(Received for publication, October 22, 1996)
From the Department of Biochemistry, Kansas State
University, Manhattan, Kansas 66506 and the ¶ Wadsworth Center for
Laboratories and Research, New York State Department of Health,
Albany, New York 12201
The dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex (PDC) consists of 60 COOH-terminal domains as an inner assemblage and sequentially via linker regions an exterior pyruvate dehydrogenase (E1) binding domain and two lipoyl domains. Mature human E2, expressed in a protease-deficient Escherichia coli strain at 27 °, was prepared in a highly purified form. Purified E2 had a high acetyltransferase activity, was well lipoylated based on its acetylation, and bound a large complement of bovine E1. Electron micrographs demonstrated that the inner core was assembled in the expected pentagonal dodecahedron shape with E1 binding around the inner core periphery.
With saturating E1 and excess dihydrolipoyl dehydrogenase (E3) but no E3-binding protein (E3BP), the recombinant E2 supported the overall PDC reaction at 4% of the rate of bovine E2·E3BP subcomplex. The lipoates of assembled human E2 or its free bilipoyl domain region were reduced by E3 at rates proportional to the lipoyl domain concentration, but those of the E2·E3BP were rapidly used in a concentration-independent manner consistent with bound E3 rapidly using a set of lipoyl domains localized nearby. Given this restriction and the need for E3BP for high PDC activity, directed channeling of reducing equivalents to bound E3 must be very efficient in the complex.
The recombinant E2 oligomer increased E1 kinase activity by up to 4-fold and, in a Ca2+-dependent process, increased phospho-E1 phosphatase activity more than 15-fold. Thus the E2 assemblage fully provides the molecular intervention whereby a single E2-bound kinase or phosphatase molecule rapidly phosphorylate or dephosphorylate, respectively, many E2-bound E1. Thus, we prepared properly assembled, fully functional human E2 that mediated enhanced regulatory enzyme activities but, lacking E3BP, supported low PDC activity.
Pyruvate dehydrogenase complexes from various sources are among
the largest enzyme systems that serve strategic roles in metabolism (1,
2). The mammalian complex has a highly organized structure in which the
dihydrolipoyl transacetylase (E2)1
component has a central role in the organization and integrated chemical reactions of the complex, and supports enhanced functioning of
dedicated kinase and phosphatase components (3-8). The other components of the mammalian complex required for the overall reaction are: the pyruvate dehydrogenase (E1) component, an
2
2 tetramer present at 20-30 copies (1);
the dihydrolipoyl dehydrogenase (E3), a homodimer present in about 6 copies (9); and the E3-binding protein (E3BP) estimated at 6-12 copies
(10, 11). Dedicated and highly regulated kinase and phosphatase
components control the conversion of E1 between an active
(nonphosphorylated) form, E1a, and an inactive (phosphorylated)
form, E1b.
Mammalian PDC-E2 subunits have four flexibly connected domains and form the core of the complex in which 60 of its COOH-terminal inner (I) domains assemble into a dodecahedron-shaped structure with its other three domains extending out around this porous surface. The icosahedral I60 inner core anchors the E3BP (12-14) and carries out the transacetylase reaction (15), probably via functional trimer units as established for the octahedral inner core of Azotobacter vinelandii PDC-E2 (16, 17). Trimer units were also shown to be important in the assembly of bovine heart E2 subunits dissociated to nonfunctional monomers in 4 M guanidinium chloride (18).
The outer globular domains of E2 consist of two lipoate-bearing domains (L1 and L2), followed by a small E1 binding (B) domain. The connecting hinge regions are 20-30 residues in length, are enriched in Ala and Pro residues (19, 20), and are highly mobile but stiffer than random coil structures (5, 7). Three-dimensional structures have been derived for examples of each of E2's domain classes from bacterial PDC-E2s (16, 21-23). However, the domains in mammalian E2 have significant differences both in structure and function (3-6). For instance, the mammalian PDC-E2 lipoyl domains appear to be larger and at least L2 has several specialized roles (see below). The small B domains of eukaryotic PDC-E2s only bind E1, whereas the structurally related domains in other PDC E2s bind only E3 (e.g. Escherichia coli PDC; Refs. 5 and 24) or bind both E3 and E1 (e.g. Bacillus stearothermophilus PDC; Refs. 5, 25, and 26). The I domains of eukaryotic PDC-E2s are apparently unique in binding the E3BP (3-7).2 E3BP is also composed of three linker-connected domains (an inner domain that binds to the I domain of E2, an E3-binding domain, and a lipoyl domain) (3, 4, 13, 27).
