From the Departments of Biochemistry and Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9038
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The E1 decarboxylase component of the human
branched-chain ketoacid dehydrogenase complex comprises two E1 (45.5 kDa) and two E1
(37.5 kDa) subunits forming an
2
2 tetramer. In patients with type
IA maple syrup urine disease, the E1
subunit is affected, resulting
in the loss of E1 and branched-chain ketoacid dehydrogenase catalytic
activities. To study the effect of human E1
missense mutations on E1
subunit assembly, we have developed a pulse-chase labeling protocol
based on efficient expression and assembly of human
(His)6-E1
and untagged E1
subunits in
Escherichia coli in the presence of overexpressed
chaperonins GroEL and GroES. Assembly of the two
35S-labeled E1 subunits was indicated by their
co-extraction with Ni2+-nitrilotriacetic acid resin. The
nine E1
maple syrup urine disease mutants studied showed aberrant
kinetics of assembly with normal E1
in the 2-h chase compared with
the wild type and can be classified into four categories of normal
(N222S-
and R220W-
), moderately slow (G245R-
), slow
(G204S-
, A240P-
, F364C-
, Y368C-
, and Y393N-
), and no
(T265R-
) assembly. Prolonged induction in E. coli grown
in the YTGK medium or lowering of induction temperature from 37 to
28 °C (in the case of T265R-
), however, resulted in the
production of mutant E1 proteins. Separation of purified E1 proteins by
sucrose density gradient centrifugation showed that the wild-type E1
existed entirely as
2
2 tetramers. In
contrast, a subset of E1
missense mutations caused the occurrence of
exclusive
dimers (Y393N-
and F364C-
) or of both
2
2 tetramers and lower molecular weight
species (Y368C-
and T265R-
). Thermal denaturation at 50 °C
indicated that mutant E1 proteins aggregated more rapidly than wild
type (rate constant, 0.19 min
1), with the T265R-
mutant E1 most severely affected (rate constant, 4.45 min
1). The results establish that the human E1
mutations in the putative thiamine pyrophosphate-binding pocket that
are studied, with the exception of G204S-
, have no effect on E1
subunit assembly. The T265R-
mutation adversely impacts both E1
folding and subunit interactions. The mutations involving the
C-terminal aromatic residues impede both the kinetics of subunit
assembly and the formation of the native
2
2 structure.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mammalian mitochondrial branched-chain -ketoacid
dehydrogenase (BCKD)1 complex
catalyzes the oxidative decarboxylation of the branched-chain
-ketoacids derived from the branched-chain amino acids, leucine, isoleucine, and valine (1). This multienzyme complex is organized around a dihydrolipoyl transacylase (E2) core, to which a
branched-chained
-ketoacid decarboxylase (E1), a dihydrolipoamide
dehydrogenase (E3), a specific kinase, and a specific phosphatase are
attached through ionic interactions (2, 3). E1 is a thiamine
pyrophosphate (TPP)-dependent enzyme comprising two E1
and two E1
subunits that assemble into an
2
2 tetramer. E2 is a 24-meric protein consisting of identical lipoic acid-bearing subunits arranged on
octahedral 4,3,2-point group symmetry. Each E2 polypeptide contains
three independently folded domains (i.e. lipoyl-bearing, E1/E3-binding, and inner core) that are highly conserved among E2
proteins of the
-ketoacid dehydrogenase complexes (4). These
complexes include pyruvate dehydrogenase,
-ketoglutarate dehydrogenase, and BCKD complexes (3). E3 is a homodimeric flavoprotein
that is common to members of the
-ketoacid dehydrogenase complexes
(3). The kinase and the phosphatase are specific for the BCKD complex
and regulate its activity through a reversible phosphorylation
(inactivation) and dephosphorylation (activation) cycle (5).
In patients with maple syrup urine disease (MSUD) or branched-chain
ketoaciduria, the activity of the BCKD complex is deficient. This leads
to clinical manifestations including often fatal ketoacidosis, neurological derangements, and mental retardation (1). The molecular
genetics of MSUD are heterogeneous as mutations in the E1, E1
,
E2, and E3 genes have been described (1, 6). Based on the locus
affected, genetic subtypes of MSUD have been proposed, with type IA
referring to mutations in the E1
gene, type IB to the E1
gene,
type II to the E2 gene, and type III to the E3 gene (1). It has been
suggested that certain type IA MSUD missense mutations, for example
Y393N-
(7) and Y368C-
(8), may impede the assembly of mutant
E1
with normal E1
subunit, resulting in the degradation of E1
subunits in patient's cells.
