ABCMdb

PMID: 18305154

Serohijos AW, Hegedus T, Aleksandrov AA, He L, Cui L, Dokholyan NV, Riordan JR
Phenylalanine-508 mediates a cytoplasmic-membrane domain contact in the CFTR 3D structure crucial to assembly and channel function.
Proc Natl Acad Sci U S A. 2008 Mar 4;105(9):3256-61. Epub 2008 Feb 27., 2008-03-04 [PubMed]

Sentences

No. Mutations Sentence Comment

61 ABCC7 p.Leu1065Pro ABCC7 p.Arg1066Cys The wild-type peptide bound strongly, whereas the two mutations (L1065P and R1066C) that prevent conformational maturation (24) reduced this binding to control levels (Fig. 2B). Login to comment 62 ABCC7 p.Gly1069Arg The third mutation (G1069R), which does not prevent maturation but impairs channel function (25), causes a lesser reduction in binding. Login to comment 71 ABCC7 p.Phe508Cys ABCC7 p.Phe508Cys ABCC7 p.Phe508Cys ABCC7 p.Phe508Cys ABCC7 p.Leu1065Cys ABCC7 p.Phe1068Cys ABCC7 p.Gly1069Cys ABCC7 p.Phe1074Cys Cross-linking of Cys pairs F508C/L1065C, F508C/F1068C, F508C/G1069C, and F508C/F1074C confirms that Phe-508 in NBD1 associates with CL4 in MSD2 (Fig. 3 and SI Fig. 7). Login to comment 72 ABCC7 p.Gln1280Cys ABCC7 p.Lys1284Cys Cross-linking of C276/Q1280C and C276/K1284C confirms interaction of CL2 and NBD2. Login to comment 73 ABCC7 p.Leu1065Pro ABCC7 p.Gly1069Arg CL4 peptide was nearly completely ablated by a folding mutation (L1065P) but only slightly diminished by one influencing channel function (G1069R). Login to comment 80 ABCC7 p.Gly1069Cys Cross-linking also occurs between cysteines substituted for another hydrophobic residue (Val-510) near Phe-508 on the NBD1 surface and G1069C in CL4 (SI Fig. 8). Login to comment 81 ABCC7 p.Leu1065Cys ABCC7 p.Gly1069Cys ABCC7 p.Trp496Cys ABCC7 p.Thr1064Cys ABCC7 p.Met498Cys ABCC7 p.Lys564Cys In addition to the Phe-508-containing NBD1 surface patch, residues in other regions of the domain also interact with CL4 residues as evidenced by cross-linking of Cys pairs involving amino acids closer to the Q loop (Gln-493), including W496C/T1064C and M498C/L1065C as well as nearer the Walker B motif (Asp-572) such as K564C/G1069C (Fig. 3A and SI Fig. 8). Login to comment 84 ABCC7 p.Phe508Cys ABCC7 p.Phe508Cys ABCC7 p.Val510Cys ABCC7 p.Phe1068Cys ABCC7 p.Gly1069Cys ABCC7 p.Gly1069Cys Nevertheless, the proximity or relative orientation of the F508C/F1068C, F508C/G1069C, and V510C/G1069C pairs permitted very little disulfide bond formation on oxidation catalyzed by copper phenanthroline, i.e., only a very small proportion of mature band was converted to cross-linked band Fig. 2. Login to comment 86 ABCC7 p.Leu1065Pro ABCC7 p.Arg1066Cys ABCC7 p.Gly1069Arg (A) Location of disease-associated mutations (L1065P, R1066C, and G1069R) at the NBD1/CL4 interface. Login to comment 97 ABCC7 p.Leu1065Cys ABCC7 p.Gly1069Cys ABCC7 p.Trp496Cys ABCC7 p.Thr1064Cys ABCC7 p.Met498Cys ABCC7 p.Lys564Cys Phe-508 participates in an apparent aromatic cluster with residues from CL4(seealsoSIFig.10).CL4alsointeractswithotherregionsinNBD1assuggested by cross-linking of residues close to the Q loop (W496C/T1064C and M498C/ L1065C) and a residue near the Walker B motif (K564C/G1069C). Login to comment 101 ABCC7 p.Gln1280Cys (Right, Bottom two) Controls showing the lack of effects of cross-linking of the Cys-less construct and individually expressed and mixed single cysteine constructs (labeled 276C ϩ Q1280C) under the same conditions (see also SI Fig. 8). Login to comment 103 ABCC7 p.Gln1280Cys (Left) There is greater propensity for disulfide bond formation at the CL2/NBD2 interface (276C/Q1280C) than at the CL4/NBD1 interface. Login to comment 113 ABCC7 p.Gln1280Cys ABCC7 p.Lys1284Cys In fact, the Q1280C and K1284C substitutions in NBD2 of Cys-less CFTR containing the native Cys-276 residue in CL2 (illustrated in Fig. 3A, Top Right) enabled cross-linking by all MTS reagents tested. Login to comment 130 ABCC7 p.Gln1280Cys (Middle) Cys-less CFTR with 276C/Q1280C (n ϭ 3). Login to comment 131 ABCC7 p.Phe508Cys ABCC7 p.Phe1068Cys (Bottom) Cys-less CFTR with F508C/F1068C (n ϭ 4). Login to comment