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

Summary What are the molecular properties of proteins that fall on the radar of protein quality control (PQC)? Here we mutate the E. coli’s gene encoding dihydrofolate reductase (DHFR), and replace it with bacterial orthologous genes to determine how components of PQC modulate fitness effects of these genetic changes. We find that chaperonins GroEL/ES and protease Lon compete for binding to molten globule intermediate of DHFR, resulting in a peculiar symmetry in their action: Over-expression of GroEL/ES and deletion of Lon both restore growth of deleterious DHFR mutants and most of the slow-growing orthologous DHFR strains. Kinetic steady-state modeling predicts and experimentation verifies that mutations affect fitness by shifting the flux balance in cellular milieu between protein production, folding and degradation orchestrated by PQC through the interaction with folding intermediates.

SUMMARY The consistent observation across all kingdoms of life that highly abundant proteins evolve slowly demonstrates that cellular abundance is a key determinant of protein evolutionary rate. However, other empirical findings, such as the broad distribution of evolutionary rates, suggest that additional variables determine the rate of protein evolution. Here, we report that under the global selection against the cytotoxic effects of misfolded proteins, folding stability (ΔG), simultaneous with abundance, is a causal variable of evolutionary rate. Using both theoretical analysis and multiscale simulations, we demonstrate that the anticorrelation between the pre-mutation ΔG and the arising mutational effect (ΔΔG), purely biophysical in origin, is a necessary requirement for abundance–evolutionary rate covariation. Additionally, we predict and demonstrate in bacteria that the strength of abundance–evolutionary rate correlation depends on the divergence time separating reference genomes. Altogether, these results highlight the intrinsic role of protein biophysics in the emerging universal patterns of molecular evolution.

The cystic fibrosis transmembrane conductance regulator (CFTR) 3 , the mutation of which causes cystic fibrosis (CF), belongs to the superfamily of ATP-binding cassette (ABC) proteins but functions as an ion channel rather than an active transporter. The chloride channel activity is crucial for maintaining salt and fluid homeostasis in epithelial tissues (1). In patients with CFTR mutations that compromise its maturation or channel activity, the airway surface liquid volume is diminished, impeding mucociliary clearance (2, 3). The absence of functional CFTR also impairs submucosal gland secretion (4).Like many other ABC family proteins, CFTR (also known as ABCC7) contains two membrane-spanning domains (MSDs) and two nucleotide-binding domains (NBDs), with an additional unique R domain (Fig. 1). While many ABC proteins are multisubunit proteins composed of two identical NBDs and MSDs, CFTR is a single polypeptide containing two distinct NBDs and MSDs. The proper folding of the individual domains and the interactions between these domains during or after protein synthesis are essential for CFTR assembly, a process that is inefficient with the majority of CFTR being degraded at the endoplasmic reticulum by the proteasome (5, 6). The most prevalent CF-causing mutation is the deletion of a phenylalanine at position 508 (⌬F508). Recent studies suggest that the folding kinetics of NBD1 and the interdomain interactions between MSDs and the NBDs are disrupted by this mutation (7-9), although the crystal structures of isolated wild-type and mutant NBD1 show no major alteration in its overall threedimensional structure (10).Control of CFTR channel activity is modulated by the phosphorylation of the R domain by protein kinase A, which allows the regulation of gating by ATP binding at the NBD1/NBD2 interface. Stable binding of ATP at NBD1 and binding and hydrolysis of ATP at NBD2, together with R domain phosphorylation, may alter allosteric interactions between these domains and impact the channel gating cycle (11-13). Although considerable progress has been made toward understanding the integrated control of CFTR channel gating by phosphorylation and ATP binding/hydrolysis (12), details at the level of interactions of specific secondary and tertiary struc-