We have recently established that chaperonins GroEL and GroES are
essential for efficient folding and assembly of the E1 tetramer in
Escherichia coli (9) and the E2 24-mer in vitro
(10). To gain insight into the biochemical basis of the apparently
impaired assembly of E1 in type IA MSUD, we have co-expressed both
mature mutant E1 and normal E1
in E. coli
co-transformed with a second plasmid overproducing chaperonins GroEL
and GroES. Pulse-chase labeling of both E1 subunits was carried out to
measure the kinetics of assembly of the mutant E1
with normal E1
in the bacterial cell. The results showed a marked reduction in the
rate of E1 assembly in certain E1
mutants compared with normal. It
was also found that a subset of E1
mutations affect the assembly
state of mutant E1 after the prolonged induction in E. coli.
Thermostability and protease digestion studies further indicated these
slowly assembled mutant E1 proteins had less stable conformations than the wild type. These results define the residues that are critical for
subunit interactions and stability of E1 and have implications for
understanding chaperonin-mediated biogenesis of hetero-oligomeric structures.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Lines and Cell Cultures-- Fibroblasts of classic MSUD patients were kindly provided by the following physicians: B.A. by Dr. David Valle, John F. Kennedy Institute, Baltimore, MD; C.Q. by Dr. Robin Casey, University of Saskatchewan, Canada; and K.U. by Dr. Selma Snyderman, New York University Medical Center, New York, NY. Fibroblasts of classic MSUD patient L.C. were obtained from Cell Repository of McGill Medical Center, Montreal, Canada. Amniocytes of the fetus-at-risk for classic MSUD (F.J.) were kindly provided by Dr. Frederico Mariona of Hutzel Hospital, Detroit, MI. Cells were cultured in Waymouth medium containing 15% fetal calf serum as described previously (11).
Construction of pHisT-E1 Prokaryotic Expression Vector--
The
5' portion of the mature human E1 cDNA sequence was amplified
from the pMAL-c2-hE1
expression vector (12) using an internal 22-mer
antisense primer with sequence 5'-GTAACAGATATCGACCCTGTT-3', and a 49-mer sense primer with sequence
5'-GGCTCTAGACTCGAGAATCTTTATTTtcaatcatctctggatgacaagc-3' to yield a 601-bp product. The sense primer adds exogenous sequence (shown in uppercase) to the 5' terminus of the mature E1
open reading frame (shown in lowercase). This exogenous sequence includes an
XbaI restriction site (shown in bold), followed by sequence encoding the first six amino acids specific for the tobacco etch virus
(TEV) protease cleavage (shown underlined). The seventh required amino
acid for the TEV protease cleavage is supplied by the N-terminal serine
of the mature E1
sequence.
Expression and Purification of Recombinant E1 Proteins-- E. coli strain CG712 (ESts) and the plasmid pGroESL, which overexpresses chaperonins GroEL and GroES (14), were kind gifts of Dr. Anthony Gatenby of DuPont Experimental Station, Wilmington, DE. CG712 cells co-transformed with GroESL and the wild-type or mutant pHisT-E1 plasmids were grown as an overnight culture at 37 °C in YTGK medium. The medium was modified from the 2× YT medium (15), and contained the following per liter: 16 g of yeast extract, 10 g of bacto-tryptone, 5 g of NaCl, 10 ml of glycerol, and 0.75 g of KCl; 100 mg of ampicillin and 12.5 mg of tetracycline. Antibiotics were added to maintain the expression plasmid (Ampr) and the F' plasmid (Tetr) that carried the lacI transcriptional repressor gene. The overnight culture was diluted 3:1000 into 1 liter of YTGK medium and grown with shaker aeration at 37 °C to a measured A550 of 0.6. The expression of (His)6-E1 was induced with 1 mM IPTG at 37 °C for 15-20 h.
