ABCMdb

PMID: 15528182

Lewis HA, Zhao X, Wang C, Sauder JM, Rooney I, Noland BW, Lorimer D, Kearins MC, Conners K, Condon B, Maloney PC, Guggino WB, Hunt JF, Emtage S
Impact of the deltaF508 mutation in first nucleotide-binding domain of human cystic fibrosis transmembrane conductance regulator on domain folding and structure.
J Biol Chem. 2005 Jan 14;280(2):1346-53. Epub 2004 Nov 3., 2005-01-14 [PubMed]

Sentences

No. Mutations Sentence Comment

40 ABCC7 p.Phe508Ala Crystallization and Data Collection-Crystallization leads at 4 °C from hNBD1-2b-F508A at 6-17 mg/ml 4 °C were optimized using microseeding. Login to comment 46 ABCC7 p.Phe508Ala ABCC7 p.Phe508Ala Structure Determination and Refinement-Structures were determined by molecular replacement using mNBD1 (for hNBD1-2b-F508A) or hNBD1-2b-F508A (for hNBD1-7a-⌬F508) as the search model with the program MolRep (16) and manually rebuilt using the program XtalView (17) over several cycles of refinement with CNX (18) and/or Refmac (16). Login to comment 48 ABCC7 p.Phe508Ala The refined model of hNBD1-2b-F508A includes residues 388-411 and 429-671 in molecule A; 389-413, 429-532, 539-541, and 547-671 in molecule B; 389-415 and 429-671 in molecule C; 388-410 and 426-672 in molecule D; 389-411 and 429-671 in molecule E; 1 ATP/ TABLE I Human NBD1 proteins Thermodynamic values are listed for those proteins analyzed in equilibrium denaturation experiments. Login to comment 73 ABCC7 p.Arg553Gln ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys We also investigated the effect of three suppressor mutations (G550E, R553Q, R555K) that have been observed to improve in vivo trafficking efficiency of STE6-CFTR chimeras containing the ⌬F508 mutation expressed in yeast (23-25). Login to comment 76 ABCC7 p.Phe508Ala Recombinant proteins harboring the F508A mutation gave higher yields than the equivalent proteins with phenylalanine at position 508, whereas constructs with the ⌬F508 mutation consistently gave lower yields. Login to comment 82 ABCC7 p.His667Arg ABCC7 p.Phe508Ala ABCC7 p.Phe508Ala ABCC7 p.Phe429Ser Crystal Structure of hNBD1 Shows That Regulatory Protein Segments Adopt Multiple Conformations Altering Access to the Active Site-High-resolution diffraction data were obtained for hNBD1-2b-F508A, containing two solubilizing mutations (F429S and H667R) in addition to the F508A substitution (Table II). Login to comment 84 ABCC7 p.Phe429Ser One (F429S) participates in intermolecular packing interactions stabilizing the lattice (data not shown). Login to comment 85 ABCC7 p.Phe508Ala With the exception of the RI and RE segments, the structure of hNBD1-2b-F508A closely matches that of mNBD1. Login to comment 86 ABCC7 p.Phe508Ala Least squares superposition of the remainder of the F1-type core and ABCbeta subdomains yields a 0.46-Å root mean square deviation (rmsd) for 127 C-␣ atoms, only slightly exceeding the 0.39-Å rmsd observed after superposition of the different molecules within the asymmetric unit of the crystal structure of hNBD1- 2b-F508A. Login to comment 91 ABCC7 p.Phe508Ala We concluded that these differences re- TABLE II Statistics of structure determination and refinement hNBD1-2b-F508A hNBD1-7a-⌬F508 Data collection Resolution range 2.25-27.3 Å 2.25-26.7 Å Space Group C2221 P43212 a 144.46 Å 59.79 Å b 154.02 Å 59.79 Å c 136.09 Å 144.40 Å Completeness (overall/outer shell) 99.9%/99.9% 99.8%/99.8% Rsym (overall/outer shell)a 8.5%/40.7% 8.3%/41.7% I/␴(I) (overall/outershell) 4.3/1.2 7.5/1.6 Refinement Resolution range 2.25-27.3 Å 2.30-25.0 Å Rfree (overall/outer shell)b 26.5%/31.0% 28.0%/32.1% R (overall/outer shell)c 21.2%/28.0% 22.0%/22.1% Waters 704 135 rmsd bond lengths 0.006 Å 0.010 Å rmsd angles 1.0° 1.3° Average B-factorsd 32.5 34.9 Core Ramachandran 92.9% 93.1% Allowed Ramachandran 6.7% 6.4% Disallowed Ramachandran 0.2% 0.0% a Rsym ϭ ⌺hkl⌺i͉Ii(hkl-)͗(I(hkl)͉͘/⌺hkl⌺iIi(hkl), where Ii is