(Received for publication, December 17, 1996, and in revised form, March 18, 1997)
From the Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9040
A growing body of evidence indicates that the
most common cystic fibrosis-causing mutation, F508, alters the
ability of the cystic fibrosis transmembrane conductance regulator
(CFTR) protein to fold and transit to the plasma membrane. Here we
present evidence that the
F508 mutation affects a step on the
folding pathway prior to formation of the ATP binding site in the
nucleotide binding domain (NBD). Notably, stabilization of the native
state with 4 mM ATP does not alter the
temperature-dependent folding yield of the mutant
F508
NBD1 in vitro. In contrast, glycerol, which promotes
F508-CFTR maturation in vivo, increases the folding yield of NBD1
F and reduces the off pathway rate in
vitro, although it does not significantly alter the free energy
of stability. 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. 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. These results demonstrate that
F508 is a
kinetic folding mutation that affects a step early in the process, and that there is a significant energy barrier between the native state and
the step affected by the mutation precluding the use of native state
ligands to promote folding. The implications for protein folding in
general are that the primary sequence may not necessarily simply define
the most stable native structure, but rather a stable structure that is
kinetically accessible.
Cystic fibrosis (CF)1 is a disease of
fatal consequence that, at the genetic level, is due to mutation of the
gene that encodes the cystic fibrosis transmembrane conductance
regulator (CFTR) (1). This protein, normally found in the apical
membrane in secretory epithelia, is of central importance for the fluid
secretion necessary for hydration of the pulmonary mucus and for
secretion of digestive enzymes in the pancreas (2). The most common CF genotype is a 3-base pair in-frame deletion in exon 10 that results in
the loss of a single phenylalanine residue at position 508 (F508) in
a nucleotide binding domain (NBD1) (1), which is important for the ATP
dependence of CFTR function (3-9). The cellular phenotype of the
F508 mutation is a defect in protein maturation and transit to the
plasma membrane (10). 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).
Previously we suggested that the defect in
F508-CFTR intracellular
trafficking was secondary to the diminished ability of the mutant NBD1
to achieve its native, functional conformation (13, 14). Studies on a
67-amino acid fragment of NBD1 (14) and intact NBD1 (5) are consistent with the notion that defective folding is responsible for the defective
maturation of the mutant protein and, thus, the lack of functional
protein.
Alteration of other residues in CFTR-NBD1 and of cellular conditions is
known to ameliorate the defective maturation and trafficking defect
caused by the F508 mutation. 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). Two
mutations at this position, R553M and R553Q, revert the mating phenotype of a
F508 STE6-CFTR chimera in yeast (16). Moreover, introduction of the second site mutations into human
F508-CFTR partially corrected the maturation defect, resulting in an increase of
functional CFTR at the membrane (16). Recently two groups have
demonstrated that when cells expressing
F508-CFTR are grown in the
presence of glycerol the maturation and functional defects can be
largely rescued (17, 18). Such a result had been predicted with the
suggestion that conditions, which stabilize protein structure during
the folding process, might restore wild-type function to the
F508-CFTR (13, 14).
Previously an in vitro folding system was developed to
quantitatively investigate the effect of the F508 mutation on the folding of NBD1 (5). These studies demonstrated that
F508 in NBD1
was a temperature-sensitive folding (tsf) mutation,
suggesting the mutation altered the folding pathway of NBD1 rather than
its native state thermodynamic stability. 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. These studies provide direct evidence that the
defect is on the folding pathway and that manipulations that correct
the maturation defect of the full-length protein in vivo
also correct the defective folding of the first nucleotide binding
domain in vitro, establishing a causal link between the two
observations.
Oligonucleotide-mediated mutagenesis (19) was used to
generate R553M, G551D, and S549R mutations in plasmid pBQ2.4 containing CFTR cDNAs. The mutant primers are as follows: R553M primer,
5-GAAATTCTTGCCATTTGACCTCCAC-3
(25 bases);
G551D primer, 5
-CTTGCTCGTTGATCTCCACTCAGTG-3
(25 bases); S549R primer,
5
-CGTTGACCTCCTCTCAGTGTGATTCC-3
(26 bases).
