CFTR regulatory region interacts with NBD1 predominantly via multiple transient helices

Abstract

The regulatory (R) region of the cystic fibrosis transmembrane conductance regulator (CFTR) is intrinsically disordered and must be phosphorylated at multiple sites for full CFTR channel activity, with no one specific phosphorylation site required. In addition, nucleotide binding and hydrolysis at the nucleotide-binding domains (NBDs) of CFTR are required for channel gating. We report NMR studies in the absence and presence of NBD1 that provide structural details for the isolated R region and its interaction with NBD1 at residue-level resolution. Several sites in the R region with measured fractional helical propensity mediate interactions with NBD1. Phosphorylation reduces the helicity of many R-region sites and reduces their NBD1 interactions. This evidence for a dynamic complex with NBD1 that transiently engages different sites of the R region suggests a structural explanation for the dependence of CFTR activity on multiple PKA phosphorylation sites.The CFTR chloride channel, the protein mutated in cystic fibrosis, is a member of the ATPbinding cassette (ABC) superfamily of proteins 1 . Like other members of the superfamily, CFTR has two membrane-spanning domains (MSD1 and MSD2) and two nucleotidebinding domains (NBD1 and NBD2). Intracellular regions between the transmembrane segments probably adopt helical structures that extend from the MSDs 2 . Unique to CFTR is the cytoplasmic intrinsically disordered 3,4 R region, of approximately 200 residues, which we refer to as a region rather than as a domain to reflect its lack of a stable, folded globular structure.

Assembly and Misassembly of Cystic Fibrosis Transmembrane Conductance Regulator: Folding Defects Caused by Deletion of F508 Occur Before and After the Calnexin-dependent Association of Membrane Spanning Domain (MSD) 1 and MSD2

“…Another likely possibility is that the deletion of the F508 residue affects the interaction of the R domain with other regions of CFTR. The R domain is thought to be largely disordered (Ostedgaard et al, 2000), but it contains ordered segments of helical structure (Baker et al, 2007), and it has been shown to interact directly with both the NBD1 domain (Baker et al, 2007), and with the N-terminal tail of CFTR (Naren et al, 1999). Translation of the R-domain is also important in ensuring that N-terminal regions of CFTR achieve a compact folded structure that can no longer be recognized by the Hsp40 Hdj-2 (Meacham et al, 1999).…”

“…Based on these data, we sought to pinpoint the first step at which the ⌬F508 mutation exerts its effect on CFTR folding, and we analyzed the stability of wild-type and mutant CFTR fragments by pulse chase ( Figure 5B). We first looked at the effect of deletion of F508 from a CFTR fragment consisting of amino acids 1-653, which stops at the NBD1 boundary before inclusion of the regulatory extension (Lewis et al, 2004;Baker et al, 2007). We found that CFTR 1-653 ⌬F508 accumulated to slightly lower levels than the wild-type fragment immediately after the labeling period (i.e., there was 27% less of 1-653 ⌬F508 in comparison with WT 1-653 at t ϭ 0).…”

“…However, the defect observed with CFTR 1-653⌬F508 did not seem to match the severity of the defect observed with the full-length protein (Ward and Kopito, 1994;Ward et al, 1995;Meacham et al, 2001) or with the steady-state levels of CFTR 1-837 ⌬F508 ( Figure 5A). Therefore, to identify other regions of CFTR that are affected by deletion of F508, we performed pulse-chase analysis on fragments containing the NBD1 plus the regulatory extension (RE) (CFTR 1-673) as well as a fragment containing the complete R domain (1-837) (Baker et al, 2007). First of all, we observed that as we included more of the R domain, the protein became more stable over the chase period (37% of 1-653 remained after 90 min, 53% of 1-673, and 71% of 1-837).…”

The CFTR Ion Channel: Gating, Regulation, and Anion Permeation

“…They observed that (1) split CFTR molecules could be cross-linked by introducing cysteines at the NBD dimer interface predicted from the crystal structures of other ABC transporters, and (2) PKA activation strongly enhanced cross-linking efficiency (confirmed by . Baker et al (2007) provided additional support for this type of mechanism in an NMR structural analysis of an R domain fragment. This fragment bound to recombinant NBD1 in solution with most of the contact points located at or near PKA sites.…”

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

“…Our goal was to generate a set of models of CFTR in the closed channel state that is consistent with geometric constraints and our experimental data and that illustrates the dynamic nature of R region interactions with the NBDs. In particular, we focused on three elements of R region, for which chemical shift data show the highest fluctuating helical propensity (23) and broadening data show significant interactions with both NBD1 and NBD2 (Fig. 1).…”