Obligate coupling of CFTR pore opening to tight nucleotide-binding domain dimerization.

Csaba Mihályi,Beáta Töröcsik,László Csanády

doi:10.7554/elife.18164

Abstract

In CFTR, the chloride channel mutated in cystic fibrosis (CF) patients, ATP-binding-induced dimerization of two cytosolic nucleotide binding domains (NBDs) opens the pore, and dimer disruption following ATP hydrolysis closes it. Spontaneous openings without ATP are rare in wild-type CFTR, but in certain CF mutants constitute the only gating mechanism, stimulated by ivacaftor, a clinically approved CFTR potentiator. The molecular motions underlying spontaneous gating are unclear. Here we correlate energetic coupling between residues across the dimer interface with spontaneous pore opening/closure in single CFTR channels. We show that spontaneous openings are also strictly coupled to NBD dimerization, which may therefore occur even without ATP. Coordinated NBD/pore movements are therefore intrinsic to CFTR: ATP alters the stability, but not the fundamental structural architecture, of open- and closed-pore conformations. This explains correlated effects of phosphorylation, mutations, and drugs on ATP-driven and spontaneous activity, providing insights for understanding CF mutation and drug mechanisms.

Highlights

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel which belongs to the superfamily of ATP binding cassette (ABC) proteins (Riordan et al, 1989)
The ion pore within the TMDs opens upon formation of a tight head-to-tail nucleotide binding domains (NBDs) dimer stabilized by two molecules of ATP occluded at the dimer interface (Vergani et al, 2005); each nucleotide is sandwiched between the conserved Walker A and B sequences of one NBD and the signature sequence of the other (Smith et al, 2002; Chen et al, 2003)
As evidence of such strict coupling between NBD and transmembrane domain (TMD) movements, a hydrogen bond between the side chains of NBD2 Walker-A threonine (T1246) and an arginine (R555) adjacent to the NBD1 signature sequence was shown to form in open, but not in closed, channels (Vergani et al, 2005). These two positions have co-evolved as a pair (Vergani et al, 2005), and in crystal structures of nucleotide-bound ABC NBD dimers the analogous side chains form a hydrogen bond, the arginine serving as hydrogen donor and the threonine/serine side chain as acceptor (Smith et al, 2002; Chen et al, 2003)

Summary

Introduction

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel which belongs to the superfamily of ATP binding cassette (ABC) proteins (Riordan et al, 1989). Pore closure accompanies NBD dimer disruption prompted by ATP hydrolysis at the composite interfacial site harbouring the Walker motifs of NBD2 (site 2) (Vergani et al, 2005): the other site (site 1) is catalytically inactive retaining ATP bound throughout multiple gating cycles (Aleksandrov et al, 2002; Basso et al, 2003) As evidence of such strict coupling between NBD and transmembrane domain (TMD) movements, a hydrogen bond between the side chains of NBD2 Walker-A threonine (T1246) and an arginine (R555) adjacent to the NBD1 signature sequence was shown to form in open, but not in closed, channels (Vergani et al, 2005).

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Conclusion