From the
We have used expression of human medium chain acyl-CoA
dehydrogenase (MCAD) in Escherichia coli as a model system for
dissecting the molecular effects of two mutations detected in patients
with MCAD deficiency. We demonstrate that the R28C mutation
predominantly affects polypeptide folding. The amounts of active R28C
mutant enzyme produced could be modulated between undetectable to 100%
of the wild-type control by manipulating the level of available
chaperonins and the growth temperature. For the prevalent K304E
mutation, however, the amounts of active mutant enzyme could be
modulated only in a range from undetectable to approximately 50% of the
wild-type, and the assembled mutant enzyme displayed a decreased
thermal stability. Two artificially constructed mutants (K304Q and
K304E/D346K) yielded clearly higher amounts of active MCAD enzyme than
the K304E mutant but were also responsive to chaperonin
co-overexpression and growth at low temperature. The thermal stability
profile of the K304E/D346K double mutant was shifted to even lower
temperatures than that of the K304E mutant, whereas that of the K304Q
mutant was closely similar to the wild-type. Taken together, the
results show that the K304E mutation affects (i) polypeptide folding
due to elimination of the positively charged lysine and (ii) oligomer
assembly and stability due to replacement of lysine 304 with the
negatively charged glutamic acid.
Medium chain acyl-CoA dehydrogenase (MCAD
Besides a series of rare point mutations, short
insertions, and deletions
(5, 6, 7) , one
prevalent point mutation
(8, 9, 10, 11) is present in more than 90% of the alleles in patients with
MCAD deficiency
(5) . This mutation is an A
To
delineate the pathological mechanism, the molecular consequences of the
K304E mutation have been investigated. We have used an approach where
mature human MCAD was expressed in Escherichia coli with and
without co-overexpression of the chaperonins GroEL and GroES, two
proteins that assist in folding of newly synthesized polypeptide chains
(for a recent review, see Ref. 14). We demonstrated that K304E mutant
MCAD can be partially rescued from misfolding in the presence of excess
GroESL and that a minor portion of the expressed K304E MCAD protein
folded into the native tetrameric structure with a specific activity in
the range of the wild-type enzyme
(15) . From these data and
comparative analysis of a MCAD variant with the artificially
constructed K304Q mutation, we proposed that the K304E mutation has a
2-fold effect: one on polypeptide folding, due to elimination of the
positive charge in the side chain, and another one on oligomer
assembly, due to the introduction of a negatively charged side chain.
The effect on oligomer assembly is supported by data from the crystal
structure of porcine MCAD
(16) , which show that lysine 304 is
situated in helix H, forming part of the subunit interface. With in
vitro translation experiments in the presence of rat liver
mitochondria, Saijo et al. (17) showed that, after
import into mitochondria, MCAD chains transiently interact with the
mitochondrial heat shock protein 70 (hsp70) and subsequently with
chaperonin hsp60, a homolog of the bacterial GroEL, before assembled
MCAD tetramers can be observed. The sequence of events was similar for
K304E mutant MCAD, but this protein remained associated with hsp60 for
longer time periods, indicating that folding and/or oligomer assembly
is impaired. These authors could not discriminate between an effect of
the mutation on monomer folding or oligomer assembly.
Another
missense mutation in the MCAD gene (T157), which has been detected in
two patients in compound heterozygosity with the G985 mutation, results
in the substitution of arginine 28 with cysteine (R28C)
(6) .
One of the patients died unexpectedly at day 3 after birth, while the
other had clinical symptoms at 8 months of age
(18) , indicating
the potential severity and variable expression of the mutation. In
COS-7 cell-based expression experiments the R28C mutant enzyme
displayed only moderately decreased MCAD activity levels (50-100%
of wild-type)
(6, 19) . Expression of this mutant
protein in E. coli revealed a strong effect of high chaperonin
levels on the produced amounts of active R28C mutant MCAD
(19) .
