(Received for publication, December 15, 1995; and in revised form, January 26, 1996)
From the
The cellular phenotype of the most common cystic
fibrosis-causing mutation, deletion of phenylalanine 508 (F508) in
the amino-terminal nucleotide binding domain (NBD1) of the cystic
fibrosis transmembrane conductance regulator (CFTR), is the inability
of the mutant protein to fold and transit to the apical membrane of
affected epithelial cells. Expressed NBD1s were purified and folded in vitro into soluble monomers capable of binding nucleotide.
Here we report that the
F508 mutation has little effect on the
thermodynamic stability of the folded NBD1. The
G
is 15.5 kJ/mol for the wild type
NBD1 and 14.4 kJ/mol for NBD1
F. In contrast, the mutation
significantly reduces the folding yield at a variety of temperatures,
indicating that Phe-508 makes crucial contacts during the folding
process, but plays little role in stabilization of the native state.
Under conditions that approximate the efficiency of maturation in
vivo, the rate off-pathway is significantly increased by the
disease causing mutation. These results establish a molecular mechanism
for most cases of cystic fibrosis and provide insight into the complex
processes by which primary sequence encodes the three-dimensional
structure.
Cystic fibrosis is a common fatal genetic disease caused by
mutations in the cystic fibrosis transmembrane conductance regulator
(CFTR) ()gene(1, 2, 3) . The
product of this gene is a plasma membrane cAMP-dependent Cl
channel gated in response to binding and hydrolysis of
ATP(4, 5, 6, 7) . Although more than
500 CF-causing mutations have been identified, the
F508 mutation
in the amino-terminal nucleotide binding domain (NBD1) is the most
prevalent, accounting for approximately 70% of the disease-causing
alleles(2, 3) . The
F508 mutation leads to
diminished amounts of mutant CFTR in the membranes of epithelial cells (8, 9, 10, 11, 12) , and,
thus, the decreased chloride conductance that is a hallmark of the
disease.
Two findings indicate that the basis of this phenotype is a
defect in protein folding. First, the mutation destabilizes the
functional conformation of a synthetic peptide containing the Phe-508
region(13) . Second, when cells expressing the F508
protein are grown at reduced temperature, the maturation defect is
partially corrected(14) . Along with the finding that the
F508 mutant protein is functional when it reaches the native
state(13, 14, 15) , this information suggests
that correcting the maturation defect may ameliorate this form of the
disease.
Recent studies suggest that the structural maturation
defect in the F508 mutant occurs at an early step in
vivo(16, 17) . In addition, these studies
indicate that the
508 mutation affects the rate of CFTR
maturation. Moreover, CF mutations cluster in the
NBDs(2, 3) , and many CF mutations identified as
maturation-defective(18) , are located in NBD1, implicating
defective folding of this domain in the vast majority of CF cases. To
quantitatively investigate the effect of the
F508 mutation on the
folding of this domain in greater detail, we have developed an in
vitro folding system. In the present study, we use the system to
examine the effects of this common CF-causing mutation on the
thermodynamic stability and folding pathway of NBD1.
Figure 1:
Expression, purification, folding,
and function of NBD1 and NBD1F. A, Coomassie-stained
Tricine SDS-PAGE of 10 µl of uninduced BL21 cell lysate (lane
1), induced NBD1 lysate (lane 2), 5 µg of purified
NBD1 (lane 3), induced NBD1
F lysate (lane 4),
and purified NBD1
F (lane 5). B, fluorescence
emission spectra of denatured and folded, purified NBD1 (solid
line) and NBD1
F (dashed line), 18 µM in
buffer B. C, HPLC gel filtration chromatography of 1 µg of
folded NBD1 (solid line) and 1 µg of folded NBD1
F (dashed line) resolved on an Alltech Macrosphere 300 GPC 7U
column with 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, and
400 mML-arginine mobile phase buffer. Size markers
are indicated by ⊤ (see ``Materials and Methods''). D, TNP-ATP binding to purified, folded NBD1 (solid
line) and NBD1
F508 (dashed line) in buffer B was
determined as described
previously(13, 21, 22) .
Denatured NBD1 and NBD1F were folded into nucleotide binding
monomers in vitro. The key to the success of the folding was
the inclusion of L-arginine in the folding
buffer(23, 24) . Possibly, the guanidinium group of
arginine increases the solubility of exposed polar side chains in the
denatured NBD1 and the amphipathic character of the amino acid protects
exposed hydrophobic interaction surfaces in the folded domains. In this
regard, it is important to note that NBD1 has been removed from the
context of the large multidomain membrane protein in which it normally
resides. Thus, surfaces that are normally involved in domain-domain
interactions in the intact protein, and which may be responsible for
the tendency of NBD1 to form polymers (21) or interact with
membranes in vitro(25) , may be shielded by arginine.
However, in the presence of 400 mM arginine, NBD1 can be
folded and maintained as a soluble, functional monomer as assessed by
intrinsic tryptophan fluorescence, size exclusion chromatography, and
TNP-ATP binding (Fig. 1, B-D).
Fluorescence
emission spectra of the wild type and F508 NBD1s reveal a
pronounced blue shift in the peak position and an increase in
fluorescence intensity upon removal of the chemical denaturant,
consistent with burial of the single tryptophan at position 496 in a
hydrophobic environment as the domain folds (Fig. 1B).
