Phenylalanine-508 mediates a cytoplasmic–membrane domain contact in the CFTR 3D structure crucial to assembly and channel function

Abstract

Deletion of phenylalanine-508 (Phe-508) from the N-terminal nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ATP-binding cassette (ABC) transporter family, disrupts both its folding and function and causes most cystic fibrosis. Most mutant nascent chains do not pass quality control in the ER, and those that do remain thermally unstable, only partially functional, and are rapidly endocytosed and degraded. Although the lack of the Phe-508 peptide backbone diminishes the NBD1 folding yield, the absence of the aromatic side chain is primarily responsible for defective CFTR assembly and channel gating. However, the site of interdomain contact by the side chain is unknown as is the high-resolution 3D structure of the complete protein. Here we present a 3D structure of CFTR, constructed by molecular modeling and supported biochemically, in which Phe-508 mediates a tertiary interaction between the surface of NBD1 and a cytoplasmic loop (CL4) in the C-terminal membrane-spanning domain (MSD2). This crucial cytoplasmic membrane interface, which is dynamically involved in regulation of channel gating, explains the known sensitivity of CFTR assembly to many disease-associated mutations in CL4 as well as NBD1 and provides a sharply focused target for small molecules to treat CF. In addition to identifying a key intramolecular site to be repaired therapeutically, our findings advance understanding of CFTR structure and function and provide a platform for focused biochemical studies of other features of this unique ABC ion channel.ABC transporter ͉ cystic fibrosis ͉ domain interactions ͉ modeling ͉ protein misfolding

Assembly and Misassembly of Cystic Fibrosis Transmembrane Conductance Regulator: Folding Defects Caused by Deletion of F508 Occur Before and After the Calnexin-dependent Association of Membrane Spanning Domain (MSD) 1 and MSD2

“…F508 of CFTR is located on the solvent-exposed surface of NBD1 (Lewis et al, 2004;Thibodeau et al, 2005), and the crystal structures of bacterial ABC transporters suggest that it makes contacts with cytosolic surface loops on MSD2 that are critical for stabilization of CFTR structure (Dawson and Locher, 2006;Serohijos et al, 2008). This supposition is supported by the observation that the F508 mutation disrupts assembly of CFTR 1-1172 (Cui et al, 2007) and makes similar CFTR fragments susceptible to cotranslational recognition by the ER-associated ubiquitin ligase RMA1 (Younger et al, 2006).…”

“…Structural studies of the related ABC transporter protein Sav1866 show that the transmembrane domains adopt a complex structure in which transmembrane domains from MSD1 cross-interact with transmembrane domains of MSD2 to form a twowinged pore structure (Dawson and Locher, 2006). A threedimensional structural model of CFTR, which is based on the Sav1866 structure, predicts that transmembrane (TM) helices 1 and 2 of CFTR pack next to TM helices 9, 10, 11, and 12 to make one wing of the pore, whereas TM helices 7 and 8 pack next to TM helices 3, 4, 5, and 6 to form the other wing of the pore (Serohijos et al, 2008). Because the CFTR glycosylation sites are found in the extracellular loop connecting TM 7 and 8, we propose that calnexin binds this segment of CFTR to assemble TM 7 and 8 into the proper wing of the pore (Figure 7).…”

“…Thus, we investigated the possibility that the ⌬F508 mutation causes similar defects in MSD2 stability. This is an important question because contact formation between F508 and intracellular loops exposed by MSD2 seems to be critical for CFTR assembly (Mendoza and Thomas, 2007;Serohijos et al, 2008). To determine whether the misfolding events caused by the deletion of F508 were similar to those caused by CAS-induced instability of CFTR, we coexpressed CFTR 1-837⌬F508 with CFTR 837-1480.…”

“…CFTR is a modular protein, and its domains can collapse to a protease-resistant conformation independently (Zhang et al, 1998). Yet, structures of related ABC transporter family members suggest that assembly of CFTR to an active conformation is a cooperative process that is dependent upon cross-contact formation between its N-and C-terminal membrane and cytosolic domains (Dawson and Locher, 2006;Mendoza and Thomas, 2007;Serohijos et al, 2008). Formation of a CFTR structure that can pass quality control occurs in cotranslational and posttranslational steps that are proposed to involve critical interactions between solvent-exposed surfaces of NBD1 and MSD2 and similar interactions between NBD2 and MSD1 (Serohijos et al, 2008).…”

“…Yet, structures of related ABC transporter family members suggest that assembly of CFTR to an active conformation is a cooperative process that is dependent upon cross-contact formation between its N-and C-terminal membrane and cytosolic domains (Dawson and Locher, 2006;Mendoza and Thomas, 2007;Serohijos et al, 2008). Formation of a CFTR structure that can pass quality control occurs in cotranslational and posttranslational steps that are proposed to involve critical interactions between solvent-exposed surfaces of NBD1 and MSD2 and similar interactions between NBD2 and MSD1 (Serohijos et al, 2008). A fragment of CFTR that contains MSD1, NBD1, the R-domain, and MSD2 (CFTR 1-1172) can fold to a conformation that can escape the ER quality control system and traffic to the plasma membrane in which it exhibits ATP-gated channel activity (Cui et al, 2007).…”