Production of mutations was verified by DNA sequencing. Expression
cassette polymerase chain reaction was employed to synthesize the
cDNA fragments of CFTR NBD1-R553M, NBD1-G551D, and NBD1-S549R
containing a 5
NdeI site, a 3
XhoI site, and a
stop codon as described previously for NBD1 and NBD1
F (5). Digested
polymerase chain reaction products were ligated into the
NdeI and XhoI sites of the pET28a plasmid. To
construct the NBD1
F-R553M expression vector, pET28a NBD1-R553M and
pET28a NBD1
F were digested with SphI. 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. The proper
construct was verified by restriction digestion and sequencing.
The NBD1 proteins were purified from expressing BL21 (DE3) Escherichia coli strains and folded in vitro as described previously (5). The thermodynamic stability of the purified NBD1s was determined by extrapolation of the GdnHCl melting profiles (5).
The temperature dependence of the folding yield experiments was
determined by monitoring the intrinsic tryptophan fluorescence at 2 µM final protein concentration (5). The fluorescence
method allows not only the amount of protein to be quantitated but
reports on the conformation as well. For the effect of ATP on the
temperature-dependent folding of NBD1F, unfolded protein
was diluted with buffer B (100 mM Tris-HCl, pH 8.0, 400 mM L-arginine·HCl, 2 mM EDTA, and 1 mM dithiothreitol) containing 4 mM ATP. An
excitation wavelength of 290 nm was used in the presence of ATP to
avoid nucleotide-dependent absorption of the incident
light.
Nucleotide binding was determined from the quenching of protein tryptophan fluorescence upon ATP binding to the NBD1 proteins. Samples containing 1.8 µM NBD1s in buffer B at the indicated concentration of ATP were excited with 295 nm of monochromatic light (2-nm band-pass). Uncorrected emission spectra were collected from 305 nm to 400 nm (4-nm band-pass). The dissociation constant (Kd) was calculated by nonlinear regression of the data according to the equation,
![]() |
(Eq. 1) |
To determine the rates of formation of
off folding pathway species, NBD1s were solubilized in 6 M
GdnHCl and rapidly diluted 30-fold with buffer B to a final protein
concentration of 18 µM at 23 °C or 2 µM
at 37 °C. Under these conditions the folding yields of wild-type and
NBD1F are much less than at 4 °C (5). Scattered light at 400 nm
was measured at a 90° angle or by turbidity to monitor the
aggregation progress. In experiments containing glycerol, 10% glycerol
was present in buffer B at the time of rapid dilution of NBD1
F.
The cDNAs for
wild-type and mutant NBD1s corresponding to exons 9-12 of the CFTR
gene (Fig. 1) were cloned into pET-based expression
vectors capable of directing the expression of this domain
(Gly404 to Ser589) as a fusion with an
amino-terminal polyhistidine tract. The expression levels of NBD1-R553M
and NBD1F-R553M are similar to NBD1 and NBD1
F (5). The expression
levels of NBD1-G551D and NBD1-S549R are approximately one-half of the
wild-type NBD1 level (data not shown). The wild-type and all mutant
proteins form inclusion bodies under these growth conditions in
E. coli. The inclusion bodies were isolated, dissolved in
GdnHCl, and purified on His-tag affinity resin under denaturing
conditions. Densitometry of the proteins resolved by 10% Tricine
SDS-polyacrylamide gel electrophoresis (20) and stained with Coomassie
Blue indicated that all samples are greater than 95% pure.
All denatured mutant NBD1s can be folded into functional nucleotide
binding conformations in vitro in buffer B by the criteria of cooperative burial of the Trp496 residue, solubility,
and the ability to bind nucleotide. The saturable binding of ATP to the
wild-type NBD1 is shown in Fig. 2. Upon interaction with
the nucleotide the domain changes to a conformation in which
Trp496 fluorescence is partially quenched. The fact that
the binding is greatly attenuated in the presence of GdnHCl indicates
that high order structure is required for interaction with the
nucleotide. None of the mutations has a dramatic effect on the affinity
of the isolated domain for ATP. The apparent Kd of
NBD1s for ATP binding are listed in Table I.
|
Fig. 3A shows the
temperature dependence of the folding processes of each of the NBD1s.
