ABCMdb

PMID: 9188468

Qu BH, Strickland EH, Thomas PJ
Localization and suppression of a kinetic defect in cystic fibrosis transmembrane conductance regulator folding.
J Biol Chem. 1997 Jun 20;272(25):15739-44., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Arg553Met Likewise a second site mutation, R553M, which corrects the maturation defect in vivo, is a superfolder which counters the effects of ⌬F508 on the temperature-dependent folding yield in vitro, but does not significantly alter the free energy of stability. Login to comment 5 ABCC7 p.Gly551Asp ABCC7 p.Ser549Arg A disease-causing mutation, G551D, which does not alter the maturation of CFTR in vivo but rather its function as a chloride channel, and the S549R maturation mutation have no discernible effect on the folding of the domain. Login to comment 12 ABCC7 p.Gly551Asp ABCC7 p.Ser549Arg Several other CF-causing mutations known to be maturation-defective, including S549R, are located in NBD1 (11) as are mutations of critical functional residues such as G551D (12). Login to comment 16 ABCC7 p.Arg553Gln ABCC7 p.Arg553Gln In a German pancreas-insufficient patient homozygous for ⌬F508 with typical gastrointestinal and pulmonary disease but sweat chloride in the normal range a second site mutation of R553Q was found on one ⌬F508-CFTR allele (15). Login to comment 17 ABCC7 p.Arg553Gln ABCC7 p.Arg553Met Two mutations at this position, R553M and R553Q, revert the mating phenotype of a ⌬F508 STE6-CFTR chimera in yeast (16). Login to comment 23 ABCC7 p.Gly551Asp ABCC7 p.Ser549Arg ABCC7 p.Arg553Met In the present study we use this system to examine the effects of the R553M second site mutation, the G551D functional mutation, the S549R maturation-defective mutation, glycerol, and ATP binding on the folding pathway and thermodynamic stability of NBD1. Login to comment 25 ABCC7 p.Gly551Asp ABCC7 p.Ser549Arg ABCC7 p.Arg553Met EXPERIMENTAL PROCEDURES Expression, Purification, and Folding of CFTR NBD1s-Oligonucleotide-mediated mutagenesis (19) was used to generate R553M, G551D, and S549R mutations in plasmid pBQ2.4 containing CFTR cDNAs. Login to comment 26 ABCC7 p.Arg553Met The mutant primers are as follows: R553M primer, 5Ј-GAAATTCTTGC- * This work was supported by National Institutes of Health Grant DK49835 and Welch Foundation Grant I-1284. Login to comment 34 ABCC7 p.Gly551Asp ABCC7 p.Ser549Arg 25, Issue of June 20, pp. 15739-15744, 1997 (c) 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. This paper is available on line at http://www.jbc.org CATTTGACCTCCAC-39 (25 bases); G551D primer, 59-CTTGCTCGTT- GATCTCCACTCAGTG-39 (25 bases); S549R primer, 59-CGTTGAC- CTCCTCTCAGTGTGATTCC-39 (26 bases). Login to comment 36 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Ser549Arg ABCC7 p.Ser549Arg ABCC7 p.Arg553Met Printed in U.S.A. This paper is available on line at http://www.jbc.org CATTTGACCTCCAC-3Ј (25 bases); G551D primer, 5Ј-CTTGCTCGTT- GATCTCCACTCAGTG-3Ј (25 bases); S549R primer, 5Ј-CGTTGAC- CTCCTCTCAGTGTGATTCC-3Ј (26 bases). Login to comment 38 ABCC7 p.Gly551Asp ABCC7 p.Ser549Arg ABCC7 p.Arg553Met ABCC7 p.Arg553Met ABCC7 p.Arg553Met Expression cassette polymerase chain reaction was employed to synthesize the cDNA fragments of CFTR NBD1-R553M, NBD1-G551D, and NBD1-S549R containing a 5Ј NdeI site, a 3Ј XhoI site, and a stop codon as described previously for NBD1 and NBD1⌬F (5). Login to comment 39 ABCC7 p.Arg553Met The larger fragment (5140 base pairs) from the pET28a NBD1-R553M plasmid digestion and the small fragment (720 base pairs) from the pET28a NBD1DF plasmid digestion were purified and ligated with T4 ligase. Login to comment 40 ABCC7 p.Arg553Met ABCC7 p.Arg553Met To construct the NBD1⌬F-R553M expression vector, pET28a NBD1-R553M and pET28a NBD1⌬F were digested with SphI. Login to comment 41 ABCC7 p.Arg553Met The larger fragment (5140 base pairs) from the