E. coli cells were collected by centrifugation and resuspended in 5 cell-pellet volumes of a wash buffer (50 mM Tris·HCl, 100 mM NaCl, 5 mM EGTA, 5 mM EDTA, pH 7.5). Rinsed cells were pelleted and resuspended in a lysis buffer (50 mM KPi, 100 mM NaCl, 2 mM MgCl2, 0.2 TPP, 0.1% (v/v) Triton X-100, 0.01% (w/v) NaN3, 0.1 mM EDTA, 0.25 mM dithiothreitol, pH 7.0) with protease inhibitors (1 mM PMSF and 1 mM benzamidine) at 10 ml of buffer per g wet cell weight. Resuspended cells were sonicated in an ice-water bath with a Bronson model 350 sonicator to eliminate most viscosity. Cell lysates clarified by centrifugation were treated with Ni2+-NTA (Qiagen, Chatsworth, CA) derivatized Sephacryl resin by batch mixing for 2 h, and the resin was packed into a column. The column was washed with 10 bed volumes of an elution buffer (100 mM KPi, pH 7.5, 500 mM KCl, 0.2% Tween 20, 2 mMPulse-Chase Labeling to Determine Kinetics of E1 and E1
Subunit Assembly--
CG712 cells were transformed with the pGroESL
plasmid and pHisT-hE1 expression vectors carrying the normal mature
E1
cDNA and either normal or mutant His-tagged mature E1
cDNA. Cells were grown at 42 °C to an
A595 of 0.8 in C2 broth minimal media. The C2
broth minimal medium was modified from the low sulfate and low amino
acid content C broth medium described by Guzman-Verduzco and
Kupersztoch (18) and contained the following per liter: 2 g of
NH4Cl, 6 g of
Na2HPO4·7H2O, 3 g of
KH2PO4, 3 g of NaCl, 6 g of yeast
extract, 40 µmol of TPP, 50 mg of carbenicillin, and 50 mg of
chloramphenicol. Cells were pelleted and resuspended in one-fifth
original volume of the same media without antibiotics and allowed to
recover with shaking for 5 min at 37 °C. Cells were subsequently
induced with 2 mM IPTG for 5 min, pulsed with 50 µCi/ml
[35S]Cys/[35S]Met (ICN Radiochemicals,
Costa Mesa, CA) for 1 min, and chased with 3 volumes of the same media
(without antibiotics) supplemented with 8 mg/ml each of non-radioactive
L-cysteine and L-methionine. At specified time
points following the chase, cell samples (1.8 ml) were taken and
quickly frozen in liquid N2. Thawed samples were lysed by
sonication, and supernatants after microcentrifugation were treated
batchwise with an excess (15 µl) of Ni2+-NTA resin. The
resin was washed three times (total volume: 2.4 ml) with 15 mM imidazole in 100 mM KPi, pH 7.5, containing 2 mM MgCl2, 0.1 mM EDTA,
0.1 mM EGTA, 0.2 mM TPP, and 2 mM
-mercaptoethanol. Bound (His)6-tagged E1
and
assembled untagged E1
polypeptides were eluted with 30 µl of
Laemmli SDS sample buffer (19) containing 50 mM EDTA.
Eluted labeled polypeptides were analyzed by SDS-PAGE, and
autoradiograms were obtained by storage phosphorimaging.
Analysis of Assembly State by Sucrose Density Gradient Centrifugation-- Wild-type and mutant E1 proteins were fractionated on a 10-25% sucrose density gradient (10 ml). Gradients were poured in 50 mM potassium phosphate, pH 7.5, 250 mM KCl, and 0.2 mM EDTA. Proteins (25-200 µg) were applied to each gradient following elution from Ni2+-NTA resin and fractionation on Sephacryl S-100 HR column. The gradients were run at 41,000 rpm (210,000 × g) in a Beckman SW-41 rotor for 18 h. Gradients were fractionated (700 µl/fraction) from top to bottom. Each fraction was treated with 10 µl of 12.5 mg/ml deoxycholate and 700 µl of 15% trichloroacetic acid. Samples were incubated on ice for 20 min and then spun in a microcentrifuge for 15 min. Each pellet was washed with 700 µl of acetone (80%), followed by a 5-min microcentrifuge spin and evacuation of the supernatant by vacuum. Pellets were resuspended in 10 µl of H2O and 5 µl of SDS-PAGE sample buffer, and the entire volume was applied to the gels for analysis by SDS-PAGE.
