ABCMdb

PMID: 18215773

Pissarra LS, Farinha CM, Xu Z, Schmidt A, Thibodeau PH, Cai Z, Thomas PJ, Sheppard DN, Amaral MD
Solubilizing mutations used to crystallize one CFTR domain attenuate the trafficking and channel defects caused by the major cystic fibrosis mutation.
Chem Biol. 2008 Jan;15(1):62-9., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC7 p.Phe429Ser ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg ABCC7 p.Gln637Arg Although F508del-NBD1 shows only minor conformational changes relative to that of wild-type NBD1, additional mutations (F494N/Q637R or F429S/F494N/Q637R) were required for domain solubility and crystallization. Login to comment 23 ABCC7 p.Arg553Gln ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys Some of these mutations represented sequence changes between human CFTR and CFTRs from other species, whereas others (G550E, R553Q, and R555K) had been previously identified as F508del-CFTR revertant mutations (Teem et al., 1996; deCarvalho et al., 2002; Chang et al., 1999). Login to comment 25 ABCC7 p.Arg553Gln Of note, one of these revertants, R553Q, was found in an F508del-homozygous patient with mild CF disease (Do¨ rk et al., 1991). Login to comment 26 ABCC7 p.Gly550Glu In addition, another revertant mutation, G550E, likely acts by altering the structure of NBD1 (Roxo-Rosa et al., 2006). Login to comment 33 ABCC7 p.Phe429Ser ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg ABCC7 p.Gln637Arg However, these new F508del-NBD1 crystal structures still required either two (F494N/Q637R; Protein Data Bank [PDB] ID code: 2BBT) or three (F429S/F494N/ Q637R; PDB ID code: 2BBS) additional mutations for domain solubility and, hence, crystal formation (Lewis, 2005). Login to comment 35 ABCC7 p.Phe429Ser ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg ABCC7 p.Gln637Arg To test these ideas, we investigated the effects of the mutations F494N/Q637R and F429S/F494N/Q637R on wt- and F508del-CFTR by studying: (1) the in vivo folding yield of NBD1, (2) the processing and trafficking of the full-length CFTR protein, and (3) the ClÀ channel function of CFTR. Login to comment 37 ABCC7 p.Phe429Ser ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg ABCC7 p.Gln637Arg RESULTS While studying the effects of F508del on the structure of NBD1 from CFTR, Lewis (2005) introduced the mutations F494N/ Q637R (double; D) and F429S/F494N/Q637R (triple; T) into NBD1 to improve domain solubility and crystallization. Login to comment 42 ABCC7 p.Phe429Ser ABCC7 p.Phe429Ser ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg ABCC7 p.Gln637Arg Solubilizing Mutations Improve wt- and F508del-NBD1 Yield To explore whether F429S, F494N, and Q637R improve the yield of soluble NBD1, wt-NBD1 and F508del-NBD1 were expressed in bacterial cells in the absence and presence of the solubilizing mutations. Login to comment 144 ABCC7 p.Phe429Ser ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg ABCC7 p.Gln637Arg Structural Implications An interesting aspect of the action of the solubilizing mutations (double, F494N/Q637R; triple, F429S/F494N/Q637R) is their remote location from that of F508. Login to comment 146 ABCC7 p.Phe508Arg ABCC7 p.Phe494Asn However, the effect produced by F494N is not completely unexpected, as structural crosstalk between the side chain of the F508 residue (e.g., F508R) and the Q loop or g-phosphate switch (e.g., residues W496 and M498) has been previously shown to occur (Massiah et al., 1999). Login to comment 148 ABCC7 p.Gln637Arg Curiously, Q637R lies in this helix, known as the RD1 region (residues 590-672) of NBD1. Login to comment 151 ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg In support of this notion, our data indicate that Q637R, at least with one additional solubilizing mutation (F494N), might contribute to the increased solubility of F508del-NBD1 and to the partial rescue of F508del-CFTR trafficking and function. Login to comment 152 ABCC7 p.Phe429Ser ABCC7 p.Phe429Ser ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg ABCC7 p.Gln637Arg The third solubilizing mutation, F429S, further promotes the revertant effect produced by the double mutant (F494N/Q637R) on F508del-CFTR, as the triple mutant (F429S/F494N/Q637R) visibly increased maturation