Biology subjects

van der Sluijs, P.

Publications and source records attributed to van der Sluijs, P..

3 recordsLinked to original sources

bioRxiv2022

Redefining hypo- and hyper-responding phenotypes of CFTR mutants for understanding and therapy.

Mutations in CFTR cause misfolding and decreased or absent ion-channel function, resulting in the disease Cystic Fibrosis. Fortunately, a triple-modulator combination therapy (Trikafta) has been FDA approved for 178 mutations, including all patients who have F508del on one allele. That so many CFTR mutants respond well to modulators developed for a single mutation is due to the nature of the folding process of this multidomain protein. We have addressed the question What characterizes the exceptions: the mutants that functionally respond either not or extremely well. A functional response is the product of the number of CFTR molecules on the cell surface, open probability, and conductivity of the CFTR chloride channel. By combining biosynthetic radiolabeling with protease-susceptibility assays, we have followed CF-causing mutants during early and late stages of folding in presence and absence of modulators. Most CFTR mutants showed typical biochemical responses for each modulator, such as a TMD1 conformational change or an increase of (cell-surface) stability, regardless of a functional response. These modulators thus should still be considered for hypo-responder genotypes. Understanding both biochemical and functional phenotypes of outlier mutations will boost our insights into CFTR folding and misfolding, and lead to improved therapeutic strategies.

biochemistry↗

bioRxiv2022

ABC-transporter CFTR folds with high fidelity through a modular, stepwise pathway

The question how proteins fold is especially pointed for large multidomain, multispanning membrane proteins with complex topologies. We have uncovered the sequence of events that encompass proper folding of the ABC transporter CFTR in live cells, by combining kinetic radiolabeling with protease-susceptibility assays. We found that CFTR folds in two clearly distinct stages. The first, co-translational, stage involves folding of the 2 transmembrane domains TMD1 and TMD2, plus one nucleotide-binding domain, NBD1. The second stage is a simultaneous, post-translational increase in protease resistance for both TMDs and NBD2 caused by assembly of these domains onto NBD1. Our technology probes every 2-3 residues (on average) in CFTR. This in-depth analysis at amino-acid level allows detailed analysis of domain folding and importantly also the next level: the assembly of the domains to native, folded CFTR. Defects and changes brought about by medicines, chaperones or mutations also are amenable to analysis. We here show that the DXD motif in NBD1 that was identified to be required for export of CFTR from the ER turned out to be required for proper domain folding and assembly instead, upstream of transport. CFTR mutated in this motif phenocopies the misfolding and degradation of the well-known disease-causing mutant F508del that established cystic fibrosis as protein-folding disease. The highly modular process of domain folding and stepwise domain assembly explains the relatively high fidelity of folding and the importance of a step-wise folding process for such complex proteins.

biochemistry↗

bioRxiv2020

Co-translational folding of the first transmembrane domain of ABC-transporter CFTR is supported by assembly with the first cytosolic domain

ABC-transporters transport a wealth of molecules across membranes and consist of transmembrane and cytosolic domains. Their activity cycle involves a tightly regulated and concerted domain choreography. Regulation is driven by the cytosolic domains and function by the transmembrane domains. Folding of these polytopic multidomain proteins to their functional state is a challenge for cells, which is mitigated by co-translational and sequential events. We here reveal the first stages of co-translational domain folding and assembly of CFTR, the ABC-transporter defective in the most abundant rare inherited disease cystic fibrosis. We have combined biosynthetic radiolabeling with protease-susceptibility assays and domain-specific antibodies. The most N-terminal domain, TMD1 (transmembrane domain 1), folds both its hydrophobic and soluble helices during translation: the transmembrane helices pack tightly and the cytosolic N- and C-termini assemble with the first cytosolic helical loop ICL1, leaving only ICL2 exposed. This N-C-ICL1 assembly is strengthened by two independent events: i) assembly of ICL1 with the N-terminal subdomain of the next domain, cytosolic NBD1 (nucleotide-binding domain 1); and ii) in the presence of corrector drug VX-809, which rescues cell-surface expression of a range of disease-causing CFTR mutants. Both lead to increased shielding of the CFTR N-terminus, and their additivity implies different modes of action. Early assembly of NBD1 and TMD1 is essential for CFTR folding and positions both domains for the required assembly with TMD2. Altogether, we have gained insights into this first, nucleating, VX-809-enhanced domain-assembly event during and immediately after CFTR translation, involving structures conserved in type-I ABC exporters.

biochemistry↗