Molecular Structure of the Human CFTR Ion Channel

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that uniquely functions as an ion channel. Here, we present a 3.9 Å structure of dephosphorylated human CFTR without nucleotides, determined by electron cryomicroscopy (cryo-EM). Close resemblance of this human CFTR structure to zebrafish CFTR under identical conditions reinforces its relevance for understanding CFTR function. The human CFTR structure reveals a previously unresolved helix belonging to the R domain docked inside the intracellular vestibule, precluding channel opening. By analyzing the sigmoid time course of CFTR current activation, we propose that PKA phosphorylation of the R domain is enabled by its infrequent spontaneous disengagement, which also explains residual ATPase and gating activity of dephosphorylated CFTR. From comparison with MRP1, a feature distinguishing CFTR from all other ABC transporters is the helix-loop transition in transmembrane helix 8, which likely forms the structural basis for CFTR's channel function.

Molecular Dynamics Flexible Fitting Simulations Identify New Models of the Closed State of the Cystic Fibrosis Transmembrane Conductance Regulator Protein

“…Indeed, such a conformation was recently reported for zebrafish and human CFTR. [15][16] Closure of the final model was not observed in the control simulations performed in the absence of ATP but without the map-derived constraints.…”

“…Backbone RMSD (Å) differences between reported CFTR models. Models [1, 2, 3 and 12], [4][5][6] and [13][14], [7][8][9], [10][11] and [15][16] 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 …”

“…[15][16]32 Efforts towards the development of such maps take advantage of impressive improvements in EM technology and the concomitant recording of medium to high resolution EM maps of CFTR and its homologues. [15][16][60][61] Moreover, recording maps in the presence and absence of known CFTR modulators could inform on their binding sites. A combination of MDFF and docking simulations could therefore be used to elucidate the binding modes of these modulators and to suggest ways to improve them, paving the way to structure-based drug design.…”

“…Yet in contrast with other ABC transporters, until very recently, a three-dimensional (3D) structure of any of CFTR's states was unavailable. This situation has changed with the publication of the 3D structure of zebrafish (Danio rerio) CFTR at a medium resolution of 3.7 Å (5UAR) from cryo electron microscopy (cryo-EM) 15 and with the subsequent release of the coordinates of the human protein (5UAK: 3.9 Å) 16 obtained with the same experimental technique. The two structures which are highly similar to one another feature an inward-facing conformation of the protein with the NBDs completely de-dimerized, which corresponds to its fully closed state.…”

Molecular dynamics of the cryo-EM CFTR structure

“…The structures of dephosphorylated zebrafish and human CFTR, which have been determined in the absence of ATP [15,16], exhibit a significant distance between the two NBDs and the two halves of the molecule show unexpected asymmetric features. In order to learn the role of these 3D features in CFTR structure and function, we assessed various properties of CFTR models and performed in-depth in silico analysis.…”