Studies with mutants in the coat and tailspike proteins of phage P22
have identified a group of so-called temperature-sensitive folding
mutations which primarily affect the yield of correctly folded
polypeptide, but not, or only to a minor extent, the stability and
functional integrity of the native structure once it is formed
(20, 21) . It has been demonstrated that
co-overexpression of the GroESL chaperonins could suppress the folding
defects of temperature-sensitive folding mutants in the phage P22 coat
protein but not in the tailspike protein
(22) .
The aim of
the present study was to distinguish the effect of the K304E and R28C
mutations on MCAD polypeptide folding from effects on oligomer assembly
and stability of the active tetramer in order to explore to what extent
the cellular folding machinery may be able to modulate manifestation of
the defect in patients harboring these mutations. Folding conditions
were optimized by growth at low temperature and co-overexpression of
the GroESL chaperonins, and the amounts of active mutant MCAD produced
as well as the temperature stability of the tetrameric enzyme variants
were analyzed. The effect of the charge reversal in the prevalent K304E
mutation was investigated further by introducing second site mutations
in charged residues that reestablish the net charge constellation
around residue 304.
For construction of
the second site mutations, plasmids pWTMCAD-2 encoding wild-type human
MCAD protein and p985 encoding K304E mutant protein, described
previously
(15, 23) , were cleaved with PstI
and HindII or PstI and BamHI respectively,
and the fragments comprising the MCAD cDNA insert were purified by gel
electrophoresis. These fragments were then used as templates for a
megaprimer PCR mutagenesis procedure described by Kuipers et al. (24) with the following modifications. The megaprimer was
amplified using primer 1327- HindIII and either one of the
mutagenic primers pri-( s)+959
(5`-CATTTATGCTGGCTAAAATGG-3`; E300K mutation) or
pri-( s)+1099 (5`-CAGTTAGCTACTAAAGCTGTGCAG-3`; D346K
mutation). After 20 cycles of PCR, the amplified fragments were
purified by ultrafiltration (Centricon 100; Amicon). A second round of
PCR was performed with each of the megaprimers, a new universal primer
pri-( s)+378 (5`-TTCTTTGGGGCAAATGCCTA-3`) and the same
templates as in the first PCR. The PCR product was precipitated,
cleaved at the internal EcoRI site and the introduced
HindIII, site and ligated into pWt cleaved with the same
enzymes. The ligation product was transformed into E. coli JM109. Clones were checked for the absence of PCR errors.
For
the wild-type protein, the amounts of active enzyme produced in the
samples, both with and without GroESL overexpression, is similar at 28,
31, and 34 °C (Fig. 1). There is only slightly more active
enzyme present at high chaperonin levels. This is also reflected by a
low amount of insoluble MCAD material in the samples without GroESL
co-overexpression. At 37 °C, the difference between the samples
with and without GroESL co-overexpression is somewhat higher, and at 40
°C GroESL co-overexpression increases the yield of active MCAD by a
factor of approximately 2, indicating that the amount of GroESL becomes
rate-limiting for the wild-type protein at these temperatures.
Concomitantly, the amounts of insoluble wild-type MCAD protein are
increased at these temperatures, and an effect of GroESL
co-overexpression on the partition between soluble and insoluble
wild-type MCAD becomes evident.
All mutant variants
display a high proportion of insoluble MCAD protein at 37 and 40
°C. The total amount of MCAD mutant material detected at these
temperatures is similar to the amount of wild-type protein, indicating
that transcription and translation rates are identical for all
variants. At low temperatures and without GroESL co-overexpression, the
amount of mutant MCAD material detected in the pellet fraction is low.
However, in some of these samples ( e.g. R28C at 34 and 31
°C) only slightly higher amounts are observed in a soluble form.
The decreased amount of insoluble MCAD material in these samples is
therefore likely to be due both to conversion of a larger proportion of
the polypeptide chains into soluble species and to increased
degradation of accumulating MCAD folding intermediates.
