Deletion of Phe508 in the first nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator increases its affinity for the heat shock cognate 70 chaperone.
The primary cause of cystic fibrosis (CF), the most frequent fatal genetic disease in Caucasians, is deletion of phenylalanine at position 508 (F508del), located in the first nucleotide-binding domain (NBD1) of the CF transmembrane conductance regulator (CFTR) protein. F508del-CFTR is recognized by the endoplasmic reticulum quality control (ERQC), which targets it for proteasomal degradation, preventing this misfolded but partially functional Cl(-) channel from reaching the cell membrane. We recently proposed that the ERQC proceeds along several checkpoints, the first of which, utilizing the chaperone heat shock cognate 70 (Hsc70), is the major one directing F508del-CFTR for proteolysis. Therefore, a detailed characterization of the interaction occurring between F508del-CFTR and Hsc70 is critical to clarify the mechanism that senses misfolded F508del-CFTR in vivo. Here, we determined by surface plasmon resonance that: (a) F508del-murine (m)NBD1 binds Hsc70 with higher affinity (K(D), 2.6 nm) than wild-type (wt) mNBD1 (13.9 nm); (b) ATP and ADP dramatically reduce NBD1-Hsc70 binding; (c) the F508del mutation increases by approximately six-fold the ATP concentration required to inhibit the NBD1-Hsc70 interaction (IC(50); wt-mNBD1, 19.7 microm ATP); and (d) the small molecule CFTR corrector 4a (C4a), but not VRT-325 (V325; both rescuing F508del-CFTR traffic), significantly reduces F508del-mNBD1 binding to Hsc70, by approximately 30%. Altogether, these results provide a novel, robust quantitative characterization of Hsc70-NBD1 binding, bringing detailed insights into the molecular basis of CF. Moreover, we show how this surface plasmon resonance assay helps to elucidate the mechanism of action of small corrective molecules, demonstrating its potential to validate additional therapeutic compounds for CF.
Scott-Ward
Toby S
TS
Universidade de Lisboa, Faculdade de Ciências de Lisboa, BioFIG, Centre for Biodiversity, Functional and integrative Genomics, Portugal.
Amaral
Margarida D
MD
D000818:Animals; D001665:Binding Sites; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D050883:HSC70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D051379:Mice; D010649:Phenylalanine / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance
D050883:HSC70 Heat-Shock Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D010649:Phenylalanine