Side chain and backbone contributions of Phe508 to CFTR folding

Department of Physiology, The University of Texas Southwestern Medical Center at Dallas, 75390 USA.
Nature Structural & Molecular Biology (Impact Factor: 13.31). 02/2005; 12(1):10-6. DOI: 10.1038/nsmb881
Source: PubMed


Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane protein, cause cystic fibrosis (CF). The most common CF-causing mutant, deletion of Phe508, fails to properly fold. To elucidate the role Phe508 plays in the folding of CFTR, missense mutations at this position were generated. Only one missense mutation had a pronounced effect on the stability and folding of the isolated domain in vitro. In contrast, many substitutions, including those of charged and bulky residues, disrupted folding of full-length CFTR in cells. Structures of two mutant nucleotide-binding domains (NBDs) reveal only local alterations of the surface near position 508. These results suggest that the peptide backbone plays a role in the proper folding of the domain, whereas the side chain plays a role in defining a surface of NBD1 that potentially interacts with other domains during the maturation of intact CFTR.

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    • "ThesechangesareaccompaniedwithaICL4-NBD1andICL2-NBD2 closecontacts(seeSupplementaryTable3),andaconsequent variationoftheinteractionenergyoftheseregions.Asexpected, thereisasubstantialincreaseofthevanderWaalscomponentof theICL4-NBD1interactionenergyduetothelackoftheresidue F508,thatcontributestochangethesurfaceoftheNBDsgrove interactingwithICL4[9] [25] [31]. "
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    ABSTRACT: The Cystic Fibrosis Transmembrane Regulator (CFTR) is a membrane protein whose mutations causes cystic fibrosis, a lethal genetic disease. We performed a molecular dynamic (MD) study of the properties of the nucleotide binding domains (NBD) whose conformational changes, upon ATP binding, are the direct responsible of the gating mechanisms of CFTR. This study was done for the wild type (WT) CFTR and for the two most common mutations, ΔF508, that produces a traffic defect of the protein, and the mutation G551D, that causes a gating defect on CFTR. Using an homology model of the open channel conformation of the CFTR we thus introduced the mutations to the structure. Although the overall structures of the G551D and ΔF508 are quite well conserved, the NBD1-NBD2 interactions are severely modified in both mutants. NBD1 and NBD2 are indeed destabilized with a higher internal energy (Ei) in the ΔF508-CFTR. Differently, Ei does not change in the NBDs of G551D but, while the number of close contacts between NBD1 and NBD2 in ΔF508 is increased, a significant reduction of close contacts is found in the G551D mutated form. Hydrogen bonds formation between NBDs of the two mutated forms is also altered and it is slightly increased for the ΔF508, while are severely reduced in G551D. A consequent modification of the NBDs-ICLs interactions between residues involved in the transduction of the ATP binding and the channel gating is also registered. Indeed, while a major interaction is noticed between NBDs interface and ICL2 and ICL4 in the WT, this interaction is somehow altered in both mutated forms plausibly with effect on channel gating. Thus, single point mutations of the CFTR protein can reasonably results in channel gating defects due to alteration of the interaction mechanisms between the NBDs and NBDs-ICLs interfaces upon ATP-binding process.
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    • "[30] Expression and purification followed procedures previously described for the NBDs from CFTR. [31], [32] E coli BL21 (DE3) cells were transformed with the expression plasmids and single colonies were picked to grow an inoculum overnight at 37°C. A one liter expression culture of either the wildtype or mutant NBD2 was inoculated and the cultures were grown at 37°C until an OD600 of ∼1.0 in LB broth. "
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    ABSTRACT: Mutations in the ABCC6 ABC-transporter are causative of pseudoxanthoma elasticum (PXE). The loss of functional ABCC6 protein in the basolateral membrane of the kidney and liver is putatively associated with altered secretion of a circulatory factor. As a result, systemic changes in elastic tissues are caused by progressive mineralization and degradation of elastic fibers. Premature arteriosclerosis, loss of skin and vascular tone, and a progressive loss of vision result from this ectopic mineralization. However, the identity of the circulatory factor and the specific role of ABCC6 in disease pathophysiology are not known. Though recessive loss-of-function alleles are associated with alterations in ABCC6 expression and function, the molecular pathologies associated with the majority of PXE-causing mutations are also not known. Sequence analysis of orthologous ABCC6 proteins indicates the C-terminal sequences are highly conserved and share high similarity to the PDZ sequences found in other ABCC subfamily members. Genetic testing of PXE patients suggests that at least one disease-causing mutation is located in a PDZ-like sequence at the extreme C-terminus of the ABCC6 protein. To evaluate the role of this C-terminal sequence in the biosynthesis and trafficking of ABCC6, a series of mutations were utilized to probe changes in ABCC6 biosynthesis, membrane stability and turnover. Removal of this PDZ-like sequence resulted in decreased steady-state ABCC6 levels, decreased cell surface expression and stability, and mislocalization of the ABCC6 protein in polarized cells. These data suggest that the conserved, PDZ-like sequence promotes the proper biosynthesis and trafficking of the ABCC6 protein.
    Full-text · Article · May 2014 · PLoS ONE
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    • "As NBD1 is the site of the dominant F508del mutation, this domain has been the subject of intensive structural, dynamic, and thermodynamic analyses. Crystal structures of NBD1 show that it is divided into the three basic regions seen in nucleotide-binding domains of other ABC transporters (Lewis et al. 2004, 2005; Thibodeau et al. 2005; Atwell et al. 2010): the b-sheet subdomain, the ATP-binding core, and the a-helical subdomain (Fig. 3 "
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    ABSTRACT: The cystic fibrosis transmembrane conductance regulator (CFTR) requires dynamic fluctuations between states in its gating cycle for proper channel function, including changes in the interactions between the nucleotide-binding domains (NBDs) and between the intracellular domain (ICD) coupling helices and NBDs. Such motions are also linked with fluctuating phosphorylation-dependent binding of CFTR's disordered regulatory (R) region to the NBDs and partners. Folding of CFTR is highly inefficient, with the marginally stable NBD1 sampling excited states or folding intermediates that are aggregation-prone. The severe CF-causing F508del mutation exacerbates the folding inefficiency of CFTR and leads to impaired channel regulation and function, partly as a result of perturbed NBD1-ICD interactions and enhanced sampling of these NBD1 excited states. Increased knowledge of the dynamics within CFTR will expand our understanding of the regulated channel gating of the protein as well as of the F508del defects in folding and function.
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