Biology subjects

Levring, J.

Publications and source records attributed to Levring, J..

4 recordsLinked to original sources

bioRxiv2026

Single-molecule dissection of CFTR folding defects and pharmacological rescue

Cystic fibrosis is a lethal genetic disorder caused by misfolding of the CFTR protein, most commonly due to the {Delta}F508 mutation. Despite extensive study, CFTRs folding process has remained inaccessible to direct observation. Here, we apply single-molecule magnetic tweezers to resolve the complete folding trajectories of wild-type and {Delta}F508 CFTR with near-amino acid resolution. We find that CFTR follows a hierarchical, template-guided folding pathway in which N-terminal domains scaffold downstream folding. This mechanism tightly couples the free energy states of intermediates, allowing {Delta}F508-induced instability to propagate across the folding pathway. Pharmacological correctors, in synergy with ATP, reshape the entire folding energy landscape by catalyzing transitions rather than simply stabilizing end states. These long-range, allosteric effects reveal a folding-embedded regulatory network. Our work provides a quantitative framework for mapping multidomain protein folding and therapeutic rescue, offering a broadly applicable strategy for interrogating rare mutations and accelerating structure-based drug discovery. Significance StatementMisfolding of CFTR underlies cystic fibrosis, and its complex, multidomain architecture makes it an ideal model for understanding how membrane proteins fold and how small molecules can restore native structure. Using single-molecule magnetic tweezers, we reveal how local instabilities propagate through CFTRs folding pathway and show that pharmacological correctors act by catalyzing specific folding transitions in addition to stabilizing the native fold. These insights establish CFTR as a paradigm for dissecting folding mechanisms in large membrane proteins and for developing general strategies to correct misfolding across diverse human diseases.

biophysics↗

bioRxiv2023

Structural basis for CFTR inhibition by CFTRinh-172

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that regulates electrolyte and fluid balance in epithelial tissues. Whereas activation of CFTR is vital to treating cystic fibrosis, selective inhibition of CFTR is a potential therapeutic strategy for secretory diarrhea and autosomal dominant polycystic kidney disease (ADPKD). Although several CFTR inhibitors have been developed by high-throughput screening, their modes of action remain elusive. In this study, we determined the structure of CFTR in complex with the inhibitor CFTRinh- 172 to 2.7 [A] resolution by cryogenic electron microscopy (cryo-EM). We observe that CFTRinh- 172 binds inside the pore near transmembrane helix 8 (TM8), a critical structural element that links ATP hydrolysis with channel gating. Binding of CFTRinh-172 stabilizes a conformation in which the chloride selectivity filter is collapsed and the pore is blocked from the extracellular side of the membrane. Single molecule fluorescence resonance energy transfer (smFRET) experiments indicate that CFTRinh-172 inhibits channel gating without compromising nucleotide-binding domain (NBD) dimerization. Together, these data show that CFTRinh-172 acts as both a pore blocker and a gating modulator, setting it apart from typical ion channel inhibitors. The dual functionality of CFTRinh-172 reconciles previous biophysical observations and provides a molecular basis for its activity. Significance statementThe pathogenesis of secretory diarrhea and autosomal dominant polycystic kidney disease involves hyperactivation of the CFTR ion channel. CFTR inhibitors, including the small-molecule CFTRinh-172, have been developed as therapeutic candidates to treat these diseases. This study offers a structural understanding of CFTRinh-172s mode of action, clarifying its dual inhibitory role as both a pore blocker and gating modulator. The molecular description of how CFTRinh-172 interacts with CFTR provides a structural foundation to its specificity and efficacy. Furthermore, the observation that CFTR inhibitors and potentiators both interact with TM8 strengthens the notion that this helix serves as an allosteric link between the ATPase site and the channel gate, and is therefore a hotspot for pharmacological modulation.

biophysics↗

bioRxiv2023

Structural identification of a selectivity filter in CFTR

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that regulates transepithelial salt and fluid homeostasis. CFTR dysfunction leads to reduced chloride secretion into the mucosal lining of epithelial tissues, thereby causing the inherited disease cystic fibrosis. Although several structures of CFTR are available, our understanding of the ion-conduction pathway is incomplete. In particular, the route that connects the cytosolic vestibule with the extracellular space has not been clearly defined, and the structure of the open pore remains elusive. Furthermore, although many residues have been implicated in altering the selectivity of CFTR, the structure of the "selectivity filter" has yet to be determined. In this study, we identify a chloride-binding site at the extracellular ends of transmembrane helices 1, 6, and 8, where a dehydrated chloride is coordinated by residues G103, R334, F337, T338, and Y914. Alterations to this site, consistent with its function as a selectivity filter, affect ion selectivity, conductance, and open channel block. The selectivity filter is accessible from the cytosol through a large inner vestibule and opens to the extracellular solvent through a narrow portal. The identification of a chloride-binding site at the intra- and extracellular bridging point leads us to propose a complete conductance path that permits dehydrated chloride ions to traverse the lipid bilayer. Significance statementCystic fibrosis is a fatal disease caused by inherited defects in the cftr gene. Understanding the structure and function of the CFTR protein is crucial for cystic fibrosis research. As an ion channel evolved from a family of ATP-driven active transporters, CFTR is structurally distinct from any other ion channel. This study describes the structure of CFTRs selectivity filter, which enables us to complete the molecular description of the CFTR pore. Moreover, it enriches our broader knowledge of ion channel physiology, with a particular focus on chloride permeation mechanisms.

biophysics↗

bioRxiv2023

Structure-based discovery of CFTR potentiators and inhibitors

The cystic fibrosis transmembrane conductance regulator (CFTR) is a crucial ion channel whose loss of function leads to cystic fibrosis, while its hyperactivation leads to secretory diarrhea. Small molecules that improve CFTR folding (correctors) or function (potentiators) are clinically available. However, the only potentiator, ivacaftor, has suboptimal pharmacokinetics and inhibitors have yet to be clinically developed. Here we combine molecular docking, electrophysiology, cryo-EM, and medicinal chemistry to identify novel CFTR modulators. We docked [~]155 million molecules into the potentiator site on CFTR, synthesized 53 test ligands, and used structure-based optimization to identify candidate modulators. This approach uncovered novel mid-nanomolar potentiators as well as inhibitors that bind to the same allosteric site. These molecules represent potential leads for the development of more effective drugs for cystic fibrosis and secretory diarrhea, demonstrating the feasibility of large-scale docking for ion channel drug discovery.

biophysics↗