ABCMdb

PMID: 22265408

Rabeh WM, Bossard F, Xu H, Okiyoneda T, Bagdany M, Mulvihill CM, Du K, di Bernardo S, Liu Y, Konermann L, Roldan A, Lukacs GL
Correction of both NBD1 energetics and domain interface is required to restore DeltaF508 CFTR folding and function.
Cell. 2012 Jan 20;148(1-2):150-63., [PubMed]

Sentences

No. Mutations Sentence Comment

26 ABCC7 p.Arg553Gln ABCC7 p.His667Arg ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys ABCC7 p.Phe429Ser ABCC7 p.Phe409Leu ABCC7 p.Phe433Leu ABCC7 p.Phe494Asn Both the R mutations (G550E, R553Q, and R555K) and S mutations (F409L, F429S, F433L, F494N, and H667R) could partially rescue the DF508 CFTR folding and functional defect (Lewis et al., 2005; Pissarra et al., 2008; Teem et al., 1993, 1996) and were assumed to stabilize the domain either alone or in combinations (1S, 3S, R, R1S, and R4S; see Figure 1B). Login to comment 69 ABCC7 p.Phe508Asp ABCC7 p.Phe508Ser ABCC7 p.Phe508Arg ABCC7 p.Phe508Gly ABCC7 p.Phe508Asn ABCC7 p.Phe508Glu These results in concert with the effect of F508E, F508R, F508G, F508S, F508D, and F508N mutations revealed that the CD4T-NBD1 PM density was proportional to the domain stability if the NBD1 Tm was >38 C (Figures 3D and S4D). Login to comment 117 ABCC7 p.Phe508* These and additional results discussed in the following section show that both folding efficiency and PM density of WT variants increased at $37- and $14-fold steeper slopes, respectively, than their DF508 or F508X counterparts as a function of NBD1 Tm (Figures 5A and 5B) and suggest that NBD1 folding energetics can define the WT but not DF508 CFTR domain-domain assembly. Login to comment 118 ABCC7 p.Phe508* These and additional results discussed in the following section show that both folding efficiency and PM density of WT variants increased at 37- and 14-fold steeper slopes, respectively, than their DF508 or F508X counterparts as a function of NBD1 Tm (Figures 5A and 5B) and suggest that NBD1 folding energetics can define the WT but not DF508 CFTR domain-domain assembly. Login to comment 121 ABCC7 p.Phe508Asn Missense mutations of F508 are depicted by colored squares (-), and F508N-3S and -R are indicated by solid triangle (:). Login to comment 122 ABCC7 p.Phe508Asn Missense mutations of F508 are depicted by colored squares (-), and F508N-3S and -R are indicated by solid triangle (:). Login to comment 125 ABCC7 p.Phe508Asn ABCC7 p.Phe508Asn (2) Although certain combinations of F508N and R or S mutations conferred thermodynamic and kinetic stability comparable with or exceeding that of the WT NBD1-1S (e.g., F508N-R; DG0 = À5.1 ± 0.1 kcal/mol, Tm z47 C and ku H20 = 0.16 10À3 secÀ1 ; Figure S5), they failed to restore WT-like folding and expression of the DF508 CFTR (Figures 5A and 5B). Login to comment 126 ABCC7 p.Phe508Asn ABCC7 p.Phe508Asn (2) Although certain combinations of F508N and R or S mutations conferred thermodynamic and kinetic stability comparable with or exceeding that of the WT NBD1-1S (e.g., F508N-R; DG0 = 5.1 &#b1; 0.1 kcal/mol, Tm z47 C and ku H20 = 0.16 103 sec1 ; Figure S5), they failed to restore WT-like folding and expression of the DF508 CFTR (Figures 5A and 5B). Login to comment 132 ABCC7 p.Arg1070Trp Remarkably, NBD1, MSD1, and NBD2 were susceptible to comparable misfolding upon disrupting the NBD1-CL4 interface by the R1070W substitution in WT CFTR. Login to comment 133 ABCC7 p.Arg1070Trp Remarkably, NBD1, MSD1, and NBD2 were susceptible to comparable misfolding upon disrupting the NBD1-CL4 interface by the R1070W substitution in WT CFTR. Login to comment 134 ABCC7 p.Val510Asp Conversely, stabilization of the NBD1-CL4 interface by the V510D substitution (see bellow) increased the WT CFTR folding efficiency by $2-fold, supporting the critical role of the NBD1-CL4 interface in the coupled domain folding of CFTR (Figure 6B; see below). Login to comment 135 