Abstract
Intrinsically disordered proteins lack persistent folded structure but are abundant and critical for regulatory protein interactions. They represent a challenge to the structure/function paradigm, particularly when significant disorder remains on interaction with partners. Although many disordered protein interactions have been described, little is known about multiple interactions of hub proteins or dependence on posttranslational modifications of the interaction network from a biophysical perspective. Cystic fibrosis transmembrane conductance regulator (CFTR), the chloride channel mutated in cystic fibrosis, is controlled by a disordered, phosphorylated regulatory region. Our detailed characterizations of phosphorylation-dependent interactions of the regulatory region and structural models of its highly dynamic complexes provide direct insight into the basis of CFTR regulation and the general binding mechanisms of disordered hub proteins.
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.