Thermal Denaturation of Normal and Mutant E1
Proteins--
Thermal aggregation was monitored by measuring
absorbance at 360 nm versus time in a Gilford response
spectrophotometer equipped with a Peltier heating device as described
previously (20). Wild-type or mutant E1 proteins (1.2 µM,
final concentration) were added to a buffer preheated to 50 °C,
which contained 50 mM KPi, 250 mM
KCl, 0.5 mM -mercaptoethanol, 0.2 mM EDTA,
and 10% glycerol at pH 7.5 in a final volume of 0.5 ml. The
temperature of samples in glass cuvettes (2 mm in width and 10 mm in
light path length) was measured using a small-bead thermocouple. The effects of cofactors on thermal denaturation and aggregation was studied by adding 2 mM TPP and 1 mM
MgCl2 to the incubation mixture.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Identification of E1 Mutations in Type IA MSUD
Patients--
Type IA (E1
-deficient) MSUD was initially suggested
by a reduced level or absence of the E1-
subunit in cells from the
patients. Missense mutations were identified by DNA sequencing with
subclones of the RT-PCR product or by direct sequencing of that
product. Y393N-
(codon TAC
AAC), F364C-
(TTC
TGC), Y368C-
(TAT
TGT), and G245R-
(GGG
AGG) mutations were reported previously
(8, 21, 22). The N222S-
(AAT
AGT) is
present in a homozygous classic MSUD patient F.J. G204S-
(GGG
AGG), R222W-
(CGG
TGG), and A240P-
(GCA
CCA) are present in one allele of
classic patients L.C., B.A., and K.U., respectively. The other allele
in these three patients is the prevalent Y393N-
mutation previously
reported to occur in homozygous Mennonite MSUD patients (23, 24). The T265R-
mutation (ACA
AGA) is in one allele
of a classic patient C.Q. The second allele in this patient is a single
C nucleotide insertion in exon 2 (the 144insC allele) that we reported
previously (8). The four novel missense mutations (G204S-
,
R220W-
, N222S-
, and A240P-
) as a cause of MSUD were studied by
transfection of the full-length human E1
cDNA carrying one of
these mutations into type IA MSUD lymphoblasts (22). Transfected cells
were unable to decarboxylate the
-keto-[1-14C]isovalerate (data not shown). As a
positive control, decarboxylation activity in type IA MSUD cells was
complemented by transfection with the normal pEBOhbE1-
plasmid
(7).
Expression and Purification of Normal (His)6-Human
E1--
In our earlier study of chaperonin-augmented expression of
mammalian E1, a maltose-binding protein (MBP) ligand was fused to the N
terminus of the E1 subunit. The presence of the MBP sequence
increases the solubility of MBP-E1 and facilitates its purification by
amylose resin affinity chromatography (12). In the present study, the
MBP ligand is replaced with a (His)6-tag, which is linked
to the N terminus of the mature E1
subunit through a TEV-protease
recognition sequence (LENLYFQ). Co-expression of (His)6-E1
and untagged E1
(the pHis T-E1 plasmid) was
carried out in an E. coli CG712 host, which contained a
second plasmid GroESL that overproduced chaperonins GroEL and GroES.
The cells grown in the YTGK medium were heat-shocked at 42 °C for
4 h, followed by induction at 37 °C with IPTG for 16 h.
The cell lysate was purified by Ni2+-NTA column
chromatography. Fig. 1 (lower
panel) shows the elution profile with an imidazole gradient. The
upper panel shows the SDS-PAGE profile of column fractions
stained with Coomassie Blue. The appearance of the
(His)6-E1 tetramer coincides with the presence of E1
activity as measured by a spectrophotometric assay with 2,6-dichlorophenol indophenol as an electron acceptor. The earlier fractions corresponding to the GroEL-E1
-E1
complex may represent a folding intermediate (see "Discussion").
|
Measurements of E1 Assembly Kinetics by Pulse-Chase
Labeling--
Our previous data suggested that the Y393N- mutation
in MSUD impedes the assembly of the mutant E1
subunit with normal
E1
, resulting in the preferential degradation of the latter subunit in patient's cells (7). In the present study, we set out to measure
the kinetics of assembly of normal and the nine MSUD mutant E1
subunits, including Y393N-
, with normal E1
by pulse-chase labeling. The method was based on the efficient expression of (His)6-E1 in the presence of excess chaperonins GroEL and
GroES as described above. CG712 cells co-expressing
(His)6-E1
, untagged E1
, GroEL, and GroES were grown
in the C2 broth minimal medium and heat-shocked at 42 °C for 4 h, followed by induction with IPTG for 5 min at 37 °C. The cells
were pulsed with
[35S]cysteine/[35S]methionine for 1 min and
then chased with unlabeled amino acids from 2 to 120 min. Lysates
prepared from cells harvested at different times were purified by
Ni2+-NTA affinity chromatography. The eluted radiolabeled
polypeptides were separated by SDS-PAGE, and autoradiograms were
obtained by storage phosphorimaging. Since the E1
subunit was
untagged, the co-purification of this subunit with the
(His)6-E1
subunit by Ni2+-NTA indicated
assembly of the two polypeptides synthesized during the 1-min pulse.
