A functional R domain from cystic fibrosis transmembrane conductance regulator is predominantly unstructured in solution

Ostedgaard, Lynda S.; Baldursson, Ólafur; Vermeer, Daniel W.; Welsh, Michael J.; Robertson, Andrew D.

doi:10.1073/pnas.100588797

Cited by 112 publications

(126 citation statements)

References 51 publications

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“…Previously, circular dichroism spectra of the isolated R domain revealed that it was mostly unstructured and that phosphorylation didn't have any effect on its secondary structure (11). But the conformation of the entire protein was not yet examined.…”

Section: Discussionmentioning

confidence: 99%

“…PKA phosphorylation relieves this inhibition. On the other hand, it has been shown that exogenous phosphorylated R domain (either residues 590 -858, 645-834, or 708 -831) increases the open probability of a CFTR channel construct missing residues 708 -835 from the R domain (7,10,11).…”

mentioning

confidence: 99%

“…Even though the role of phosphorylation on CFTR activity has been widely studied, the only structural information regarding CFTR phosphorylation that has been obtained so far concerned soluble R domains (11,18). In the present study, we reconstituted active CFTR in proteoliposomes and analyzed the * The costs of publication of this article were defrayed in part by the payment of page charges.…”

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confidence: 99%

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Phosphorylation-induced Conformational Changes of Cystic Fibrosis Transmembrane Conductance Regulator Monitored by Attenuated Total Reflection-Fourier Transform IR Spectroscopy and Fluorescence Spectroscopy

Grimard

Ramjeesingh

et al. 2004

Journal of Biological Chemistry

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Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ABC protein superfamily. Phosphorylation of a regulatory domain of this protein is a prerequisite for activity. We analyzed the effect of protein kinase A (PKA) phosphorylation on the structure of purified and reconstituted CFTR protein. 1 H/ 2 H exchange monitored by attenuated total reflection Fourier transform IR spectroscopy demonstrates that CFTR is highly accessible to aqueous medium. Phosphorylation of the regulatory (R) domain by PKA further increases this accessibility. More specifically, fluorescence quenching of cytosolic tryptophan residues revealed that the accessibility of the cytoplasmic part of the protein is modified by phosphorylation. Moreover, the combination of polarized IR spectroscopy with 1 H/ 2 H exchange suggested an increase of the accessibility of the transmembrane domains of CFTR. This suggests that CFTR phosphorylation can induce a large conformational change that could correspond either to a displacement of the R domain or to long range conformational changes transmitted from the phosphorylation sites to the nucleotide binding domains and the transmembrane segments. Such structural changes may provide better access for the solutes to the nucleotide binding domains and the ion binding site.Cystic fibrosis is caused by a mutation in the membrane chloride channel CFTR 1 (1). CFTR is a member of the ABC superfamily. As the other members of this family, CFTR contains two transmembrane domains and two nucleotide binding domains (NBD) responsible for ATP hydrolysis. In addition to the common ABC structure, CFTR possesses an R domain (regulatory domain) that contains several consensus phosphorylation sites. Phosphorylation of this domain followed by ATP binding and hydrolysis by the NBDs is necessary to induce chloride permeability (2, 3). Yet, an alternative activation mode has been proposed recently, where glutamate induces chloride channel activity in the absence of PKA and ATP (4), but it still needs further investigation.CFTR contains 10 dibasic (R(R/K)X(S/T)) consensus sequences for PKA phosphorylation as well as several monobasic and low affinity sites, most of them located in the R domain (1). Although the overall sequence identity of CFTR R domains is low, the phosphorylation sites are remarkably conserved among species (5). It is believed that the R domain combines both inhibitory and stimulatory effects. Effectively, deletion of the residues 708 -835 from the R domain (6, 7), and even of the residues 760 -783 (8), generates constitutively active channels. Moreover, overexpression or addition of the unphosphorylated R domain, encompassing residues 590 -858, inhibits chloride transport (9). PKA phosphorylation relieves this inhibition. On the other hand, it has been shown that exogenous phosphorylated R domain (either residues 590 -858, 645-834, or 708 -831) increases the open probability of a CFTR channel construct missing residues 708 -835 from the R domain (7,10,11).Nevertheless, the effect of the ph...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Phosphorylation-induced Conformational Changes of Cystic Fibrosis Transmembrane Conductance Regulator Monitored by Attenuated Total Reflection-Fourier Transform IR Spectroscopy and Fluorescence Spectroscopy

Grimard

Ramjeesingh

et al. 2004

Journal of Biological Chemistry

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“…Another likely possibility is that the deletion of the F508 residue affects the interaction of the R domain with other regions of CFTR. The R domain is thought to be largely disordered (Ostedgaard et al, 2000), but it contains ordered segments of helical structure (Baker et al, 2007), and it has been shown to interact directly with both the NBD1 domain (Baker et al, 2007), and with the N-terminal tail of CFTR (Naren et al, 1999). Translation of the R-domain is also important in ensuring that N-terminal regions of CFTR achieve a compact folded structure that can no longer be recognized by the Hsp40 Hdj-2 (Meacham et al, 1999).…”

Section: Discussionmentioning

confidence: 99%

Assembly and Misassembly of Cystic Fibrosis Transmembrane Conductance Regulator: Folding Defects Caused by Deletion of F508 Occur Before and After the Calnexin-dependent Association of Membrane Spanning Domain (MSD) 1 and MSD2