ABCC7 p.Val510Asp Conversely, stabilization of the NBD1-CL4 interface by the V510D substitution (see bellow) increased the WT CFTR folding efficiency by 2-fold, supporting the critical role of the NBD1-CL4 interface in the coupled domain folding of CFTR (Figure 6B; see below). Login to comment 136 ABCC7 p.Arg1070Trp ABCC7 p.Val510Asp R1070W or V510D substitutions at the interfaces restored the proximity of the DF508 NBD1 and CL4 as shown by Cys crosslinking. Login to comment 137 ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp ABCC7 p.Val510Asp ABCC7 p.Val510Asp The NBD1-CL4 interface stabilization can be accomplished by filling the cavity created by the DF508 with the bulky hydrophobic side chain of R1070W or by salt bridge formation between V510D in NBD1 and R1070 in CL4 (Figure S6B) (He et al., 2010; Loo et al., 2008, 2010; Thibodeau et al., 2010). Login to comment 138 ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp ABCC7 p.Val510Asp ABCC7 p.Val510Asp R1070W, similar to V510D, alone modestly increased the DF508 CFTR folding efficiency and cellular and PM expression (Figures 6A-6E), in part confirming previous reports (He et al., 2010; Thibodeau et al., 2010; Loo et al., 2010). Login to comment 139 ABCC7 p.Arg1070Trp ABCC7 p.Val510Asp R1070W, similar to V510D, alone modestly increased the DF508 CFTR folding efficiency and cellular and PM expression (Figures 6A-6E), in part confirming previous reports (He et al., 2010; Thibodeau et al., 2010; Loo et al., 2010). Login to comment 140 ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp At the individual domain level, the combination of R1S and R1070W, but not R1S alone, largely restored domain assembly, as indicated by the WT-like trypsin resistance of the MSD1, MSD2, and NBD2 in DF508-CFTR-R1S-R1070W (Figure 5C). Login to comment 141 ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp ABCC7 p.Val510Asp Similarly, the DF508 CFTR folding and expression were synergistically rescued by combining the DF508-NBD1 stabilizing mutation DRI with R1070W or V510D (Figures 6F-6H and S7B), ruling out nonspecific effects of second-site mutations. Login to comment 142 ABCC7 p.Arg1070Trp ABCC7 p.Val510Asp ABCC7 p.Val510Asp Direct energetic stabilization of the DF508-NBD1-0S and -3S by the V510D mutation was marginal (Figure S5). Login to comment 143 ABCC7 p.Val510Asp Direct energetic stabilization of the DF508-NBD1-0S and -3S by the V510D mutation was marginal (Figure S5). Login to comment 144 ABCC7 p.Arg1070Trp ABCC7 p.Val510Asp Accordingly, substantially increased folding efficiency of DF508-CFTR-1218X was only achieved by synergistic stabilization of the NBD1-CL4 interface (R1070W or V510D) and NBD1 (3S, -3R, or -R1S) (Figures 7A- 7C, and S7C). Login to comment 145 ABCC7 p.Arg1070Trp ABCC7 p.Val510Asp Accordingly, substantially increased folding efficiency of DF508-CFTR-1218X was only achieved by synergistic stabilization of the NBD1-CL4 interface (R1070W or V510D) and NBD1 (3S, -3R, or -R1S) (Figures 7A- 7C, and S7C). Login to comment 147 ABCC7 p.Arg600Gly ABCC7 p.Ala559Glu CF-associated nonconserved amino acid substitutions (A559E or R600G) that are solvent exposed and distant from the NBD1-CL4 interface prevented the expression of soluble NBD1 in E. coli and CFTR processing in mammalian cells (Figure S7D; data not shown). Login to comment 148 ABCC7 p.Arg1070Trp ABCC7 p.Arg600Gly ABCC7 p.Ala559Glu Likewise, disrupting the NBD1-CL4 interface by the R1070W mutation compromised both WT CFTR and CFTR-1218X folding (Figures 6B, 6C, 6E, left panel, and 7C), as well as the NBD1, NBD2, and MSD1 conformation, which was partly rescued by stabilizing the NBD1 with 3S, R, or R1S (Figure S6A). Login to comment 149 ABCC7 p.Arg1070Trp Likewise, disrupting the NBD1-CL4 interface by the R1070W mutation compromised both WT CFTR and CFTR-1218X folding (Figures 6B, 6C, 6E, left panel, and 