The data for wild-type and NBD1F were previously published (5) and
are presented here for comparison. At 37 °C, 63% of the wild-type
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). Thus, the R553M mutation
significantly enhances the folding yield of NBD1. For the double mutant
NBD1
F-R553M the folding yield is indistinguishable from that of the
wild-type. Thus, the second site mutation R553M effectively suppresses
the
F508 mutation defective folding yield in vitro.
The rate of formation of the off pathway conformer was assessed by
light scattering at 18 µM protein concentration and
23 °C. A lag phase followed by an increase in light intensity was
observed (Fig. 3B). 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. Once again
the double mutant NBD1F-R553M dramatically increases the lag time
and decreases the rate change in light scattering in comparison with
NBD1
F.
Two other mutations, S549R and G551D, do not affect the domain folding
yield compared with the wild-type NBD1 in vitro (Fig. 4A). 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. Moreover, neither
mutation affects the rate of formation of the off pathway conformer of
this domain (Fig. 4B).
Thermodynamic Stability of NBD1s
The GdnHCl-induced
cooperative unfolding of the domains is shown in Fig. 5.
The concentration of GdnHCl required is similar for all NBD1s. The
Cm values are between 1.2 M and 1.5 M (Table II). The GD
of the NBD1s, indicating the domains' thermodynamic stability, can be
calculated from this reversible two-state transition (Fig. 5,
inset). Compared with the wild-type NBD1, the maximum mutant
GD,0 is only 1.4 kJ/mol (Table II). In
comparison, the
GD,0 of the native state
wild-type NBD1 is 15.5 kJ/mol. Thus, the effect of the
F508 mutation
on the stability of NBD1 under these conditions is minimal. The
cooperativity of the transition is reflected in the
mGdnHCl values (the slope of the natural log of
the fraction folded versus the denaturant concentration)
(Fig. 5, inset). 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. 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.
|
As shown in Fig. 6, the formation of off
pathway species is inhibited by the presence of 10% glycerol. In
addition, the temperature-dependent folding yield is
increased by this osmolyte (data not shown). These results provide an
in vitro explanation for the observation that when glycerol
is added to cultured cells expressing CFTR the amount of mutant protein
that is successfully trafficked to the plasma membrane is increased.
The in vitro results here suggest that the mechanism by
which glycerol works is to assist the mutant nucleotide binding domain
in its folding. GdnHCl unfolding of NBD1F in the presence of 10%
glycerol (data not shown) yields a
GD,0 of 14.3 kJ/mol representing a
GD,0 of 0.1 kJ/mol
compared with the
GD,0 value reported for the
mutant in the absence of glycerol (5). Therefore, the presence of 10%
glycerol does not have a significant effect on the Gibb's free energy
of stability of the domain.
Folding of NBD1
Since the F508
mutant form of the protein is functional when folded, it had been
suggested that native state ligands might serve to stabilize this
conformation and overcome the folding defect (13, 14). Recently such an
approach has been effective in promoting the folding of mutant forms of
transthyretin and the CFTR homologue P-glycoprotein (21, 22). To test
this possibility we examined the effect of the natural ligand ATP on
the folding yield and off pathway rate of NBD1
F. The folding
reaction and off pathway reaction were performed in the presence of 4 mM ATP. This concentration is at least 40 times the
KD of NBD1
F for ATP. Although the binary complex
is more stable than NBD1
F alone, the presence of the nucleotide did
not increase the folding yield or decrease the off pathway rate (Fig.
7).
An emerging body of evidence indicates that many genetic diseases have their basis in the defective folding of the protein product of the mutant gene (23). For example, in the case of cystic fibrosis, deletion of a single phenylalanine residue at position 508 in the 1480-amino acid CFTR protein leads to a reduction in the folding yield in vitro (5) and of the efficiency of maturation and transit to the plasma membrane in vivo (10). Understanding the molecular pathology of this disease should provide not only a means of developing therapeutic strategies, but also aid in elucidating the relationships between primary structure and the process of folding a protein sequence into its functional three-dimensional structure.
