ABCMdb

PMID: 19944699

Lewis HA, Wang C, Zhao X, Hamuro Y, Conners K, Kearins MC, Lu F, Sauder JM, Molnar KS, Coales SJ, Maloney PC, Guggino WB, Wetmore DR, Weber PC, Hunt JF
Structure and dynamics of NBD1 from CFTR characterized using crystallography and hydrogen/deuterium exchange mass spectrometry.
J Mol Biol. 2010 Feb 19;396(2):406-30. Epub 2009 Nov 26., 2010-02-19 [PubMed]

Sentences

No. Mutations Sentence Comment

32 ABCC7 p.Arg553Gln ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys Comparing these hNBD1 structures to each other and to wild-type murine NBD1 (mNBD1) suggested that the overall fold of NBD1 was retained in the ΔF508 mutant and that structural changes were localized near the site of the deleted phenylalanine residue.5 However, three of these solubility-enhancing mutations (G550E/ R553Q/R555K) were known to be in vivo suppressors of the trafficking defect caused by the ΔF508 mutation.51-53 Compared to mNBD1, no significant structural perturbations were observed in the vicinity of these suppressor mutation sites in the crystal structures of hNBD1, suggesting that they act indirectly to suppress the effects of the ΔF508 mutation.5 Indeed, folding studies of a wider variety of hNBD1 variants, including several without trafficking-suppressor mutations, indicated that these mutations increase the thermodynamic stability of NBD1,5 which could account for improved folding and maturation in vivo. Login to comment 41 ABCC7 p.Arg553Gln ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys This structure was obtained from the same hNBD1-7a protein construct that yielded the previously reported ΔF508 structure, which has seven solubilizing mutations including the three trafficking-suppressor mutations G550E/R553Q/R555K. Login to comment 48 ABCC7 p.Phe429Ser ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg These constructs have two solubilizing mutations selected because they are sequence variations naturally present in more soluble NBD1 variants from other vertebrate species [F494N in the γ- phosphate switch from several fish species (unpublished results) and Q637R in the RE from mouse].5,37 One newly reported ΔF508 structure has only these two mutations (PDB ID 2BBT, construct hNBD1-2f- ΔF508, Rwork =23.2 and Rfree =29.5 at 2.30 Å), while the other has an additional F429S mutation in a disordered region of the RI (PDB ID 2BBT, construct hNBD1-3-ΔF508, Rwork =22.6 and Rfree =29.1 at 2.05 Å). Login to comment 53 ABCC7 p.Phe429Ser Therefore, the 2BBS and 2BBT structures permit assessment of the effects of these differences in solvent environment on hNBD1 conformation, because the only sequence difference between them is the F429S mutation in the disordered region of the RI. Login to comment 114 ABCC7 p.Phe508Ala Taking this possibility into account, the available structures can be clustered into three maximally independent groups-the four hNBD1 structures from crystals grown in high-molecular-weight PEG precipitants at pH ~7.5-9.0 (1XMJ, 2BBO, 2BBS, and 2BBT), the single hNBD1-F508A structure from a crystal grown in a low-molecular-weight PEG precipitant at pH ~4.5 (1XMI), and the nine mNBD1 structures from crystals grown using 3.5 M sodium acetate as the precipitant at pH 7.5. Login to comment 132 ABCC7 p.Phe508Ala ABCC7 p.Phe508Ser ABCC7 p.Phe508Arg Bright green and magenta represent human F508 and F508A structures, respectively, shades of red/orange represent human ΔF508 human structures, and shades of blue/cyan represent murine structures (F508, F508S, or F508R). Login to comment 133 ABCC7 p.Arg553Gln ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys The structures harboring the G550E/R553Q/ R555K suppressor mutation set are shown in bright green (F508) and bright red (ΔF508). Login to comment 139 ABCC7 p.Arg553Gln ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys This region contains F508, the LSGGQ signature sequence (at residues 548-552), and the G550E/ R553Q/R555K suppressor mutation sites. Login to comment 156 ABCC7 p.Phe508Ala Other than these variations in domain orientation and loop conformation, no significant structural variations are observed elsewhere even in the most divergent structures in the ensemble (PDB IDs 1XMI and 1R0Z, containing hNBD1-2b-F508A and mNBD1, respectively). Login to comment 294 ABCC7 p.Val510Asp Notably, a V510D mutation has been reported to suppress the trafficking defect caused by ΔF508, restoring export of a significant fraction of ΔF508-CFTR to the cell surface.73 The conformational change caused by the ΔF508 mutation dramatically alters the surface topography and chemical characteristics in the immediate vicinity of the 509-511 loop (Fig. 9) but not elsewhere in NBD1 (Figs. 3a and 8a). Login to comment 300 ABCC7 p.Arg553Gln ABCC7 p.Arg555Lys The G550E/R553Q/ R555K mutation set has been demonstrated to suppress the defective trafficking of ΔF508-CFTR (Fig. 11).51-53 Based on detailed structural, dynamic, and thermodynamic considerations presented in section ST12 in the Supplementary Information, we conclude that these suppressor mutations are unlikely to directly reverse a cryptic structural Fig. 9. Login to comment 305 ABCC7 p.His667Arg ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg Equivalent structural analyses conducted on the F494N, Q637R, and H667R solubilizing mutations are described in detail in sections ST13-14 in the Supplementary Information. Login to comment 318 ABCC7 p.Val510Asp The efficacy of V510D in rescuing the ΔF508 trafficking defect suggests that this suppressor mutation changes the conformation of the 509-511 loop, stabilizes the folding of the ABCα subdomain, or blocks chaperone interactions, because it seems unlikely to directly improve NBD1 binding to the TMDs. Login to comment 325 ABCC7 p.Phe429Ser ABCC7 p.Phe409Leu ABCC7 p.Phe433Leu Not shown here are three residues that were mutated in various constructs but located in the disordered region of the RI (i.e., the F429S mutation present in constructs 2b, 3, and 7a and the F409L and F433L mutations present in construct 7a). Login to comment 352 ABCC7 p.Arg553Gln ABCC7 p.Arg555Lys The trafficking-suppressor mutations (G550E/R553Q/R555K) overlap the LSGGQ signature sequence located at residues 548-552 in hNBD1. Login to comment