Fig. 2 shows that the assembly of normal
E1
with normal (His)6-E1
occurs as early as 10 min in
the chase and reaches a plateau at 30 min. Similar results were
obtained with N222S-
(second panel from the
top) and R220W-
(data not shown). These two mutations are
in the category of a normal assembly with the E1
subunit. A second
group of mutations represented by the G245R-
showed that significant
assembly with E1
did not occur until 30 min into the chase and
plateaued at 60 min. Mutations that produce this sluggish assembly
kinetics belong to the category of moderately slow assembly. A third
group of E1
mutations comprising G204S-
, A240P-
, F364C-
,
Y368C-
and Y393N-
did not generate detectable assembly with
normal E1
during the 2-h chase. This is indicated by the absence of
the E1
subunit in the autoradiogram. This group of mutations are
classified as slow assembly, as they produce the assembled mutant E1
only after a prolonged growth of the transformed cells at 37 °C for
16 h (see below). The fourth category of no assembly is
represented by the T265R-
subunit, which is not soluble when cells
are grown at 37 °C, as indicated by the rapid disappearance of the
mutant E1
subunit in the chase. This resulted in a complete absence
of assembly with the normal E1
subunit, even after a prolonged
growth at 37 °C. However, a significant amount of the assembled
T265R-
E1 was produced, when the induction temperature was lowered
from 37 to 28 °C (see below).
|
Measurements of Total and Soluble E1 and E1
Subunits--
The levels of total recombinant E1
and E1
polypeptides in E. coli cells were measured by Western
blotting. Cells co-transformed with pHisT-E1 and pGroESL plasmids and
grown in the C2 broth minimal medium were heat-shocked as described
above and induced with IPTG for 12 h. Cells were harvested and
total lysates prepared by sonication followed by solubilization in an
SDS-PAGE sample buffer. After SDS-PAGE, the samples were subjected to
Western blotting using the antibody to E1
or E1
as a probe. Fig.
3A shows that the levels of
normal and mutant E1
are comparable. More significantly, the levels
of normal E1
in cells expressing normal or mutant E1
are
relatively constant. The data rule out the possibility that the reduced
level or absence of normal E1
assembled with the mutant E1
subunits (Fig. 2) is caused by aberrant E1
expression. Fig.
3B shows Western blotting of total soluble E1
(normal or mutant) and E1
(normal) subunits in E. coli cells when
co-expressed at 37 °C. Five (G204S-
, R220W-
, N222S-
,
A240P-
, and G245R-
) of the nine E1
mutants studied are
associated with wild-type levels of soluble E1
and E1
subunits.
In contrast, the level of T265R-
is markedly reduced with a
concomitant near-absence of the normal E1
subunit. The results
support the conclusion drawn from pulse-chase labeling (Fig. 2), which
indicates that the mutant T265R-
is largely insoluble at 37 °C
and fails to assemble with the normal E1
subunit. The unassembled
E1
subunit, while expressed at a normal rate (Fig. 3A),
became aggregated and was removed from the supernatant after
centrifugation. As for the F364C-
, Y368C-
, and Y393N-
mutants
(Fig. 3B), the levels of both soluble mutant E1
subunits
and the soluble E1
subunit are decreased, compared with the
wild-type E1
.
|
Expression and Assembly State of Wild-type and Mutant E1
Proteins--
The co-purification of (His)6-E1 with
untagged E1
cannot discern the assembly state of assembled E1
subunits. To address this question, E. coli cells in the C2
broth medium which expressed wild-type E1 subunits were grown for
20 h. E1 subunits in cells harvested at different induction times
were purified by Ni2+-NTA resin and subsequently subjected
to size fractionation on a TSK-G3000SWXL column by HPLC.
The molecular weight of assembled E1 subunits species is inversely
proportional to the retention time on HPLC. Fig.