7C), as well as the NBD1, NBD2, and MSD1 conformation, which was partly rescued by stabilizing the NBD1 with 3S, R, or R1S (Figure S6A). Login to comment 153 ABCC7 p.Arg1070Trp Red and blue circles depict the combined effect of R1070W or V5010D interface and 3S, R, or R1S stabilizing mutations on the DF508 CFTR expression. Login to comment 154 ABCC7 p.Arg1070Trp Red and blue circles depict the combined effect of R1070W or V5010D interface and 3S, R, or R1S stabilizing mutations on the DF508 CFTR expression. Login to comment 158 ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp resistance of NBD1, MSD1, and NBD2 (Figure S6A) as well as restored folding and processing upon NBD1 stabilization by 3S, R, R1S, or DRI in the CFTR-R1070W and CFTR-1218X- R1070W (Figures 6B, 6C, 6E, and 7C). Login to comment 159 ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp resistance of NBD1, MSD1, and NBD2 (Figure S6A) as well as restored folding and processing upon NBD1 stabilization by 3S, R, R1S, or DRI in the CFTR-R1070W and CFTR-1218X- R1070W (Figures 6B, 6C, 6E, and 7C). Login to comment 166 ABCC7 p.Arg1070Trp (D and E) Correction of the DF508 CFTR gating defect by the combination of the 3S and R1070W mutations. Login to comment 167 ABCC7 p.Arg1070Trp (D and E) Correction of the DF508 CFTR gating defect by the combination of the 3S and R1070W mutations. Login to comment 174 ABCC7 p.Arg1070Trp ABCC7 p.Val510Asp WT-like domain assembly and stabilization of DF508 CFTR were achieved by a combination of NBD1 and NBD1-CL4 interface-stabilizing mutations (e.g., 3S and R1070W or V510D). Login to comment 175 ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp ABCC7 p.Val510Asp M2* and red line depict the R1070W mutation. Login to comment 176 ABCC7 p.Arg1070Trp M2* and red line depict the R1070W mutation. Login to comment 178 ABCC7 p.Arg1070Trp The 3S and R1070W mutations increased the DF508 CFTR Po by 27% and 54%, respectively. Login to comment 179 ABCC7 p.Arg1070Trp The 3S and R1070W mutations increased the DF508 CFTR Po by 27% and 54%, respectively. Login to comment 197 ABCC7 p.Arg1070Trp Although interdomain interactions likely stabilize NBD1 conformationally in WT CFTR, as suggested by the enhanced NBD1 protease susceptibility upon destabilizing the MSD1 or NBD2 by CF-causing point mutation (Cui et al., 2007; Du and Lukacs, 2009; Du et al., 2005; Xiong et al., 1997), or the NBD1-CL4 interface by R1070W (Figure S6A), these stabilizing factors are compromised due to coupled misfolding of MSD1, MSD2, and NBD2 in DF508 CFTR and other mutants (Cui et al., 2007; Du and Lukacs, 2009; Du et al., 2005; Xiong et al., 1997). Login to comment 198 ABCC7 p.Arg1070Trp Although interdomain interactions likely stabilize NBD1 conformationally in WT CFTR, as suggested by the enhanced NBD1 protease susceptibility upon destabilizing the MSD1 or NBD2 by CF-causing point mutation (Cui et al., 2007; Du and Lukacs, 2009; Du et al., 2005; Xiong et al., 1997), or the NBD1-CL4 interface by R1070W (Figure S6A), these stabilizing factors are compromised due to coupled misfolding of MSD1, MSD2, and NBD2 in DF508 CFTR and other mutants (Cui et al., 2007; Du and Lukacs, 2009; Du et al., 2005; Xiong et al., 1997). Login to comment 203 ABCC7 p.Arg1070Trp S, R, or DRI mutations increased the WT channel folding efficiency (by 20%-80%) in proportion with NBD1 stabilization, a tendency that is also observed in the background of NBD1-CL4 destabilization in R1070W-CFTR (Figures 4B and 6B). Login to comment 204 ABCC7 p.Arg1070Trp ABCC7 p.Val510Asp Stabilization of the NBD1-CL4 interface by salt bridge formation between V510D and R1070 also improved WT CFTR biogenesis (Figure 6B). Login to comment 205 ABCC7 p.Val510Asp Stabilization of the NBD1-CL4 interface by salt bridge formation between V510D and R1070 also improved WT CFTR biogenesis (Figure 6B). Login to comment