pET28a NBD1-R553M plasmid digestion and the small fragment (720 base pairs) from the pET28a NBD1⌬F plasmid digestion were purified and ligated with T4 ligase. Login to comment 56 ABCC7 p.Arg553Met ABCC7 p.Arg553Met The expression levels of NBD1-R553M and NBD1DF-R553M are similar to NBD1 and NBD1DF (5). Login to comment 57 ABCC7 p.Gly551Asp ABCC7 p.Ser549Arg The expression levels of NBD1-G551D and NBD1-S549R are approximately one-half of the wild-type NBD1 level (data not shown). Login to comment 58 ABCC7 p.Arg553Met ABCC7 p.Arg553Met The expression levels of NBD1-R553M and NBD1⌬F-R553M are similar to NBD1 and NBD1⌬F (5). Login to comment 59 ABCC7 p.Gly551Asp ABCC7 p.Ser549Arg The expression levels of NBD1-G551D and NBD1-S549R are approximately one-half of the wild-type NBD1 level (data not shown). Login to comment 80 ABCC7 p.Arg553Met The Kd for ATP binding to the other NBD1s determined in similar experiments (data not shown) are presented in Table I. NBD1 and only 38% of the NBD1DF fold into the soluble conformation, whereas 96% of NBD1-R553M assumes the folded conformation at this temperature (Fig. 3A). Login to comment 81 ABCC7 p.Arg553Met Thus, the R553M mutation significantly enhances the folding yield of NBD1. Login to comment 82 ABCC7 p.Arg553Met ABCC7 p.Arg553Met The Kd for ATP binding to the other NBD1s determined in similar experiments (data not shown) are presented in Table I. NBD1 and only 38% of the NBD1⌬F fold into the soluble conformation, whereas 96% of NBD1-R553M assumes the folded conformation at this temperature (Fig. 3A). Login to comment 83 ABCC7 p.Arg553Met ABCC7 p.Arg553Met Thus, the R553M mutation significantly enhances the folding yield of NBD1. Login to comment 84 ABCC7 p.Arg553Met For the double mutant NBD1⌬F-R553M the folding yield is indistinguishable from that of the wild-type. Login to comment 85 ABCC7 p.Arg553Met Thus, the second site mutation R553M effectively suppresses the ⌬F508 mutation defective folding yield in vitro. Login to comment 86 ABCC7 p.Arg553Met Significantly the R553M mutation increases the length of the lag phase and decreases the rate of change in light scattering, indicating that the rate of formation of the off pathway conformer is dramatically reduced in this mutant. Login to comment 87 ABCC7 p.Arg553Met Once again the double mutant NBD1DF-R553M dramatically increases the lag time and decreases the rate change in light scattering in comparison with NBD1DF. Login to comment 88 ABCC7 p.Gly551Asp ABCC7 p.Ser549Arg ABCC7 p.Arg553Met Significantly the R553M mutation increases the length of the lag phase and decreases the rate of change in light scattering, indicating that the rate of formation of the off pathway conformer is dramatically reduced in this mutant. Login to comment 89 ABCC7 p.Gly551Asp ABCC7 p.Ser549Arg ABCC7 p.Ser549Arg ABCC7 p.Arg553Met Once again the double mutant NBD1⌬F-R553M dramatically increases the lag time and decreases the rate change in light scattering in comparison with NBD1⌬F. Login to comment 90 ABCC7 p.Gly551Asp ABCC7 p.Ser549Arg Two other mutations, S549R and G551D, do not affect the domain folding yield compared with the wild-type NBD1 in vitro (Fig. 4A). Login to comment 91 ABCC7 p.Gly551Asp ABCC7 p.Ser549Arg ABCC7 p.Ser549Arg In vivo studies indicate that the S549R mutation but not G551D is maturation-defective in the full-length protein (11); however, the NBD1-S549R folding yield is not decreased in vitro indicating that it does not act by altering the ability of the domain itself to fold. Login to comment 99 ABCC7 p.Ser549Arg The mGdnHCl values are similar for the NBD1s except for S549R, which has a somewhat larger value indicating that the change in solvent-accessible surface area upon folding is appropriate for a protein of this size. Login to comment 100 ABCC7 p.Ser549Arg ABCC7 p.Arg553Met These results indicate that the inability of