA, Promega) and was monitored by recording a series of two-dimensional 1 H-15 N TROSY-HSQC spectra at 30°C.…”

“…Chemical shifts for each spectrum were referenced to 4,4-dimethyl-4-silapentane-1-sulfonic acid (35). Spectra were pro-cessed using NMRPipe/NMRDraw (36) and analyzed with NMRView (37 15 N-labeled only on specific amino acids (Gly, Leu, Ser, and Val) (31,41,42) or 14 N-labeled on specific amino acids (Ala, Arg, His, Lys, Met, and Thr) and 15 N-labeled at all other positions (43). Spectra of the specifically labeled NBD1-⌬N samples were recorded at 25°C at 600 MHz.…”

Cystic Fibrosis Transmembrane Conductance Regulator (ABCC7) Structure

“…These terminal sequences are likely disordered except when interacting with specific protein-binding partners. Similarly, the R domain is predominantly disordered (Ostedgaard et al 1997;Baker et al 2007; Kanelis et al 2010; Lewis et al 2010), at least in isolation from the other domains of CFTR, which has led to the proposal that it should be renamed the R region rather than the R domain. Low-resolution electron microscopy data on CFTR molecules with the R domain labeled with 1.8-nm immunogold spheres has identified a relatively well-defined location for some of its residues relative to the other domains in full-length CFTR (Zhang et al , 2010, as discussed further below.…”