Article

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

ABSTRACT

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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    • "[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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    • "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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