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.

Figure 1

R-region phosphorylation. (a) Schematic showing sites of R-region phosphorylation by PKA. (b) ¹H-¹⁵N HSQC NMR spectrum of the isolated human R region, comprising CFTR residues 654–838. Nonphosphorylated R region (black) and phosphorylated R region (red) are superimposed. Owing to the spectral width, the apparent chemical shift of the single tryptophan side chain peak is moved (aliased) to the top left corner (marked with blue asterisk). The true ¹⁵N chemical shift is 129.7 p.p.m. Apparent chemical shifts of arginine side chain resonances are also aliased and appear at lower right (dashed blue ellipse). More arginine side chain resonances were observed for the phosphorylated R region, probably because interactions between phosphates and arginine guanidinium groups led to reduced solvent exchange. A dashed blue rectangle marks glutamine and asparagine side chain resonances at upper right. (c) Calculated ¹H chemical shift difference between nonphosphorylated and phosphorylated R region resonances (nonphosphorylated minus phosphorylated). Red circles indicate phosphorylation sites.

Figure 2

Structural properties of the free R region. (a) SSP values calculated for the free phosphorylated or nonphosphorylated R region and averaged over a sliding window of 5 residues. Positive SSP values reflect the fractional helical structure for each residue in a weighted average of R-region conformations. Negative SSP values reflect the fractional β-strand structure. Red circles indicate phosphorylation sites. (b,c) R₂ relaxation rates for nonphosphorylated (b) and phosphorylated (c) R region. Error bars indicate propagation of errors calculated from the covariance matrix of the least-squares fit of R₁ and R_1ρ values. Black arrows indicate residues with decreased R₂ relaxation rates upon phosphorylation.

Figure 3

Interaction of the R region with NBD1. (a) Superposition of a selected portion of the ¹H-¹⁵N HSQC spectra of nonphosphorylated ¹⁵N,¹³C-labeled R region in the free state (black) and in the presence of ATP-bound NBD1 (red). Resolved peaks are labeled with their assignments. Minor chemical shift changes with NBD1 binding were readily tracked in HNCO spectra. (b) Slices at 115.36 p.p.m. (¹⁵N frequency) of the HNCO spectra of free and bound R region, showing the Ser790 peak, the only one that maintains intensity in this experiment upon addition of NBD1. (c) Slices at 115.07 p.p.m. (¹⁵N frequency) of the HNCO spectra, showing the Thr760 peak, which is weaker upon NBD1 binding, indicating interactions with NBD1. In b and c, a trace through the approximate center of the peak (dashed line represents slice through 3D data) is shown at the bottom of each spectrum, illustrating the lineshape.

Figure 4

Analysis of R-region interactions with NBD1. (a–d) Graphs plot ratios of ¹⁵N,¹³C-labeled R-region peak intensities with and without NBD1, from HNCO experiments. Shown are interactions of nonphosphorylated or phosphorylated R region with apo-NBD1 or ATP-bound NBD1. Red circles indicate phosphorylation sites. Bar colors reflect secondary structural properties of the R region in the absence of NBD1 (Fig. 2a). Stretches of at least three consecutive residues with SSP values above 5% (indicating fractional α-helical structure) or below –5% (indicating fractional β-strand structure) are colored blue or yellow, respectively, with the remaining residues colored gray. Error bars are derived from error propagation of the noise level of the spectrum. Bound peak intensities were calculated at the chemical shifts of the bound peak, as some peaks had small chemical shift changes upon interaction with NBD1. Absence of bar indicates that data could not be analyzed for that residue, except for some residues that had ratios of bound to free peak intensity equal to 0, including residues 765, 771 and 774 in a, and 763, 766, 771, 772 and 775 in c.

Figure 5

Schematic illustrating how phosphorylation-induced structural changes in the R region lead to a redistribution of binding equilibria with various regulatory interaction partners. R region is shown as red curve, with multiple helices reflecting the fractional helical structure in portions of the sequence. Gray ellipses, putative binding surfaces of interaction partners; green arrows, potential tertiary interactions within the R region; gold arrows, interactions with binding partners (multiple arrows represent dynamic exchange of both multiple R-region binding sites and multiple interaction interfaces, without implying involvement of specific R-region segments). Other components of CFTR are also shown, including membrane-spanning domains (MSDs), intracellular domains (ICDs), nucleotide-binding domains (NBDs) and cytoplasmic, helical N terminus (N tail). (a,b) Interactions favored by nonphosphorylated (a) and phosphorylated (b) R region. Upon phosphorylation, R region has less helical structure and reduced interactions with NBD1 and possibly NBD2, as well as within the R region; NBD1-NBD2 dimerization leads to channel opening.