This
result was further confirmed by a qualitative determination of the
ratio between wild-type and K304E MCAD material in the cells
heterozygous for the K304E mutation. We have previously shown that
wild-type and K304E mutant MCAD can be separated by two-dimensional gel
electrophoresis. Two spots each, one modified and one nonmodified form,
could be observed for wild-type and K304E MCAD, respectively
(27) . The results are depicted in Fig. 3. Clearly, the
steady-state amount of K304E MCAD relative to the wild-type protein is
higher in the cells cultivated at 34 °C than at 37 or 39 °C.
This demonstrates that the relative amount of K304E MCAD protein is
temperature-sensitive in these cells.
The mutants were expressed at 28 and 37 °C in
E. coli with and without co-overexpression of the GroESL
chaperonins. As shown in Fig. 5 A, the E300K single and
the E300K/K304E double mutant variants yield very low or undetectable
amounts of MCAD activity when produced at 37 °C, although
considerable amounts of soluble MCAD protein were observed in the
samples with GroESL co-overexpression (data not shown). When the cells
were grown at 28 °C, increased amounts of active E300K single and
E300K/K304E double mutant MCAD could be observed, with the double
mutant displaying lower levels than the single mutant. Analysis of
these extracts by native PAGE/Western blotting revealed an amount of
tetramer roughly corresponding to the MCAD activity measured (data not
shown). This means that the effect of the E300K mutation increases
rather than compensates the effects of the K304E mutation. The E300K
and E300K/K304E mutant enzymes were very unstable as indicated by a
dramatically decreased MCAD enzyme activity level after only one
freeze/thaw cycle of the extracts (data not shown).
The thermal
stability profile of the K304E/D346K double mutant was determined and
is displayed in Fig. 5 B. The profiles for wild-type and
K304E MCAD are replotted from Fig. 2for comparison. The profile
for the K304E/D346K double mutant is shifted to even lower temperatures
than the K304E single mutant. We also attempted to determine the
profile for the D346K single mutant. It turned out that this mutant
protein was very unstable in the handling. While it was not possible to
obtain a reliable profile, it was apparent that the D346K enzyme was
inactivated at temperatures above 30 °C. This shows that the D346K
mutation itself strongly compromises enzyme stability, and that this
effect is to some extent compensated by the K304E mutation in the
K304E/D346K double mutant.
In the present study we have exploited both chaperonin
co-overexpression and growth at low temperatures in an E. coli model system to maximize the folding yield and thereby overcome
the effect of mutations in MCAD which impair folding of the
polypeptide. The percentage of wild-type level to which the mutant MCAD
variants could be restored by these treatments was assessed, and this
was used to evaluate to what extent the folding environment can
modulate the steady state level of active MCAD of two disease-causing
mutant variants.
Our experiments demonstrate that the R28C mutation
primarily affects polypeptide folding rather than the stability of the
assembled enzyme. This mutation may thus be compared to the group of
temperature-sensitive folding mutations investigated in detail for the
phage P22 tailspike and coat proteins, which are impaired in kinetics
of folding but not in the stability of the folded state. Manipulation
of the growth temperature and the availability of chaperonins in the
E. coli model system made it possible to modulate the relative
amounts of active R28C MCAD from undetectable to levels in the range of
the wild-type. In a parallel study we had shown that expression of the
R28C mutant in eucaryotic COS-7 cells produced 50-100% of the
amounts observed for wild-type MCAD
(6, 19) , indicating
that a particularly high capacity of the folding machinery in COS-7
cells may camouflage the folding problem. This mutant protein may thus
be expected to be strongly sensitive to factors affecting the folding
capacity of the cell. Arginine 28 is situated in helix A and forms a
salt bridge with glutamic acid 86 in the neighboring helix D. Both
arginine 28 and glutamic acid 86 are exposed on the surface of the
tetramer
(6) . This salt bridge does apparently not contribute
significantly to the thermal stability of the native tetramer. It has
been observed for other proteins that salt bridges located at the
surface of the molecule often are not critical for stability
(36) . It may be noted here that a mutation (R22W) at the
homologous position in short chain acyl-CoA dehydrogenase has been
detected in a patient with short chain acyl-CoA dehydrogenase
deficiency
(37) , suggesting a similar molecular mechanism also
in this case.
