Crystallographic and single-particle analyses of native- and nucleotide-bound forms of the cystic fibrosis transmembrane conductance regulator (CFTR) protein

Abstract

Cystic fibrosis, one of the major human inherited diseases, is caused by defects in the CFTR (cystic fibrosis transmembrane conductance regulator), a cell-membrane protein. CFTR acts as a chloride channel which can be opened by ATP. Low-resolution structural studies of purified recombinant human CFTR are described in the present paper. Localization of the C-terminal decahistidine tag in CFTR was achieved by Ni 2+ -nitriloacetate nanogold labelling, followed by electron microscopy and single-particle analysis. The presence of the gold label appears to improve the single-particle-alignment procedure. Projection structures of CFTR from twodimensional crystals analysed by electron crystallography displayed two alternative conformational states in the presence of nucleotide and nanogold, but only one form of the protein was observed in the quiescent (nucleotide-free) state.The ABC (ATP-binding cassette) proteins are a superfamily of active transporter membrane proteins and are composed of approx. 50 functionally diverse classes of prokaryotic and eukaryotic transmembrane proteins [1]. Despite the differences in the type of substances that are transported by each protein, most members of the ABC proteins have a similar pattern of two NBDs (nucleotide-binding domains) and two TMDs (transmembrane domains) [2]. The CFTR (cystic fibrosis transmembrane conductance regulator), also termed ABCC7, is a unique member of the ABC superfamily of transporter proteins, which works as an ion channel and has an extra domain called the regulatory domain (R) located between NBD1 and TMD2 [3]. Channel activity is affected by phosphorylation (by protein kinases A and C) and by ATP. CFTR regulates secretion and reabsorption of ions at epithelial surfaces [4]. Defects in the channel function and/or folding and processing lead to cystic fibrosis [5].Structural data for many ABC transporter proteins suspected to be responsible for human diseases are mostly lacking. Structural studies of ABC proteins were successful in determining the high-resolution structures of MsbA (bacterial lipid transporter) [6,7] and BtuCD (bacterial vitamin B 12 transporter) [8] and the low-resolution structures

Insight in eukaryotic ABC transporter function by mutation analysis

“…In contrast, high-resolution structures for eukaryotic ABC transporters are not available. However, low-resolution structures for Pgp [266,267], CFTR [268] and the Kir6.2-SUR1 complex [269] allow an overall survey of the ABC transporter molecules and demonstrate structural homology. Furthermore, Pgp undergoes large structural rearrangements of the TM helices upon nucleotide binding reflecting coupling between ATP hydrolysis and drug binding [266,267].…”

NMR evidence for differential phosphorylation-dependent interactions in WT and ΔF508 CFTR

“…These interactions are via short helical segments, or coupling helices, located at the ends of the ICDs. Electron microscopy (EM) studies of human CFTR are consistent with similar interactions of the NBDs and the coupling helices in CFTR (Rosenberg et al, 2004;Awayn et al, 2005;Zhang et al, 2009). However, at 21-35 A ˚the EM data do not provide information regarding the specific residues involved in these interactions.…”

Dynamics Intrinsic to Cystic Fibrosis Transmembrane Conductance Regulator Function and Stability

“…Electron microscopy images obtained from single-particle analysis of noncrystalline as well as two-dimensional crystals of CFTR in a nucleotide-free state and a nucleotide-bound phosphorylated state lend some support to CFTR having inward-and outward-facing conformations (Rosenberg et al 2004;Awayn et al 2005;Zhang et al 2009). The images suggest that in the absence of nucleotide the helical bundles have a greater separation on the cytoplasmic side, whereas in the nucleotide-bound form the helical bundles are closer to the outward-facing model.…”