the DF508 and S549R CFTR to transit to the apical membrane and the effect of the R553M suppressor cannot be explained simply by a reduction or enhancement in the free energy of stability of the mutant proteins. Login to comment 101 ABCC7 p.Ser549Arg The mGdnHCl values are similar for the NBD1s except for S549R, which has a somewhat larger value indicating that the change in solvent-accessible surface area upon folding is appropriate for a protein of this size. Login to comment 102 ABCC7 p.Ser549Arg ABCC7 p.Arg553Met These results indicate that the inability of the ⌬F508 and S549R CFTR to transit to the apical membrane and the effect of the R553M suppressor cannot be explained simply by a reduction or enhancement in the free energy of stability of the mutant proteins. Login to comment 109 ABCC7 p.Gly551Asp ABCC7 p.Ser549Arg ABCC7 p.Arg553Met ABCC7 p.Arg553Met Protein NBD1 NBD1DF NBD1-R553M NBD1DF-R553M NBD1-S549R NBD1-G551D Kd (mM) 91 88 89 87 81 61 FIG. 3. Login to comment 110 ABCC7 p.Arg553Met The effects of the R553M mutation on the temperature-sensitive folding of NBD1 and the rate of formation of off pathway conformers. Login to comment 111 ABCC7 p.Gly551Asp ABCC7 p.Ser549Arg ABCC7 p.Arg553Met ABCC7 p.Arg553Met ABCC7 p.Arg553Met Protein NBD1 NBD1#2c;F NBD1-R553M NBD1⌬F-R553M NBD1-S549R NBD1-G551D Kd (␮M) 91 88 89 87 81 61 FIG. 3. Login to comment 112 ABCC7 p.Arg553Met ABCC7 p.Arg553Met ABCC7 p.Arg553Met The effects of the R553M mutation on the temperature-sensitive folding of NBD1 and the rate of formation of off pathway conformers. Login to comment 113 ABCC7 p.Arg553Met A, the effects of the R553M mutation on the folding yield of wild-type and NBD1⌬F were determined as described under "Experimental Procedures." Login to comment 114 ABCC7 p.Arg553Met ABCC7 p.Arg553Met Temperature-dependent folding of NBD1 (q), NBD1⌬F (E), NBD1-R553M (ç), and NBD1⌬F-R553M (É). Login to comment 115 ABCC7 p.Arg553Met ABCC7 p.Arg553Met NBD1 (solid line), NBD1DF (dotted line), NBD1-R553M (dashed line), and NBD1DF-R553M (dotted and dashed line). Login to comment 117 ABCC7 p.Arg553Met ABCC7 p.Arg553Met NBD1 (solid line), NBD1⌬F (dotted line), NBD1-R553M (dashed line), and NBD1⌬F-R553M (dotted and dashed line). Login to comment 126 ABCC7 p.Gly551Asp ABCC7 p.Ser549Arg The effects of the S549R and G551D mutations on the temperature-sensitive folding of NBD1 and the rate of formation of off pathway conformers. Login to comment 128 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Ser549Arg ABCC7 p.Ser549Arg The effects of the S549R and G551D mutations on the temperature-sensitive folding of NBD1 and the rate of formation of off pathway conformers. Login to comment 129 ABCC7 p.Gly551Asp ABCC7 p.Ser549Arg B, formation of off pathway conformers of NBD1 (solid line), NBD1DF (dotted line), NBD1-G551D (dashed line), and NBD1-S549R (dotted and dashed line). Login to comment 130 ABCC7 p.Gly551Asp ABCC7 p.Ser549Arg "A,temperature-dependent folding of NBD1 (q), NBD1⌬F (E), NBD1-G551D (ç), and NBD1-S549R (É). Login to comment 131 ABCC7 p.Gly551Asp ABCC7 p.Ser549Arg B, formation of off pathway conformers of NBD1 (solid line), NBD1⌬F (dotted line), NBD1-G551D (dashed line), and NBD1-S549R (dotted and dashed line). Login to comment 135 ABCC7 p.Gly551Asp ABCC7 p.Ser549Arg ABCC7 p.Arg553Met ABCC7 p.Arg553Met The 1.8 mM folded NBD1 (cf;), NBD1DF (E), NBD1-R553M (&#e7;), NBD1DF-R553M (&#c9;), NBD1-G551D (f), and NBD1-S549R (M) in 30 mM Tris-HCl, pH 8.0, 40 mM arginine, 0.2 mM EDTA, and 0.1 mM dithiothreitol were incubated with GdnHCl at the indicated concentration for 2 h. The sample was excited at 282 nm, and fluorescence emission spectra were collected. Login to comment 137 ABCC7 p.Gly551Asp ABCC7 p.Ser549Arg ABCC7 p.Arg553Met ABCC7 p.Arg553Met The 1.8 ␮M folded NBD1 (q), NBD1⌬F (E), NBD1-R553M (ç), NBD1⌬F-R553M (É), NBD1-G551D (f), and NBD1-S549R (Ⅺ) in 30 