For the K304E mutant variant optimization of the
folding conditions by lowering the growth temperature and
co-overexpressing the GroESL chaperonins resulted in a maximum level of
approximately 40-50% of the wild-type. This yield was obtained at
34, 31, and 28 °C, indicating that a further improvement of the
folding conditions had no further effect. The observed decreased
thermal stability indicated that the mutation does not confer a
classical temperature-sensitive folding phenotype, but rather impairs
both polypeptide folding and the thermal stability of the folded and
assembled mutant tetramer.
The data obtained previously with the
K304E and the artificially constructed mutant K304Q suggested that
replacement of lysine 304 with either glutamic acid or glutamine
resulted in impaired folding kinetics. Based on the discrepancy
observed between the properties of the K304Q and the K304E mutant, we
proposed
(15) that charge reversal in the K304E variant may
induce a structural distortion in the monomer that impairs tetramer
assembly. Using an in vitro translation system in the presence
of mitochondria, Yokota et al. (38) observed that a
K304R mutant was converted efficiently into tetramers, whereas a K304D
mutant behaved like the K304E mutant. This also highlights the
importance of the charge in the side chain of residue 304. In our
present work we addressed the question about this charge effect on
stability and tetramer assembly by introduction of second site
mutations replacing negatively charged residues in the vicinity of
glutamic acid 304 in the folded structure. The second site mutant
E300K/K304E did not rescue any of the effects of the K304E mutation,
rather the effects of the mutations were additive. This indicates that
disruption of the salt bridge between glutamic acid 300 and arginine
383 in the other subunit has strong effects on subunit assembly in
itself and that the charge interaction between the side chains of
residues 304 and 300 is less important.
Introduction of the D346K
second site mutation reestablishes a high oligomer assembly efficiency.
This is evident from the clearly higher amounts of active MCAD
produced. The yield of active mutant enzyme was still clearly increased
by improving the folding conditions, demonstrating that the effect on
folding is not suppressed. The rescuing effect of the D346K mutation
thus indicates that a charge repulsion effect between the carboxyl
groups of glutamic acid 304 and aspartic acid 346 in the K304E mutant
is responsible for a distortion in the monomer structure which results
in a surface unfavorable for subunit docking. The fact that the K304E
mutant, with a lower but observable efficiency, does form oligomers
suggests that the unfavorable alignment of the side chains can be
overcome. This may require a rearrangement which demands a high energy
intermediate state making it kinetically unfavorable. The K304Q mutant,
with a constellation where there is neither charge attraction nor
repulsion between residues 304 and 346, also displays a relatively high
oligomer assembly competence, further supporting the notion of a
deleterious effect of the charge repulsion. The thermal stability
results obtained for the D346K and K304E/D346K mutants indicate that
aspartic acid 346 in itself is critical for the thermal stability.
The contribution of the observed decreased thermal stability of
K304E MCAD enzyme to the low level of K304E MCAD observed in patient
cells is at present not clear. The temperature range at which the
enzyme becomes inactivated is well above physiological temperatures.
However, it reflects a higher plasticity of the mutant tetramers and is
thus expected to result in a decreased half-life of the K304E MCAD
enzyme. In the light of the fact that the protein degradation
machineries in E. coli and eucaryotic mitochondria are
homologous
(39, 40) , the finding that levels of up to
80% of the wild-type control could be obtained in E. coli for
the K304E/D346K double mutant which displays a lower thermal stability
than the K304E mutant, lead us to suggest that the effect of the K304E
mutation on stability is of minor pathophysiological importance.
