Structural changes of CFTR R region upon phosphorylation: a plastic platform for intramolecular and intermolecular interactions

Abstract

Chloride channel gating and trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) are regulated by phosphorylation. Intrinsically disordered segments of the protein are responsible for phospho-regulation, particularly the regulatory (R) region that is a target for several kinases and phosphatases. The R region remains disordered following phosphorylation, with different phosphorylation states sampling various conformations. Recent studies have demonstrated the crucial role that intramolecular and intermolecular interactions of the R region play in CFTR regulation. Different partners compete for the same binding segment, with the R region containing multiple overlapping binding elements. The non-phosphorylated R region interacts with the nucleotide binding domains and inhibits channel activity by blocking heterodimerization. Phosphorylation shifts the equilibrium such that the R region is excluded from the dimer interface, facilitating gating and processing by stimulating R region interactions with other domains and proteins. The dynamic conformational sampling and transient binding of the R region to multiple partners enables complex control of CFTR channel activity and trafficking.

Keywords: IDP; binding; disordered; hub; post-translational modification; regulation.

Figure 1

Key elements of CFTR R region in (A) the non‐phosphorylated and (B) the phosphorylated states. Bar graphs represent the secondary structure propensity (SSP). Positive values correspond to helical structure sampling, while negative values refer to extended conformations. Horizontal bars represent R region binding segments to NBD1 and 14‐3‐3 10. The lengths of 14‐3‐3 interaction segments are set to six residues to match mode I binding to 14‐3‐3. Kinase phosphorylation sites of PKA (star), PKC (circle) and AMPK (triangle) are marked. Both the secondary structure and binding experiments were carried out on non‐phosphorylated (open symbols) and PKA‐phosphorylated (solid symbols) isolated R region (654–838).

Figure 2

Heterogeneity of CFTR R region:NBD1 interactions. N‐terminus of the R region (red, residues 638–671) in different NBD1 crystal structures (A, 1R0X; B, 1XMI) oriented to show the NBD dimerization interface 15. The core NBD1 (surface representation) is colored as a gold gradient reflecting the proximity to atoms in the other NBD1 molecule within the NBD1:NBD1 homodimer structure (2PZE). The flexible regulatory insertion (RI) of NBD1 that is in different conformations in the two structures is shown as gray helices and is not part of the surface representation.

Figure 3

Schematic models of (A) non‐phosphorylated/closed channel and (B) PKA‐phosphorylated/open channel states of CFTR. Shown are the N‐terminal segment (blue), membrane‐spanning domains (dark green), the intracellular domains (yellow‐green), NBD1 (yellow), NBD2 (gold), R region (red) and the C‐terminal segment (purple). Disordered elements (N‐terminus, R region and C‐terminus) are shown as a superposition of multiple possible conformations. Helix 9 of NBD1 (residues 635–643, yellow) and the C‐terminal elbow helix (residues 842–855, green) are also not in a fixed position to demonstrate that they can sample multiple conformations and may be considered part of the R region. The dashed line (in B) schematically illustrates the open channel pore, the membrane is represented as a gray bar and the R region sampled space is colored as a gradient (red of various intensities). PKA phosphorylation sites are not marked. R region sampling in the two phosphorylation states is similar, although the phosphorylated R region samples space farther from the core of CFTR as it is excluded from the NBD interface.