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Intermolecular Interaction Between R Domains of Cystic Fibrosis Transmembrane Conductance Regulator

Departments of Pediatrics and Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio

Address correspondence to: Pamela B. Davis, Department of Pediatrics, Case Western Reserve University at Rainbow Babies and Children's Hospital, Biomedical Research Bldg, Rm 831, 2109 Adelbert Road, Cleveland, OH 44106-6006. E-mail: pbd{at}po.cwru.edu

The function of the R domain of cystic fibrosis transmembrane conductance regulator (CFTR) has not yet been fully established. The cis-trans proline isomerase cyclophilin A stimulates channel activity, and stimulation depends on the presence of highly conserved prolines at positions 740, 750, and 759. When the prolines at these positions, which normally exist in the cis conformation, are locked into the trans conformation by mutation to alanine (the P3A mutant), the open probability of P3A is high and is not further increased by cyclophilin A. We speculated that one mechanism by which this could occur was by promoting CFTR dimerization, which has been shown to increase open probability, and that the P3A-CFTR might favor dimerization more strongly than the native sequence. To test the hypothesis that R–R interaction occurs and is stronger in the P3A-R mutants, we investigated R–R interactions. GST-R and StrepII-R proteins expressed in Escherichia coli could interact with R domain protein translated in vitro as well as with full-length CFTR. In similar assays, the P3A mutant of R domain also interacts with R domain and P3A-R. The P3A-R–P3A-R interaction is stronger than the R–R interaction, which corroborates our data from the channel study and supports our hypothesis. Studies of deletion constructs of the isolated R domain and of full-length CFTR localize the region of interaction to the C-terminal portion of R (after amino acid 708). Particularly, the last 22 a.a. residues (838–859) of R are essential for this binding. R–R interaction possibly plays a role in channel gating.

Abbreviations: amino acid, a.a. • ATP-binding cassette, ABC • circular dichroism, CD • cystic fibrosis transmembrane conductance regulator, CFTR • glutathione sulfo-transferase, GST • phosphate-buffered saline, PBS • polymerase chain reaction, PCR • protein kinase A (cAMP-dependent), PKA • open probability, Po • single chain Fv, scFv