In
an attempt to check the relevance of the results obtained in the
bacterial expression system we analyzed the effect of cultivation at
low temperature on the relative amounts of K304E mutant enzyme produced
in lymphoblastoid cells from patients. Our results indicate that
cultivation at low temperature has a small but measurable positive
effect on the relative amount of the K304E mutant protein in these
cells. The temperature effect can most likely be ascribed to an
improvement of the folding conditions. An increase in the steady state
level of functional protein at low cultivation temperature has also
been observed for the prevalent
The possibility of being able to modulate
the yield of correctly folded and assembled mutant protein may thus
provide clues to explain the variable phenotypical manifestation of
MCAD deficiency. On the one hand, clinical symptoms usually occur only
under conditions of fasting stress, and on the other hand, the
frequency studies indicate that only a minority of the individuals
homozygous for the K304E mutation are symptomatic
(13) . In most
cases, attacks are precipitated by fasting stress accompanied by
feverish infections
(4) . This means that the lipid metabolism
is activated and synthesis of
MCAD enzyme activity in extracts from
lymphoblastoid cells cultivated at 34, 37, or 39 °C. Cells (normal
controls, cells heterozygous for the K304E mutation and cells from a
patient homozygous for the K304E mutation) were disrupted by sonication
and centrifuged, and MCAD enzyme activity was measured with the mass
spectrometric product formation assay (26). Both values from
measurements in duplicates are given in nanomoles of OH-octanoic acid
formed per h per mg of soluble protein.
;
(
)
EC 1.3.99.3) is one of four related enzymes with different
chain length specificity that catalyze the initial step in
mitochondrial
-oxidation of straight chain fatty acids
(1, 2) . The active enzyme consists of four identical
subunits, each containing one molecule of FAD. MCAD has received
particular interest, since MCAD deficiency is by far the most
frequently detected inheritable defect in mitochondrial
-oxidation
(3) . Disease manifestation in clinically affected patients
presents with life-threatening attacks induced by fasting stress, often
in connection with viral infections
(4) . Patients present with
clinical symptoms typically at the age of 1-4 years, and
approximately 20% of the patients detected so far died during the first
attack
(4) . When fasting is avoided, patients are usually
asymptomatic.
G transition
at position 985 of the cDNA sequence (G985), leading to replacement of
lysine 304 in the mature protein with glutamic acid (K304E). The
calculated homozygote frequency of the G985 mutation in Northwest
European and North American Caucasians is in the range of 1/6,400 to
1/40,000
(12) . This frequency is much higher than the actual
number of clinically detected symptomatic cases, suggesting that many
homozygotes are clinically asymptomatic
(12, 13) . The
reason for the phenotypic variation from sudden unexpected death in
childhood to asymptomatic presentation
(4) is not clear.
Plasmids
The previously used plasmid
pBMCK2(8) , which carries a gene encoding the
mature part of human MCAD preceded by an artificial initiator
methionine under control of the lac promoter, was modified in
order to allow straightforward subcloning of PCR amplified MCAD cDNA
derived from patient material. For this purpose pBMCK2
was modified in a series of steps involving standard PCR and
cloning techniques in the following way. A HindIII site
included in the PCR primer 1327- HindIII
(5`-TTTTCTGAAGCTTAAACAGTGGCTTGTGTTC-3`)
(7) was introduced at
the 3` end of the cDNA insert; the linker fragment of the parent vector
(pBluescriptKS
; Stratagene) between the
HindIII and BamHI sites was reintroduced followed by
deletion of the sites between EcoRV and SmaI by
cleavage with these enzymes and religation. The final product was
designated pWt. The K304E, R28C, and K304Q mutations were introduced
into pWt by subcloning of appropriate fragments from plasmids described
previously
(6, 15) producing a set of plasmids which
only differ in their respective mutation sites.