Four additional lines of evidence argue that both NBD1s are folded.
First, they are soluble in the absence of denaturant. Second, both
elute from a molecular sizing column intermediate to carbonic anhydrase
(29 kDa) and aprotinin (6.5 kDa), a position consistent with a globular
monomer with a predicted molecular weight of 22,000 (Fig. 1C). Third, as has been described previously for
expressed soluble NBD1(15, 21) , in vitro folded NBD1 and NBD1
F bind the nucleotide TNP-ATP (Fig. 1D). The apparent K
of both
NBD1s for TNP-ATP is 3 µM under these conditions in
agreement with previous results(21) . Finally, denaturation of
the folded NBD1s is highly cooperative, indicating disruption of an
ordered structure (Fig. 2A, inset).
Figure 2:
Denaturation of NBD1 and NBD1F. The
native structures of folded NBD1 and NBD1
F were denatured by
addition of the chemical denaturant GdnHCl or by increasing the
temperature. In the absence of GdnHCl, the denatured domains form
insoluble associations that scatter 400 nm light. A,
denaturation of NBD1 resulted in decreased tryptophan fluorescence and
a red shift in the emission maximum as the single tryptophan, Trp-496,
is exposed to solvent (see Fig. 1B). 1.8 µM folded wild type NBD1 (
-
) and NBD1
F
(
- - -
) in buffer B were incubated
with GdnHCl at the indicated concentration for 2 h. The sample was
excited at 282 nm and fluorescence emission spectra were collected. The
equilibrium dependence of the fluorescence emission peak position on
the denaturant concentration (inset) reveals cooperative
unfolding of the domain. As this is a reversible process which
approximates a two-state conversion, the free energy of denaturation
(
G
) can be calculated from the
fraction folded over the transition region. Extrapolation to the
absence of denaturant indicates that
G
between NBD1 and NBD1
F
is 1.1 kJ/mol. B, thermal denaturation of folded NBD1 and
NBD1
F. NBD1 (0.9 µM, solid line) and
NBD1
F (0.9 µM, dashed line) in buffer B were
heated from 6 to 75 °C at a rate of 0.5 °C/min. Scattered light
was measured at 400 nm at an angle of
90°.
Figure 3:
Kinetic competition between folding and
off-pathway steps. The effects of the F508 mutation on the folding
yield and rate of formation of off-pathway conformers were determined in vitro. A, temperature-dependent folding of NBD1 (solid
lines) and NBD1
F (dashed lines). Unfolded domains in
6 M GdnHCl were diluted 30-fold with buffer B to final protein
concentrations of 2 µM (closed symbols) or 18
µM (open symbols) and incubated at the indicated
temperature for 14 h. The relative folding yield was determined as the
fraction of soluble protein determined from the fluorescence emission
intensity. B, kinetics of formation of off-pathway conformers
of NBD1 (solid line) and NBD1
F (dashed line) (18
µM) at 23 °C in buffer B. The concentration of GdnHCl
was rapidly reduced by dilution, and scattered light was monitored as
in Fig. 2B.
Previous results demonstrated that a
peptide fragment of NBD1, P67, containing the A consensus region and a
region of homology around Phe-508, capable of binding ATP, is
destabilized by the F508 mutation(13) . These results
suggested that conditions counteracting the destabilizing effects may
allow maturation of the mutant CFTR and rescue of the disease
phenotype(13, 28) . It was subsequently observed that
reduction of the temperature at which expressing cells were grown
increased the efficiency of CFTR maturation(14) . Moreover,
functional CFTR chloride channels could be observed in the plasma
membrane at the sensitive temperature of 35 °C after growth at the
permissive temperature of 26 °C. These findings are consistent with
the current results which indicate that a step on the folding pathway,
rather than the stability of the native state is affected by the
mutation. Thus, destabilization of the peptide P67 by the
F508
mutation suggests it may provide a model of a kinetically trapped
folding intermediate (28) .
The fact that Phe-508 is
critical for interactions that direct folding, but makes little
contribution to the stability of the native state is not unique. For
example, a large number of mutations of the P22 tail spike protein
affect its folding but not its native state stability, indicating that
non-native state interactions may be important for directing the
folding pathway(29, 30) . These mutations are, thus,
temperature-sensitive for folding (tsf), as is F508.
Interestingly, global second site suppressor mutants of these tsf mutations have been isolated(30) . It remains an
intriguing possibility that intragenic suppressors of the
F508
phenotype (31, 32) may act by correcting the folding
defect.
Understanding the interactions that take place on the CFTR folding pathway may have profound importance for describing the mechanisms of both the disease process and how primary sequence determines the final native structure of proteins. Therapies directed at correcting the folding defect deserve further consideration as treatments for cystic fibrosis. Potentially, alteration of molecular chaperone expression or of cellular conditions to increase the on-pathway rate or decrease the off-pathway rate may prove useful. However, it is important to remember that simply inhibiting the final proteolytic off-pathway step is apparently not adequate to correct the disease phenotype (26, 27) as might be expected if the steps prior to proteolysis were in equilibrium with the native state. More likely, positive impact on the disease state will require intervention at the initial off-pathway steps.