Rosser

Grove

Chen

et al. 2008

MBoC

115

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Cystic fibrosis transmembrane conductance regulator (CFTR) is a polytopic membrane protein that functions as a Cl ؊ channel and consists of two membrane spanning domains (MSDs), two cytosolic nucleotide binding domains (NBDs), and a cytosolic regulatory domain. Cytosolic 70-kDa heat shock protein (Hsp70), and endoplasmic reticulum-localized calnexin are chaperones that facilitate CFTR biogenesis. Hsp70 functions in both the cotranslational folding and posttranslational degradation of CFTR. Yet, the mechanism for calnexin action in folding and quality control of CFTR is not clear. Investigation of this question revealed that calnexin is not essential for CFTR or CFTR⌬F508 degradation. We identified a dependence on calnexin for proper assembly of CFTR's membrane spanning domains. Interestingly, efficient folding of NBD2 was also found to be dependent upon calnexin binding to CFTR. Furthermore, we identified folding defects caused by deletion of F508 that occurred before and after the calnexin-dependent association of MSD1 and MSD2. Early folding defects are evident upon translation of the NBD1 and R-domain and are sensed by the RMA-1 ubiquitin ligase complex. INTRODUCTIONCystic fibrosis transmembrane conductance regulator (CFTR) is a membrane glycoprotein that is localized to the apical surface of epithelial cells that line ducts of glands and airways. CFTR functions as an ATP-gated Cl Ϫ channel that is critical for proper hydration of the mucosal layer that lines lung airways (Welsh and Smith, 1993). Individuals who inherit two mutant forms of CFTR have exceedingly viscous mucous and, due to chronic lung infections, develop cystic fibrosis and often die from lung failure. CFTR is a member of the ATP-binding cassette (ABC) transporter superfamily (Hyde et al., 1990) and is a 1480-amino acid protein that contains two membrane spanning domains (MSDs), MSD1 and MSD2; two cytosolic nucleotide binding domains (NBDs), NBD1 and NBD2; and a regulatory (R) domain (Riordan et al., 1989). The proper folding and assembly of CFTR subdomains in the endoplasmic reticulum (ER) is required for CFTR to engage the COPII machinery and be packaged into vesicles for transport to the plasma membrane (Kopito, 1999;Wang et al., 2004). The folding pathway of this complex polytopic membrane protein has been a topic of great interest, because misfolding results in premature recognition of CFTR by the ER quality control system (ERQC) and degradation by the ubiquitin proteasome system (Skach, 2000). In fact, the most common disease-causing mutation of CFTR, ⌬F508CFTR, results in almost complete degradation of the protein by the ERQC system, which gives rise to a loss of function phenotype and lung disease (Ward and Kopito, 1994).The assembly of CFTR into an ion channel is complicated because it requires the coordinated folding and assembly of its membrane and cytoplasmic domains into a functional unit (Du et al., 2005;Riordan, 2005;Cui et al., 2007). CFTR is a modular protein, and its domains can collapse to a protease-resistant conformation i...

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“…One model to explain this mode of regulation involves the binding of multiple phosphorylation sites, potentially with different affinities and effects, to multiple CFTRbinding surfaces, so that increased phosphorylation leads to increased channel activity 3 . Understanding the dynamic, multisite interactions involved in this model at the molecular level requires specific consideration of the R region as an intrinsically disordered protein segment.…”

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CFTR regulatory region interacts with NBD1 predominantly via multiple transient helices

Baker

Hudson

Kanelis

et al. 2007

Nat Struct Mol Biol

267

327

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The regulatory (R) region of the cystic fibrosis transmembrane conductance regulator (CFTR) is intrinsically disordered and must be phosphorylated at multiple sites for full CFTR channel activity, with no one specific phosphorylation site required. In addition, nucleotide binding and hydrolysis at the nucleotide-binding domains (NBDs) of CFTR are required for channel gating. We report NMR studies in the absence and presence of NBD1 that provide structural details for the isolated R region and its interaction with NBD1 at residue-level resolution. Several sites in the R region with measured fractional helical propensity mediate interactions with NBD1. Phosphorylation reduces the helicity of many R-region sites and reduces their NBD1 interactions. This evidence for a dynamic complex with NBD1 that transiently engages different sites of the R region suggests a structural explanation for the dependence of CFTR activity on multiple PKA phosphorylation sites.The CFTR chloride channel, the protein mutated in cystic fibrosis, is a member of the ATPbinding cassette (ABC) superfamily of proteins 1 . Like other members of the superfamily, CFTR has two membrane-spanning domains (MSD1 and MSD2) and two nucleotidebinding domains (NBD1 and NBD2). Intracellular regions between the transmembrane segments probably adopt helical structures that extend from the MSDs 2 . Unique to CFTR is the cytoplasmic intrinsically disordered 3,4 R region, of approximately 200 residues, which we refer to as a region rather than as a domain to reflect its lack of a stable, folded globular structure.

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A functional R domain from cystic fibrosis transmembrane conductance regulator is predominantly unstructured in solution

Cited by 112 publications

References 51 publications

Phosphorylation-induced Conformational Changes of Cystic Fibrosis Transmembrane Conductance Regulator Monitored by Attenuated Total Reflection-Fourier Transform IR Spectroscopy and Fluorescence Spectroscopy

Phosphorylation-induced Conformational Changes of Cystic Fibrosis Transmembrane Conductance Regulator Monitored by Attenuated Total Reflection-Fourier Transform IR Spectroscopy and Fluorescence Spectroscopy

Assembly and Misassembly of Cystic Fibrosis Transmembrane Conductance Regulator: Folding Defects Caused by Deletion of F508 Occur Before and After the Calnexin-dependent Association of Membrane Spanning Domain (MSD) 1 and MSD2

CFTR regulatory region interacts with NBD1 predominantly via multiple transient helices

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