4A shows that
(His)6-E1
and untagged E1
subunits form mostly
dimers with molecular mass in the 80-kDa range at the 1-h induction
time. The Ni2+-NTA extractable
dimers are the
predominant species with the appearance of a minor species of
2
2 tetramers (165 kDa) when induced for
2 h. Conversely, at the 3- and 20-h induction times the major
species is
2
2 tetramers and the minor
species
dimers. The data indicate that (His)6-E1
and E1
subunits initially form the
dimers which later slowly
dimerize to produce
2
2 tetramers. The
fractions collected at different retention times from HPLC were
analyzed for E1 activity using an assay mixture containing added excess
lipoylated recombinant E2 and recombinant E3, which allowed one to
measure reconstituted BCKD activity. This radiochemical assay for BCKD
activity is 15-fold more sensitive than the one measuring the E1
component activity alone. As shown in Fig. 4B, the fraction
collected at the 9-min retention time, which corresponds to the
2
2 tetramer in the 20-h induction lysate,
contains the peak enzyme activity. The fractions corresponding to
dimers with a retention time of 10.3 min in 2- and 3 h-induction
lysates do not have enzyme activity.
|
|
|
Activity Levels and Stability of Wild-type and Mutant E1
Proteins--
Wild-type and mutant E1 proteins produced in E. coli grown on the YTGK medium were purified by
Ni2+-NTA affinity column and gel filtration on Sephacryl
S-100 column. The E1 activity of normal and mutant proteins was assayed
by the radiochemical method with 2,6-dichlorophenol indophenol as an electron acceptor. As shown in Table I, only the mutant E1 carrying N222S- or G245R-
has residual E1 activity (1.37 and 2.66% of normal activity, respectively). The mutant E1 proteins containing each
of the remaining seven E1
mutations do not have detectable E1
catalytic activity.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The major focus of this investigation is to characterize the
effect of human E1 mutations in type IA MSUD on the assembly and
stability of mutant E1 proteins. For these studies, we have developed
efficient bacterial expression systems for folding and assembly of E1
2
2 tetramers. We showed previously that
co-expression of mature MBP-E1
and E1
sequences of the human BCKD
complex in the same E. coli cells is essential for MBP-E1
assembly; however, the yield was very low (12). Co-transformation of a
second plasmid that overexpressed GroEL and GroES into the same
E. coli cell resulted in a 500-fold increase in the yield of
active MBP-E1 tetramers (9). In the present study, a (His)6
affinity tag is fused to the mature E1
N terminus through a TEV
protease recognition site. Co-transformation with the pGroESL plasmid
was also found necessary and sufficient for productive folding and
assembly of (His)6-E1. The results argue against the
suggestion that the dependence of human E1 on chaperonins for a high
yield is due to the presence of the MBP sequence in the chimeric E1
polypeptide (23). Our recent in vitro refolding results
indicate that the reconstitution of untagged E1, MBP-E1, and
(His)6-E1 show the same chaperonin-dependent kinetics.2 The findings
further established that productive folding and assembly of mature
human E1 have an absolute requirement for enrichment for chaperonins
GroEL and GroES and are not affected by the presence of affinity tags.
Thus, the pulse-chase labeling protocol developed in this study
provides the first approximation of the rate of E1 subunit assembly
under optimal conditions through the augmentation of bacterial
chaperonins that are homologue of mitochondrial chaperonins Hsp60 and
Hsp10, respectively.
The wild-type and mutant human E1 and the wild-type E1
subunits
are expressed at relatively equal efficiencies and are stable, as
indicated by Western blotting of the total crude lysates prepared 12 h after induction (Fig. 3A). This allows one to
follow the fates of E1
and E1
subunits synthesized during the
1-min window of pulse-labeling. The presence of the
(His)6-tag in the wild-type and mutant E1
subunits
facilitates the isolation of the 35S-labeled subunit. One
can measure the kinetics of the E1
assembly with the wild-type and
mutant E1
subunits by the co-purification of the untagged E1
with
(His)6-E1
with the Ni2+-NTA resin as a
function of time. The equally strong signals of E1
and E1
subunits during the 2-h chase (Fig. 2A) indicate that both
subunits are efficiently synthesized. The total numbers of cysteine and
methionine residues in the E1
and E1
subunits are similar, which
are 16 and 15, respectively. The autoradiogram of the co-purified E1
subunits as separated by SDS-PAGE cannot discern the assembly state of
the associated subunits. However, size fractionation of the pulse-chase
labeled products by HPLC show that the wild-type E1
and E1
assemble during the 2-h chase occur predominantly as inactive
dimers, which are later converted to active
2
2 tetramers (Fig. 4, A and
B). It is noteworthy that a significant amount of the
wild-type
dimeric intermediate was observed only when E. coli cells were grown on the C2 broth minimal medium. When the
bacterial cells were cultured on the YTGK medium, wild-type E1 was
expressed predominantly as
2
2 tetramers
(Fig. 5) with little or no accumulation of
dimers during the
16-h induction period (data no shown). The factors responsible for the
apparent effects of culture media on the accumulation of the wild-type
dimeric intermediate during E1 assembly are currently unknown. The
major differences between the two bacterial culture media lie in the
fact that the C2 minimal medium is low in the content of
SO42
and amino acids when compared
with the YTGK medium (18). Possible effects of these ingredients on the
dimerization of wild-type
dimers are under investigation.
