Computational Studies Reveal Phosphorylation-dependent Changes in the Unstructured R Domain of CFTR

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent chloride channel that is mutated in cystic fibrosis, an inherited disease of high morbidity and mortality. The phosphorylation of its approximately 200 amino acid R domain by protein kinase A is obligatory for channel gating under normal conditions. The R domain contains more than ten PKA phosphorylation sites. No individual site is essential but phosphorylation of increasing numbers of sites enables progressively greater channel activity. In spite of numerous studies of the role of the R domain in CFTR regulation, its mechanism of action remains largely unknown. This is because neither its structure nor its interactions with other parts of CFTR have been completely elucidated. Studies have shown that the R domain lacks well-defined secondary structural elements and is an intrinsically disordered region of the channel protein. Here, we have analyzed the disorder pattern and employed computational methods to explore low-energy conformations of the R domain. The specific disorder and secondary structure patterns detected suggest the presence of molecular recognition elements (MoREs) that may mediate phosphorylation-regulated intra- and inter-domain interactions. Simulations were performed to generate an ensemble of accessible R domain conformations. Although the calculated structures may represent more compact conformers than occur in vivo, their secondary structure propensities are consistent with predictions and published experimental data. Equilibrium simulations of a mimic of a phosphorylated R domain showed that it exhibited an increased radius of gyration. In one possible interpretation of these findings, by changing its size, the globally unstructured R domain may act as an entropic spring to perturb the packing of membrane-spanning sequences that constitute the ion permeability pathway and thereby activate channel gating.

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

“…The unphosphorylated R domain inhibits channel opening by several reported mechanisms including disrupting NBD dimerization (10) and restricting the motions or flexibilities of the ICLs and TMs (11,12). The latter mechanism is supported by our earlier observation that PKA phosphorylation of the R domain markedly increases the channel activity of the NBD2 deletion construct, K978C/⌬1198-CFTR (12).…”

“…Site 1 is catalytically inactive but binds ATP tightly; thus, most cycles of channel opening and closing are temporally linked to ATP binding and subsequent hydrolysis at site 2 (6 -9). R domain phosphorylation controls channel gating by unknown mechanisms that may include regulating both NBD dimerization (10) and the conformations or flexibilities of the TMs (11,12).…”

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).…”

Cooperative Assembly and Misfolding of CFTR Domains In Vivo

“…The dispensable nature of the R domain C-terminal region in CFTR biogenesis is conceivably due to its intrinsically disordered structure and low-affinity intramolecular interactions, indicated by in vitro, in vivo, and in silico studies (Winter and Welsh, 1997;Ostedgaard et al, 2000b;Baker et al, 2007;Hegedus et al, 2008).…”