Growth of Bacterial Cells and Analysis of
Extracts
JM109 cells transformed with the respective MCAD
plasmid and pGroESL encoding the GroESL
(25) proteins or the
control plasmid pCaP
(15) were grown at 28, 31, 34, 37, or 40
°C in a shaking water bath until an ODof
0.7-1.0 was reached. The cells were then induced with 1
mM isopropyl-
-D-thiogalactopyranoside for 3 h
and harvested. Disruption of cells, SDS-PAGE, native PAGE, and Western
blotting were performed as described elsewhere
(15) . MCAD
enzyme activity in bacterial lysates was determined with the
ferricenium ion-based colorimetric assay as described by Lehman et
al.
(26) .
Thermal Stability Profiles
For determination of
the thermal stability profiles, aliquots of 12-22 µl of
extracts from cells expressing the respective MCAD variant and the
GroESL chaperonins were incubated at various temperatures for 10 min
and chilled on ice. MCAD enzyme activity was subsequently measured with
the colorimetric ferricenium ion assay as above.
Cultivation of Lymphoblastoid Cells and Two-dimensional
Gel Electrophoresis
Epstein-Barr virus-transformed
lymphoblastoid cells were cultivated at various temperatures for 14
days using RPMI 1640 medium supplemented with 10% fetal calf serum.
Preparation of samples and two-dimensional gel electrophoresis followed
by Western blotting were carried out as previously described
(27) . MCAD enzyme activity in extracts from lymphoblastoid
cells was measured with the mass spectrometric product formation assay
(28) .
Influence of Temperature and Amount of Chaperonins on
Solubility and Yield of Active Wild-type and Mutant MCAD
Variants
The mature part of wild-type MCAD and the R28C, K304E,
and K304Q mutant variants were expressed in E. coli with and
without co-overexpression of the GroESL chaperonins. Cells were
cultured at a range of temperatures between 28 and 40 °C.
Figure 1:
Solubility and relative MCAD
enzyme activity of MCAD variants expressed at different culture
temperatures in E. coli with or without co-overexpression of
the GroESL chaperonins. E. coli JM109 cells transformed with
plasmids containing the cDNA sequence for the mature part of wild-type,
R28C, K304E, or K304Q MCAD were grown and induced for 3 h at the
temperature indicated. Cells were harvested, disrupted, and split into
soluble ( s) and insoluble ( p) fractions as described
under ``Experimental Procedures.'' Left panel, MCAD
enzyme activity in the soluble fraction was measured with the
colorimetric assay using the ferricenium ion as electron acceptor. The
scale is in micromoles of ferricenium reduced per h per mg of total
soluble protein. Columns represent the average from two measurements
( open columns, without GroESL co-overexpression; closed
columns, with GroESL co-overexpression). Right panel,
aliquots containing 1.25 µg of total protein of the soluble
fraction and a corresponding aliquot of the solubilized pellet fraction
were subjected to SDS-PAGE followed by Western blotting with rabbit
antiporcine MCAD antibodies. (+) with co-overexpression of the
GroESL genes, () without co-overexpression of the GroESL
genes.
For all three mutant proteins
investigated, GroESL co-overexpression has a strong positive effect on
the amounts of active mutant enzyme produced. The chaperonin effect is
most dramatic at 34 °C and becomes smaller at lower temperatures.
An exception is the K304Q mutant which displays a strong chaperonin
effect at 34, 37, and 40 °C. With GroESL co-overexpression, R28C
and K304Q MCAD reach relative activity levels in the range of the
wild-type enzyme at 34 °C. The activity level of the K304E mutant
MCAD with GroESL co-overexpression increases to approximately half of
the wild-type activity at 34 °C and remains at this level at 31 and
28 °C. This suggests that this is a limit which cannot be surpassed
by further improving the folding conditions.
Thermal Stability of the Mutant Enzymes
In order
to investigate whether the mutations affect the stability of the
assembled enzyme, we determined the thermal inactivation profiles of
the mutant MCAD enzymes (Fig. 2). K304E MCAD shows a temperature
stability profile clearly shifted to lower temperatures, indicating
that this mutation has an effect on the stability of the tetramer once
it is formed. Almost identical curves could be observed for wild-type,
R28C, and K304Q MCAD with the curve for the K304Q mutant being
marginally but consistently steeper. This indicates that folded and
assembled R28C MCAD is as stable, and the K304Q mutant only slightly
less stable than the wild-type enzyme.