It is of interest that a weak GroEL signal co-purifies with wild-type
(His)6-E1 and E1
at 120 min into the chase (Fig.
2A). This apparent ternary complex is also observed in the
early fractions of the Imidazole gradient during purification of
wild-type (His)6-E1 (Fig. 1). The GroEL-E1
-E1
ternary
complex is a productive intermediate at a later step of the
chaperonin-mediated assembly of E1
2
2 tetramers.2 Only a GroEL-E1
binary complex is observed
in F364C-
because the assembly of E1
with mutant E1
did not
occur within the 2-h chase. The weak and sub-stoichiometric signal of
GroEL relative to E1 subunits is a result of isotopic dilution by the
overabundance of unlabeled GroEL in E. coli.
The assembled mutant dimers were produced in E. coli
grown on the YTGK medium after the 16-h induction with "slow
assembly" E1
mutants including Y393N-
and F364C-
. The
results indicate that this group of mutations not only reduces the rate
of the assembly of the mutant E1
with normal E1
but also prevents
conversion of the dimeric assembly intermediate into the stable
2
2 structure of wild-type E1. The
production of these mutant
dimers apparently is not affected by
growth media, as the expression of mutant E1 carrying these mutations
in E. coli grown in the C2 broth minimal medium also
resulted in the expression of exclusive dimers. In vitro
reconstitution of the 6 M urea-denatured mutant Y393N-
E1 in the presence of chaperonins GroEL and GroES also resulted exclusively in mutant
dimers.2 The unstable
Y393N-
dimeric intermediate as demonstrated by its propensity for
thermal aggregation and proteolytic digestion compared with the
wild-type tetramer explains the markedly reduced levels E1
and E1
subunits in cells from Mennonite MSUD patients homozygous for the
Y393N-
mutation (24, 25). The present study establishes that
C-terminal aromatic residues (F364-
, Y368-
, and Y393-
) in the
E1
subunit are crucial for proper E1 assembly. The important roles
of the C terminus in subunit assembly and protein interactions have
been demonstrated. For example, the C-terminal 25 amino acid residues
of the herpes simplex virus type 1 UL26.5 protein are required for the
assembly of the icosahedral capsid shell (26). In the case of the E2
core of the related pyruvate dehydrogenase complex from
Azotobacter vinelandii, the C-terminal residues 632-637
comprise a 310-like helix (H6) which acts as a
"hydrophobic knob" that fits into a "hole" in the 2-fold related subunit to produce the 24-mer cubic assembly (27). Introduction of a polyhistidine tag into the C terminus of BCKD-E2, which is highly
homologous to the bacterial pyruvate dehydrogenase-E2, results in the
formation of stable trimers instead of the native 24-mer structure
(data not shown).
The T265R- subunit, when expressed at 37 °C, was largely
insoluble even in the presence of excess chaperonins GroEL and GroES. This was indicated by the rapid disappearance of the mutant E1
signal in the soluble fraction during the 2-h chase (Fig.
2A); however, the level of the T265R-
subunits in total
crude lysates was comparable to that of wild type (Fig. 3A).