Prokaryotic lipoyl domains (22, 23) are flattened barrel structures
with the lipoate attached to a specific lysine residue located in tight
turn protruding from one end. Building on this framework, larger
mammalian PDC-E2 lipoyl domains have added structure at their
COOH-terminal ends that may contribute to performing their unique
roles. High mobility of the connecting hinge regions allows the lipoyl
domains to be delivered to the E1, E2, and E3 components where the
prosthetic group extends into active site channels (8). Specific
interaction of the lipoyl domain with E1 is essential for efficient E1
catalysis and probably aids the E2 and E3 reactions (5, 28,
29).34 The L2 domain
binds the E1a kinase (30) and the E1b phosphatase (31). Binding of a
kinase dimer probably engages two L2 domains plus their lipoyl
prosthetic groups (30), while the binding of the phosphatase to L2
requires Ca2+ (31). Furthermore, the L2 domain has a direct
role in mediating regulatory stimulations of the kinase by the PDC
products, NADH and acetyl-CoA (32), and the marked enhancement of
phosphatase activity by the Ca2+-facilitated binding to L2
constitutes an important means of second messenger activation (33,
34).
Although recombinant lipoyl domain structures of human E2 have proved useful for determining binding sites and characterizing component reactions, some catalytic and regulatory processes require a higher level of structural complexity. For instance, markedly activated functioning of the kinase and the phosphatase, when bound to the complex, is not supported by these isolated domains and apparently requires the full oligomeric E2 structure, which can bind multiple copies of the E1 substrate as well as a regulatory enzyme and can facilitate a sequence of steps needed to produce enhanced activity. The capacity of the mammalian E2 core to support the overall PDC reaction has been previously evaluated only when a major portion of E3BP has been removed from the E2 core under highly chaotropic conditions (35, 36), making uncertain the status of the residual E2. Recombinant expression of assembled E2 would overcome these problems and allow development of altered structures in subsequent work.
Successful expression and recovery of assembled recombinant E2 could
not be expected on the basis of previous studies, in which bacterial
(E. coli and A. vinelandii) or
Saccharomyces cerevisiae PDC-E2s were recovered only upon
co-expression in E. coli of the cognate E1 or E1 + E3 (13,
37, 38). The successful expression of assembled but lipoyl-deficient E2
core of the bovine branched-chain -keto acid dehydrogenase complex
was encouraging (39), as well as evidence for the independent folding
and assembly of unfolded bovine E2 subunits (18). Here, we have
developed conditions for expressing fully assembled mature human E2 in
E. coli and for obtaining highly purified preparations
that have the expected structure and known catalytic functions. We
have evaluated how well this E3BP-deficient E2 supports the PDC
reaction, E3 catalysis, and the enhanced catalytic reactions of
the kinase and the phosphatase.
Bovine kidney PDC (40), E1 component, E2·E3BP-kinase subcomplex (41), and the recombinantly expressed bilipoyl domain region of human E2 (42) were prepared as described previously. Porcine heart E3 was from Boehringer Mannheim. The original pBTA vector that coded for all of mature human E2 as well as a portion of its leader sequence was kindly provided by M. E. Gershwin (University of California, Davis). The expression vector pSE420 and its recommended E. coli host strain, Top10, were from Invitrogen; and the host E. coli strain BL21(DE3), lacking the lon protease and ompT outer membrane protease, was from Novagen. PEG-8000 was from J.T. Baker Co., and Pluronic F-68 was from BASF Corp. Other chemicals and reagents were as described by Liu et al. (42).
Vector Construction and ExpressionThe plasmid pBTA
harboring the human E2 cDNA insert was cut with restriction enzymes
SfaNI and BamHI to produce a DNA fragment lacking
the coding region for the leader sequence and first 7 amino acids of
mature E2. Two phosphorylated synthetic oligonucleotides (sequences in
Fig. 1) were hybridized to produce a small double-stranded DNA fragment
that coded for this remaining portion of mature E2 preceded by the ATG
start codon and containing compatible splicing sequences for a
NcoI site at the 5-end of the coding sequence and a
SfaNI at its 3
-terminus. This synthetic structure, the pBTA
restriction fragment, and the pSE420 vector digested with NcoI and BamHI were ligated to produce the
expression vector coding for mature E2 preceded by a start codon (Fig.
1). The ligated plasmid was transformed into E. coli strains
by electroporation using Transfector 300 BTX under the conditions
recommended in the instrument manual. The expression of E2 was first
confirmed by dot blots and then Western blots using a mixture of
monoclonal antibodies 150.2 and 157.2 (42), which react with the first and second lipoyl domain, respectively. Clones were designated pShE2
(pS for pSE420 vector, h for human, E2 for coding for full-sized mature
E2). The DNA sequencing was conducted using a series of synthetic
primers and the dideoxynucleotide sequencing procedure using the
Sequenase Version 2.0 kit from U. S. Biochemical Corp.