Figure 2:
Thermal inactivation profiles of mutant
MCAD enzymes. Aliquots from the soluble fractions of cells expressing
wild-type, R28C, K304E, or K304Q MCAD were incubated at various
temperatures for 10 min, and MCAD enzyme activity was measured
afterward. Residual enzyme activity (percentage of the activity
measured at 40 °C) is plotted versus the incubation
temperature. The values from two independent experiments each with
extracts from induction experiments performed at 28 or 31 °C are
shown. The total protein concentrations in the extracts were in the
range of 2.6-2.9 mg/ml (28 °C) and 2.1-2.5 mg/ml (31
°C). The profile obtained with an extract from cells expressing
wild-type MCAD is shown for comparison.
Temperature-sensitive Yield of K304E Mutant MCAD in
Patient Cells
To test the relevance of the results obtained in
the E. coli model system, we analyzed the steady state amount
of K304E MCAD in lymphoblastoid cells as a function of the temperature
at which the cells were cultivated. MCAD enzyme activity was measured
in lymphoblastoid cells cultivated at 34, 37, or 39 °C,
respectively. Cells homozygous or heterozygous for the K304E mutation
and normal controls were used. shows that the activity
values for the wild-type control cells are constant between 34 and 37
°C and decreased to less than half at 39 °C. A similar picture
is observed for the cells heterozygous for the K304E mutation. The
respective activity levels are about half of those for the wild-type
control, indicating that the contribution of the mutant enzyme to the
activity level is insignificant. The cells homozygous for the K304E
mutation display very low levels at 39 and 37 °C and an
approximately 3-fold increased relative activity at 34 °C.
Figure 3:
Two-dimensional gel electrophoresis of
lymphoblastoid cells cultivated at different temperatures.
Lymphoblastoid cells carrying the K304E mutation on one and wild-type
MCAD on the other allele were grown at 34, 37, or 39 °C,
respectively, harvested, and solubilized in lysis buffer. Aliquots
corresponding to 127 µg ( 34 °C), 111 µg ( 37
°C), and 183 µg ( 39 °C) total protein were
subjected to two-dimensional gel electrophoresis followed by Western
blotting with rabbit antiporcine MCAD antibodies as described elsewhere
(27). Sections of the gels are shown and the spots corresponding to
unmodified ( large arrowhead) and modified ( small
arrowhead) wild-type ( upward arrowheads) and K304E mutant
( downward arrowheads) MCAD are
marked.
Analysis of Interactions of Lysine 304 with Other Charged
Residues in the Native Structure
If the effect of the K304E
mutation on polypeptide folding is distinct from those on oligomer
assembly and stability, it should be possible to compensate for this
effect separately by reconstituting the charge interaction which is
distorted in the K304E mutant. The crystal structure of porcine MCAD is
available at 2.4 Å resolution
(16) . Fig. 4 A gives an enlarged view of the vicinity of lysine 304, showing
distances between relevant atoms. There are two negatively charged
groups, aspartic acid 300 and aspartic acid 346 in the vicinity of the
amino group of lysine 304. Aspartic acid 300 forms a salt bridge with
arginine 383 in the neighboring subunit. Lysine 304 forms a hydrogen
bond with the -carbonyl oxygen of glutamine 342. A similar
hydrogen bond can also be formed with the
-amido-NH
group of glutamine 342, if lysine 304 is replaced by glutamic
acid
(16) . The distances between the positively charged group
of lysine 304 and the other charged atoms are greater than expected for
genuine salt bridge interactions. All possible interactions of lysine
304 with other charged side chains are within the monomer, and there is
no indication that lysine 304 directly interacts with residues of the
neighboring subunit. Preliminary results with the human enzyme
demonstrate that the structure is highly similar to that of porcine
MCAD.