the intensity of the observation and ͗I͘ is the mean intensity of the reflection. Login to comment 98 ABCC7 p.Phe508Ala Although the conformation of the regulatory segments observed in the crystal structure of hNBD1-2b-F508A would preclude formation of a canonical ATP-sandwich complex with NBD2 because of a steric overlap at the interface, their dramatic change in conformation compared with the crystal structure of mNBD1 confirms our prediction that these segments of NBD1 are conformationally dynamic. Login to comment 100 ABCC7 p.Arg553Gln ABCC7 p.His667Arg ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys ABCC7 p.Phe429Ser ABCC7 p.Phe409Leu ABCC7 p.Phe433Leu Crystal Structure of ⌬F508 hNBD1 Shows Minimal Conformational Changes but Substantive Changes in Surface Topography at the Putative Site of MSD1 Interaction-Crystals diffracting to a resolution of 2.3 Å were obtained for hNBD1-7a- ⌬F508, which contains seven mutations (F409L, F429S, F433L, G550E, R553Q, R555K, H667R) in addition to the deletion of Phe-508 (see Table II). Login to comment 101 ABCC7 p.Phe508Ala It shows only minor differences compared with hNBD1-2b-F508A except in the immediate vicinity of the deletion of Phe-508 (Fig. 2) and at the regulatory seg- FIG. 1. Login to comment 109 ABCC7 p.Phe508Ala Regions with conformational differences are shown in cyan for hNBD1-2b-F508A (molecule E), blue for hNBD1-7a-⌬F508, and gold for mNBD1 (molecule B). Login to comment 113 ABCC7 p.Phe508Ala Superposition of the F1-type core and ABCbeta subdomains with those in hNBD1-2b-F508A gives an rmsd of 0.51 Å for 127 C-␣ atoms, similar to the deviations observed between the different protomers in that structure. Login to comment 114 ABCC7 p.Phe508Ala The ABC␣ subdomain is rotated by 6° relative to its position in hNBD1-2b-F508A but is largely conserved in structure, exhibiting an rmsd of 0.87Å for the superposition of 49 C-␣ atoms. Login to comment 116 ABCC7 p.Phe508Ala However, the position of ␣-helix 9b in the RE is very similar in the structures of hNBD1-7a-⌬F508 and mNBD1 (and different from the position observed in the structure of hNBD1-2b-F508A), suggesting that this may represent a preferred conformation of the dynamically flexible RE. Login to comment 119 ABCC7 p.Phe508Ala A, stereo image of conformation of Phe-508 loop region in mNBD1 (gold), hNBD1-2b-F508A (cyan), and hNBD1-7a-⌬F508 (blue). Login to comment 120 ABCC7 p.Phe508Ala B and C, worm diagrams of hNBD1-2b-F508A (B) and hNBD1-7a-⌬F508 (C). Login to comment 125 ABCC7 p.Phe508Ala D and E, surface properties of hNBD1-2b-F508A (D) and hNBD1-7a-⌬F508 (E) in same orientations as in B and C. Login to comment 126 ABCC7 p.Phe508Ala Residues 507-510 in hNBD1-2b-F508A structure have been replaced with those from the mNBD1 structure to provide an image representative of the wild-type human protein. Login to comment 133 ABCC7 p.Phe508Ala The superposition of the three available NBD1 crystal structures (mNBD1, hNBD1-2b-F508A, and hNBD1-7a-⌬F508) based on least squares alignment of ␣-helices 3 and 4 demonstrates that the conformation is extremely similar even in the immediate vicinity of the deletion, consistent with the quantitatively similar folding parameters observed either in the absence or presence of the ⌬F508 mutation. Login to comment 138 ABCC7 p.His667Arg ABCC7 p.Phe429Ser ABCC7 p.Phe409Leu ABCC7 p.Phe433Leu Of the seven solubilizing mutations present in the ⌬F508 form of hNBD1, three (F409L, F429S, F433L) occur in disordered regions and therefore likely interact with solvent, whereas residue H667R is only minimally solvent-exposed on the surface of ␣-helix 9b in the RE. Login to comment 139 ABCC7 p.Arg553Gln ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys The three suppressor mutations (G550E, R553Q, R555K) occur either in or immediately following the LSGGQ signature sequence at the N terminus of ␣-helix 5. Login to comment