Regulatory R region of the CFTR chloride channel is a dynamic integrator of phospho-dependent intra- and intermolecular interactions

Abstract

Intrinsically disordered proteins play crucial roles in regulatory processes and often function as protein interaction hubs. Here, we present a detailed characterization of a full-length disordered hub protein region involved in multiple dynamic complexes. We performed NMR, CD, and fluorescence binding studies on the nonphosphorylated and highly PKA-phosphorylated human cystic fibrosis transmembrane conductance regulator (CFTR) regulatory region, a ∼200-residue disordered segment involved in phosphorylation-dependent regulation of channel trafficking and gating. Our data provide evidence for dynamic, phosphorylation-dependent, multisite interactions of various segments of the regulatory region for its intra-and intermolecular partners, including the CFTR nucleotide binding domains 1 and 2, a 42-residue peptide from the C terminus of CFTR, the SLC26A3 sulphate transporter and antisigma factor antagonist (STAS) domain, and 14-3-3β. Because of its large number of binding partners, multivalent binding of individually weak sites facilitates rapid exchange between free and bound states to allow the regulatory region to engage with different partners and generate a graded or rheostat-like response to phosphorylation. Our results enrich the understanding of how disordered binding segments interact with multiple targets. We present structural models consistent with our data that illustrate this dynamic aspect of phospho-regulation of CFTR by the disordered regulatory region.protein interaction network | fuzzy complex

Molecular dynamics of the cryo-EM CFTR structure

“…The nonphosphorylated zfCFTR and hCFTR in the absence of ATP exhibit an inward-facing conformation with widely open NBDs. Some level of opening, even larger than that of transporters, is not unexpected, since the unphosphorylated R domain has been envisioned to exert its inhibitory role by intertwining between the NBDs [33]. However, this intertwining could also be realized in the case of the bottom-closed TM287/288-like conformation with partially opened domains (Supplementary Discussion and Figures S3 and S4).…”

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

“…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). As observed for CFTR, phosphorylated SUR NBDs bearing disease-causing mutations may also possess a different conformation from phosphorylated wild type NBDs.…”

“…Analysis of the homology model of SUR2B NBD1 indicates that phosphorylation affects residues that are up to ϳ40 Å away disordered proteins or disordered residues of a folded protein with multiple binding sites has been observed for many intrinsically disordered proteins (30,72).…”

An Electrostatic Interaction at the Tetrahelix Bundle Promotes Phosphorylation-dependent Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Opening

“…Phosphorylation of its regulatory (R) domain by a cyclic-dependent protein kinase (e.g. PKA) is also required for channel activity (4,5). ATP-induced dimerization of the NBDs appears to link ATP binding to subsequent conformational changes of the transmembrane domains (TMs) to promote channel opening.…”