(
)
Comparison of the conservation of these
residues in the sequences of other acyl-CoA dehydrogenases
(Fig. 4 B) reveals that the negative charge of residue
300 as well as arginine 383 are conserved in all enzymes. A negative
charge at position 346 is present in all cases where there is a
positively charged residue at position 304. It may therefore be
speculated that a charge repulsion between glutamic acid 304 and
glutamic acid 300 and/or aspartic acid 346 in the human K304E MCAD
mutant could propagate a structural distortion within the monomer.
Figure 4:
A, an
enlarged view of the vicinity of lysine 304 of a monomer of porcine
MCAD. Helices H and I are shown in ribbons and side chain atoms of
lysine 304, aspartic acid 346, glutamine 342, aspartic acid 300, and
the main chain carbonyl atoms of glutamine 342 are shown in solid
balls. The side chain of arginine 383 of the neighboring monomer
is represented by open ball-and-stick. Distances between polar
atoms in are shown with dotted lines. The
figure was drawn using the program Molscript (29). B,
conservation of residues 300, 304, 342, 346, and 383 (MCAD numbering)
in the acyl-CoA dehydrogenase gene family. Nomenclature (cDNA sequence
reference in parentheses): LCAD, long chain acyl-CoA
dehydrogenase (30, 31); MCAD (32, 33); SCAD, short chain
acyl-CoA dehydrogenase (30, 34), IVD, isovaleryl-CoA
dehydrogenase (30, 35); small letters refer to the species ( h,
human; r, rat; p,
porcine).
To determine whether such charge interactions are relevant for the
effect of the K304E mutation on oligomer assembly and stability, we
constructed the second site mutants K304E/D346K and E300K/K304E,
thereby reconstituting opposite charge constellations in the vicinity
of the carboxyl group of glutamic acid 304. The single mutants D346K
and E300K were also constructed in order to analyze the effect of the
mutations as such.
Figure 5:
A,
enzyme activity levels of MCAD variants with second site mutations.
E. coli JM109 cells transformed with a plasmid encoding the
mature part of the respective MCAD mutant variants as indicated and
either the plasmid encoding the GroESL ( closed columns) genes
or the control plasmid ( open columns) were grown and induced
at 28 or 37 °C, respectively. Cells were harvested, disrupted, and
split into soluble and insoluble fractions as described under
``Experimental Procedures.'' MCAD enzyme activity in the
soluble fractions was measured with the colorimetric assay using the
ferricenium ion as electron acceptor. The scale is in micromoles of
ferricenium reduced per h per mg of total soluble protein. Columns
represent the average from two measurements. B, thermal
inactivation profile of the K304E/D346K double mutant. Aliquots from
extracts expressing K304E/D346K mutant MCAD were incubated at various
temperatures for 10 min, and MCAD enzyme activity was measured
subsequently. Residual enzyme activity expressed as the percentage of
the activity measured after 10 min of incubation at 30 °C is
plotted versus the incubation temperature. The profiles for
wild-type and K304E MCAD determined in the same way (see Fig. 2) are
shown for comparison.
Strikingly, the
amount of active K304E/D346K double mutant protein is clearly increased
in comparison to both the K304E and the D346K single mutants. The
effect is most remarkable in the 37 °C series. The amounts of
active K304E/D346K MCAD produced are strongly responsive to chaperonin
co-overexpression, indicating that the effect on folding is not
overcome. The characteristics of the K304E/D346K double mutant are
similar to those of the K304Q mutant (see Fig. 2).
F508 mutation in cystic fibrosis
(41, 42) .
-oxidation enzymes is up-regulated
under conditions of elevated temperature. It can well be imagined that
this poses high demands on the capacity of the folding machinery, and
proteins with folding defects will be less competitive in this
situation. Although this has to be substantiated further, it may be a
common pattern in many inherited diseases. Such
``conditional'' mutations may be retained in the population
as they do not abolish the function of the protein under normal
conditions and only become detrimental under conditions of stress for
the folding machinery.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.