The intrinsically disordered nonphosphorylated and phosphorylated R region of CFTR and its interactions with NBD1 and SLC26A3 STAS have been characterized at residue-specific resolution, primarily using NMR. Limited chemical shift dispersion indicates that the R region is intrinsically disordered in solution and that no global folding event occurs upon phosphorylation. Chemical shifts of backbone nuclei and sidechain carbons were assigned. SSP values indicate that phosphorylation acts as a structural switch, with a reduction in helical propensity in multiple nonphosphorylated R region segments.
Free nonphosphorylated and phosphorylated R region were characterized using a variety of structural probes. Fast timescale motion indicates the presence of structural contacts in many R region segments. Hydrodynamic radii are intermediate to those expected for fully folded or denatured proteins, with the phosphorylated R region being slightly more compact. The nonphosphorylated R region was further characterized, including measurements of molecular dimensions, N-H bond vector orientation and inter-residue distances from 6 spin label sites. Using these parameters as input to the program ENSEMBLE enabled calculation of a representative pool of nonphosphorylated R region conformations, indicating the presence of transient contacts that could not be directly discerned from the input data.
Examining labeled R region with the addition of unlabeled NBD1 provided evidence that multiple segments of nonphosphorylated R region bind and are released from NBD1 with varying affinities in a highly dynamic equilibrium. Phosphorylation relieves these interactions, with the exception of limited R region interactions near S768 when NBD1 is ATP-bound. Largely similar nonphosphorylated R region residues bind both ATP-bound ΔF508 and wild-type NBD1. Addition of unlabeled R region to labeled ATP-bound NBD1 caused spectral changes indicative of a direct interaction with more than one surface or conformational changes within NBD1 that are transmitted from one binding surface to other surface(s). Binding of unlabeled SLC26A3 STAS domain to labeled phosphorylated R region was also monitored and indicated that similar R region segments bind NBD1 and STAS, suggesting a direct competition between these two domains for binding. A model is proposed where the R region acts as a regulatory hub, integrating interactions with a variety of partners to regulate channel function.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/19315 |
| Date | 05 March 2010 |
| Creators | Baker, Jennifer May Reta |
| Contributors | Forman-Kay, Julie Deborah |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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