The results presented here support our earlier studies which suggested
that the defective folding of F508 CFTR is due to alteration of the
process of folding rather than to destabilization of the native state
of NBD1 (5). First, the R553M suppressor mutation effectively corrects
the
F508 folding defect in vitro but does not
significantly alter the free energy of stability. Like
F508, R553M
may exert its effect on an intermediate formed during the process of
folding. 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. 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). 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.
Altered function may then be due to deficient catalysis or coupling
between the nucleotide site and other parts of the protein. The fact
that in the in vitro folding system the ATP binding function
of the double mutant is normal and the suppression is complete, as
opposed to the partial function and suppression in vivo
(16), indicates that although NBD1 folding reliably reports the
fundamental effects of the mutations the situation is more complex in
the cell, and subtle secondary effects may not always be evident in the
in vitro system. For example, the reduction of the folding
yield by the
F508 mutation is not as pronounced as in
vivo, perhaps indicating that cellular mechanisms for identifying
malfolded conformers are more efficient than in vitro
aggregation.
A second line of evidence that the F508 folding defect is kinetic in
nature is that addition of the native state ligand has no discernible
effect on the folding yield or on the rate of formation of off pathway
conformers (Fig. 7). Although not all of the binding energy contributes
to the stabilization of the native state, as some is expended to affect
the conformational change, the binary complex would be expected to be
significantly more stable than the native state in the absence of
nucleotide. The fact that the addition of ATP does not increase the
folding yield is, thus, consistent with the notion that the
intermediate affected by the mutation is kinetically isolated from the
native state by a significant energy barrier during the process of
folding.
The other CF-associated mutations tested here have no significant
effects on either the temperature-dependent folding yield or the off pathway kinetics under our experimental conditions. The
G551D mutation, which has no observable effect on the maturation of
CFTR in vivo (11) but rather alters the ability of the
mature protein to function (7), has no effect on the ability of NBD1 to
fold as would be expected. In addition, G551D does not significantly alter the binding affinity of NBD1-G551D for ATP. The effect of the
mutation may be due to altered domain-domain interactions or the
reduced ability of NBD1-G551D to hydrolyze ATP (7). 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). The S549R mutation may alter a
surface important for interaction with other CFTR domains or
other proteins during the process of conformational maturation.
We suggested previously that alcohols and other compounds that are
known to stabilize protein and peptide structures might restore
wild-type like folding to the F508-CFTR (13, 14). In this regard, it
has been demonstrated recently that such a compound, glycerol, corrects
the defective maturation of
F508 in vivo (17, 18).
In vitro, 10% glycerol inhibits the off pathway reaction
and significantly enhances the temperature-dependent folding yield of
F508 NBD1. Glycerol does not significantly affect the free energy of stability of the native state. Therefore, its effects may be to stabilize the putative mutationally sensitive intermediate or transition state proposed here, or to inhibit the off
pathway association reaction of partially folded chains. Notably, this
intermediate is not significantly populated under equilibrium
conditions as the denaturation curves fit a two-state approximation.
With these results in mind, our previous suggestion for therapeutic
ligands should be modified to either the special case of compounds that
lower a kinetic barrier by stabilizing a crucial transition or those
that specifically interfere with the formation of off pathway
species.
The results presented here indicate that mutational and environmental
effects on the folding of NBD1 of CFTR in vitro in many ways
parallel their effects on the folding and maturation of the full-length
protein in vivo. As such, this study demonstrates that a
defect in protein folding is the biochemical basis of CF. Thus,
although commonly referred to as a trafficking defect, the F508
mutation is primarily a protein folding defect with altered protein
trafficking or kinesis secondary to altered folding. The distillation
of the pathology of CF to a single soluble protein domain is a step
toward generating a molecular description of the disease process. In
addition, since most cases of CF are due to defective folding and
maturation, this information is necessary for the logical design of
novel therapeutics directed at ameliorating the disease in humans.
The plasmids pBQ4.7 and pCOFF508
containing CFTR cDNAs for wild-type and
F508 were a generous
gift from Dr. J. Rommens. We thank Yu Zeng for technical
assistance and Dr. S. Muallem and the members of the Thomas and
Rizo-Rey laboratories for helpful discussions.