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.

Dynamics of Bacteriorhodopsin in the Dark-Adapted State from Solution Nuclear Magnetic Resonance Spectroscopy.

To achieve efficient proton pumping in the light-driven proton pump bacteriorhodopsin (bR), the protein must be tightly coupled to the retinal to rapidly convert retinal isomerization into protein structural rearrangements. Methyl group dynamics of bR embedded in lipid nanodiscs were determined in the dark-adapted state, and were found to be mostly well ordered at the cytosolic side. Methyl groups in the M145A mutant of bR, which displays only 10 % residual proton pumping activity, are less well ordered, suggesting a link between side-chain dynamics on the cytosolic side of the bR cavity and proton pumping activity. In addition, slow conformational exchange, attributed to low frequency motions of aromatic rings, was indirectly observed for residues on the extracellular side of the bR cavity. This may be related to reorganization of the water network. These observations provide a detailed picture of previously undescribed equilibrium dynamics on different time scales for ground-state bR.

Backbone and methyl assignment of bacteriorhodopsin incorporated into nanodiscs.

Resonance assignments are challenging for membrane proteins due to the size of the lipid/detergent-protein complex and the presence of line-broadening from conformational exchange. As a consequence, many correlations are missing in the triple-resonance NMR experiments typically used for assignments. Herein, we present an approach in which correlations from these solution-state NMR experiments are supplemented by data from 13C unlabeling, single-amino acid type labeling, 4D NOESY data and proximity of moieties to lipids or water in combination with a structure of the protein. These additional data are used to edit the expected peaklists for the automated assignment protocol FLYA, a module of the program package CYANA. We demonstrate application of the protocol to the 262-residue proton pump from archaeal bacteriorhodopsin (bR) in lipid nanodiscs. The lipid-protein assembly is characterized by an overall correlation time of 44 ns. The protocol yielded assignments for 62% of all backbone (H, N, Cα, Cß, C') resonances of bR, corresponding to 74% of all observed backbone spin systems, and 60% of the Ala, Met, Ile (δ1), Leu and Val methyl groups, thus enabling to assign a large fraction of the protein without mutagenesis data. Most missing resonances stem from the extracellular half, likely due intermediate exchange line-broadening. Further analysis revealed that missing information of the amino acid type of the preceding residue is the largest problem, and that 4D NOESY experiments are particularly helpful to compensate for that information loss.