of F508del-CFTR as measured by the higher maturation yield at steady state of F508delT-CFTR compared with that of F508delD-CFTR (Figure 1C). Login to comment 154 ABCC7 p.Phe429Ser This suggests that F429S might lie on the surface of NBD1 in a position where it likely interacts with the environment (Lewis et al., 2004, 2005). Login to comment 155 ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys ABCC7 p.Arg516Lys ABCC7 p.Arg29Lys ABCC7 p.Arg766Lys Comparison with Other Revertants Using the same cellular system employed to investigate the solubilizing mutations, we recently examined the mechanism of action of two other F508del-CFTR revertants, G550E and 4RK, the simultaneous mutation of four arginine-framed tripeptides (AFTs), R29K, R516K, R555K, and R766K (Roxo-Rosa et al., 2006). Login to comment 157 ABCC7 p.Gly550Glu G550E likely alters the conformation of NBD1, whereas 4RK allows F508del-CFTR to escape endoplasmic reticulum retention/retrieval mediated by AFTs. Login to comment 158 ABCC7 p.Gly550Glu We also showed that both G550E and 4RK affected wt-CFTR (Roxo-Rosa et al., 2006). Login to comment 160 ABCC7 p.Gly550Glu ABCC7 p.Gly550Glu Moreover, whereas the revertants 4RK and G550E restored the iodide efflux of F508del-CFTR to wt levels (Roxo-Rosa et al., 2006), the magnitude of iodide efflux elicited by F508delD- and F508delT-CFTR was less than that of wt-CFTR. This indicates that the solubilizing mutations are not as effective as 4RK and G550E at augmenting either the cell-surface expression or function of F508del-CFTR. Login to comment 161 ABCC7 p.Gly550Glu However, at steady state, F508delD and F508delT produced amounts of band C similar to those of G550E- or 4RK-F508del (present study and A.S. and M.D.A., unpublished data). Login to comment 165 ABCC7 p.Gly550Glu By contrast, both revertant mutations markedly prolonged the MBD of F508del-CFTR, with G550E exceeding that of wt-CFTR by 4-fold (Roxo-Rosa et al., 2006). Login to comment 166 ABCC7 p.Gly550Glu Moreover, only G550E suppressed the prolonged IBI of F508del-CFTR (Roxo-Rosa et al., 2006), reducing it to a level similar to that achieved here with F508delT-CFTR. Login to comment 167 ABCC7 p.Gly550Glu We interpret these data to suggest that the conformation state(s) that solubilizing mutations achieve is closer to that of wt-CFTR than the conformation produced by the revertant G550E (Roxo-Rosa et al., 2006). Login to comment 168 ABCC7 p.Gly550Glu Consistent with this interpretation, the revertants, especially G550E, extended the MBD and elevated the Po of wt-CFTR (Roxo-Rosa et al., 2006), whereas wt-CFTR containing the solubilizing mutations had the same MBD, IBI, and Po as wt-CFTR. Login to comment 169 ABCC7 p.Gly550Glu We thus suggest that the solubilizing mutations act specifically on F508del-CFTR (in contrast to G550E) to abrogate its gating defect, possibly through correction of NBD1 and/or CFTR folding. Login to comment 175 ABCC7 p.Phe429Ser ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg ABCC7 p.Gln637Arg The available crystal structure of F508del-NBD1 was determined after the introduction of additional mutations (F494N/Q637R or F429S/ F494N/Q637R) to help domain solubilization and crystal formation. Login to comment 182 ABCC7 p.Phe429Ser ABCC7 p.Phe429Ser ABCC7 p.Phe429Ser ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg ABCC7 p.Gln637Arg ABCC7 p.Gln637Arg ABCC7 p.Gln637Arg Site-Directed Mutagenesis, Cells, and CFTR Expression To introduce the solubilizing mutations F494N/Q637R and F429S/F494N/ Q637R into wt- and F508del-CFTR cDNAs in the pNUT expression vector, we used the primers F429S, 50 -GGTGATGACAGCCTCTCCTTCAGTAATTTC TCA-30 ; F494N, 50 -CATTCTGTTCTCAGAATTCCTGGATTATGCCTGG-30 ; Q637R, 50 -GAACTCCAAAATCTAAGGCCAGACTTTAGCTC-30 and the QuikChange site-directed mutagenesis kit (Stratagene). Login to comment 187 ABCC7 p.Phe429Ser ABCC7 p.Phe429Ser ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg ABCC7 p.Gln637Arg ABCC7 p.Gln637Arg ABCC7 p.Gln637Arg Cell lines expressing different solubilizing mutations are referred to as follows: wtD-CFTR, F494N-Q637R-CFTR; F508delD-CFTR, F494N- F508del-Q637R-CFTR; wtT-CFTR, F429S-F494N-Q637R-CFTR; and F508delT-CFTR, F429S-F494N-F508del-Q637R-CFTR. Login to comment