mM Tris-HCl, pH 8.0, 40 mM arginine, 0.2 mM EDTA, and 0.1 mM dithiothreitol were incubated with GdnHCl at the indicated concentration for 2 h. The sample was excited at 282 nm, and fluorescence emission spectra were collected. Login to comment 147 ABCC7 p.Gly551Asp ABCC7 p.Ser549Arg ABCC7 p.Arg553Met ABCC7 p.Arg553Met Protein DGD,0 DDGD,0 Cm m kJ/mol kJ/mol M kJ/mol/M NBD1 15.5 1.5 10.3 NBD1DF 14.4 21.1 1.3 11.2 NBD1-R553M 16.6 1.1 1.4 11.7 NBD1DF-R553M 14.1 21.4 1.4 10.1 NBD1-S549R 16.7 1.2 1.2 13.4 NBD1-G551D 16.6 1.1 1.4 11.7 reduction in the folding yield in vitro (5) and of the efficiency of maturation and transit to the plasma membrane in vivo (10). Login to comment 149 ABCC7 p.Gly551Asp ABCC7 p.Ser549Arg ABCC7 p.Arg553Met ABCC7 p.Arg553Met Protein ⌬GD,0 ⌬⌬GD,0 Cm m kJ/mol kJ/mol M kJ/mol/M NBD1 15.5 1.5 10.3 NBD1⌬F 14.4 -1.1 1.3 11.2 NBD1-R553M 16.6 1.1 1.4 11.7 NBD1⌬F-R553M 14.1 -1.4 1.4 10.1 NBD1-S549R 16.7 1.2 1.2 13.4 NBD1-G551D 16.6 1.1 1.4 11.7 reduction in the folding yield in vitro (5) and of the efficiency of maturation and transit to the plasma membrane in vivo (10). Login to comment 150 ABCC7 p.Arg553Met First, the R553M suppressor mutation effectively corrects the DF508 folding defect in vitro but does not significantly alter the free energy of stability. Login to comment 151 ABCC7 p.Arg553Met Like DF508, R553M may exert its effect on an intermediate formed during the process of folding. Login to comment 152 ABCC7 p.Arg553Met ABCC7 p.Arg553Met First, the R553M suppressor mutation effectively corrects the ⌬F508 folding defect in vitro but does not significantly alter the free energy of stability. Login to comment 153 ABCC7 p.Arg553Met ABCC7 p.Arg553Met Like ⌬F508, R553M may exert its effect on an intermediate formed during the process of folding. Login to comment 154 ABCC7 p.Arg553Met ABCC7 p.Arg553Met In both cases, the results indicate that the mutations are kinetic in nature but with opposite characteristics; R553M is a superfolder, whereas ⌬F508 is an ineffective folder. Login to comment 155 ABCC7 p.Arg553Met Results in vivo indicate that the R553M/⌬F508 CFTR double mutant only partially corrects the ⌬F508 maturation defect, and the fully mature mutant protein is only partially functional (16). Login to comment 156 ABCC7 p.Arg553Met The current results indicate that the diminished function of R553M observed in vivo is not due to a loss of the ability of NBD1 to bind ATP. Login to comment 162 ABCC7 p.Gly551Asp The G551D mutation, which has no observable effect on the maturation of CFTR in vivo (11) but rather alters the FIG. 6. Login to comment 164 ABCC7 p.Gly551Asp The G551D mutation, which has no observable effect on the maturation of CFTR in vivo (11) but rather alters the FIG. 6. Login to comment 174 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp In addition, G551D does not significantly alter the binding affinity of NBD1-G551D for ATP. Login to comment 175 ABCC7 p.Gly551Asp The effect of the mutation may be due to altered domain-domain interactions or the reduced ability of NBD1-G551D to hydrolyze ATP (7). Login to comment 176 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Ser549Arg In addition, G551D does not significantly alter the binding affinity of NBD1-G551D for ATP. Login to comment 177 ABCC7 p.Gly551Asp ABCC7 p.Ser549Arg The effect of the mutation may be due to altered domain-domain interactions or the reduced ability of NBD1-G551D to hydrolyze ATP (7). Login to comment 178 ABCC7 p.Ser549Arg Similarly, the S549R mutation, which like ⌬F508 inhibits the formation of mature functional CFTR in vivo (11), has no discernible effect on the ability of NBD1 to fold (Fig. 4). Login to comment 179 ABCC7 p.Ser549Arg The S549R mutation may alter a surface important for interaction with other CFTR domains or other proteins during the process of conformational maturation. Login to comment