These results strongly suggest that the T265R-
residue is important
for proper folding of the E1
subunit. It is also of interest that
lowering of the induction temperature from 37 to 28 °C resulted in
the production of a significant amount of assembled mutant E1 protein
carrying the T265R-
mutation. The yield of the mutant E1 was 5 mg/liter culture at 28 °C compared with 20-40 mg/liter culture for
the wild-type E1 expressed at 37 °C (data not shown). The finding is
consistent with the thesis that lowering the expression temperature slows the folding kinetics of the nascent peptide, thereby reducing the
probability of the off-pathway folding reactions, as demonstrated by
the expression of rabbit muscle phosphorylase (28) and the human E1
(13). However, the assembled T265R-
mutant E1 has a grossly altered
conformation, which renders it very unstable as indicated by its most
rapid thermal aggregation at 50 °C among the nine E1
mutants
studied. This unstable conformation is also manifested by the apparent
dissociation of the mutant tetramers to lower molecular weight species
as detected by sucrose density gradient centrifugation. The current
data indicate that the T265R-
residue also plays a key role in
subunit interactions and are consistent with the location of this
residue at the putative subunit-interaction site conserved between BCKD
and pyruvate dehydrogenase E1 proteins (29).
The crystal structure of the E1 2
2 has
not been solved, but structures are known for the related
TPP-dependent proteins transketolase (30) and pyruvate
decarboxylase (31) from Saccharomyces cerevisiae and
pyruvate oxidase from Lactobacillus plantarum (32). The
transketolase is a homodimer, whereas the human E1 is a tetramer made
up of two non-identical subunits. Sequence alignment between the two
enzymes shows that the highly conserved TPP-binding pocket in E1 is
composed of residues from both E1
and E1
subunits (33, 34).
Aromatic residues from E1
form a hydrophobic pocket to accommodate
the pyrimidium and thiazolium rings of cofactor TPP. On the other hand,
the highly conserved TPP-binding motif
GDG(X)22-28NN, which was first described by
Hawkins et al. (35) and is essential for binding the
pyrophosphate moiety, is located in the E1
subunit (Fig.
8). It should be mentioned that a D440E
mutation introduced via mutagenesis into this motif in pyruvate
decarboxylase from Zymomonas mobilis yielded a homodimeric
enzyme with reduced affinity for TPP, in contrast to the wild-type
enzyme which exists as a homotetramer (36). It was proposed that
deficient TPP binding may have caused a failure in the conversion of
the mutant dimeric forms into native tetramers. However, the occurrence
of
dimers in the mutant E1 carrying Y393N-
and F364C-
substitutions is likely through a different mechanism, since these
residues are not involved in TPP binding and are located in the
C-terminal region. G204S-
, R220W-
, and N222S-
that are
affected in type IA MSUD are residues within the TPP-binding motif
(Fig. 8). Specifically, N222S-
aligns with Asn-187 in the yeast
transketolase and provides a ligand to this pentameric coordination
involving the Mg2+ cation. The N222S-
mutation
conceivably disrupts the pentameric coordination, resulting in the
inability of E1 to bind the pyrophosphate moiety of TPP and the loss of
E1 catalytic function. However, the N222S-
mutation is without
effect on the assembly of the mutant E1
with normal E1
, as
determined by pulse-chase labeling (Fig. 2A). The R220W-
mutation, which is also located in the pyrophosphate moiety binding
site, also has no adverse effect on E1 subunit assembly. In contrast,
G204S-
mutation, which is presumably located at the interface
between the two non-identical subunits of E1, based on the yeast
transketolase structure (37), impedes the assembly of the mutant E1
subunit with the normal E1
unit.
|
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grant DK26758 from the National Institutes of Health, Grant I-1286 from the Welch Foundation, and Grant 95G-074 from American Heart Association, Texas Affiliate.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Medical Scientist Trainee supported by National Institutes of
Health Grant 5-P32 GM08014 and by the Perot Family Foundation.
§ To whom correspondence should be addressed: Dept. of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9038. Tel.: 214-648-2457; Fax: 214-648-8856; E-mail: chuang01{at}utsw.swmed.edu.
1
The abbreviations used are: BCKD, branched-chain
-ketoacid dehydrogenase; E1, branched-chain
-ketoacid
decarboxylase; E2, dihydrolipoyl transacylase; E3, dihydrolipoamide
dehydrogenase; HPLC, high performance liquid chromatography; Hsp, heat
shock protein; IPTG, isopropyl
-D-thiogalactopyranoside;
KPi, potassium phosphate; MBP, maltose-binding protein;
MSUD, maple syrup urine disease; NTA, nitrilotriacetic acid; PMSF,
phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel
electrophoresis; TEV, tobacco etch virus; TPP, thiamine
pyrophosphate.
2 J. L. Chuang, R. M. Wynn, and D. T. Chuang, manuscript in preparation.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|