Experiments to optimize expression used Western blot analyses employing not only the lipoyl domain-specific monoclonal antibodies (above) but also a polyclonal antibody that reacts with the inner domain of E2. With different host strains, induction at different stages of growth, and expression for variable periods, Western blots were used to detect production and cleavage of E2 when intact bacterial cells were extracted with hot SDS-sample buffer, soluble and particulate fractions were prepared from different host strains, and clarified lysates were incubated for various times.
Purification of the Recombinant E2 OligomerAll steps were
performed at 4 °C; PEG and
(NH4)2SO4 fractionations involved
continuous stirring with centrifugation conducted 20 min after
additions of these precipitants were completed. E2 activity was
measured in a rapid continuous assay (below) with corrections in the
case of crude extracts for E. coli PDC-E2 activity made
assuming the same ratio of E2 to PDC activity as measured in the
nontransformed host strain. (The expression of human E2 had no effect
on the E. coli PDC activity.) Cells (12-14 g) harvested from 1.5 liters were thawed and resuspended at 12.5% w/v in 50 mM potassium phosphate (pH 7.2), containing 0.5 mM EDTA, 1 µg/ml aprotinin and leupeptin (buffer A) and
immediately ruptured using Vibra-cell high intensity ultrasonic
processor (Sonics and Materials Inc.) equipped with a 0.5-inch probe at
a setting of 4 using 10 1-min bursts. Particulate material was removed
by centrifugation at 15,000 × g for 20 min. PEG-8000
(50% w/v) was added dropwise to the clarified lysate to 8% (v/v), and
precipitated protein, removed by centrifugation, was discarded. An
additional 8% (v/v) was added followed by centrifugation. This 8-16%
PEG pellet was dissolved in buffer A. The protein concentration was
diluted to 2-3 mg/ml, and solid
(NH4)2SO4 was added to 25%
saturation. The E2-containing precipitate was removed by centrifugation
and dissolved in 4 ml of buffer A containing 0.2 mg/ml Pluronic F-68.
After 12 h, the fraction was clarified by centrifugation and
applied to 2.7 × 75-cm bed volume Sephacryl S-400 column
equilibrated with 40 mM sodium phosphate, 0.5 mM EDTA, 1 µg/ml aprotinin, and leupeptin, and 0.2 mg/ml
Pluronic F-68 (buffer B). Fractions (4 ml) were assayed, and those
containing high E2 activity were clarified by centrifugation at
15,000 × g for 20 min and then simultaneously analyzed
by SDS-PAGE (43) with silver staining (44) and concentrated 10-12 fold
by centrifugation in Centricon 10 units. Concentrated fractions were
then stored in aliquots at 80 °C.
The purified E2 preparations were analyzed to identify cleavage products, establish the NH2-terminal sequences of all E2 bands, and determine the proportion of intact E2 subunits. Putative intact E2 and cleaved E2-polypeptides, initially identified by immunoblotting were blotted onto ProBlott, stained with 0.1% Coomassie Blue R250 in 1% acetic acid, 40% methanol for 1 min, and destained in 50% methanol, and then NH2-terminal sequencing was conducted as described previously (42). To estimate the proportion of E2-protein and relative levels of intact versus cleaved E2, quantitative area scanning of silver-stained patterns was performed following SDS-PAGE separation of 0.4, 0.8, 1.6, and 2.4 µg of purified human E2 and 0.6, 1.2, 2.0, and 3.2 µg of bovine E2·E3BP loaded into lanes. Alternatively, 0.48, 0.96, 1.6, 2.4 and 4.0 µg of human E2 and 0.8, 1.2, 2.4, 5.0 µg of bovine E2·E3BP were analyzed in Coomassie Brilliant Blue R-250-stained patterns. Band densities were evaluated by scanning with an Epson ES-800c (Model 2) scanner, acquiring area density data with Adobe Photoshop program, and analyzing the data using NIH Image software. Each series gave a linear increase in area with the amount loaded (data not shown).
E2 Activity AssaysRoutine E2 activity assays monitored
acetyl-dihydrolipoamide production by recording the
A232 nm with time (45). Reaction mixtures
contained 30 mM Tris·HCl (pH 7.4), 1 mM
dihydrolipoamide, 1 mM acetyl-phosphate, 5.0 µM CoA (prepared as concentrate 1:1 with cysteine), and 1 unit of phosphotransacetylase in a 1-ml cuvette at 30 °C. After the
absorbance became constant due to formation of acetyl-CoA (~10 s),
the E2 source was added and increased absorbance at 232 nm measured.
The activities are recorded simply as absorbance/min because the
precise extinction coefficient of the immediate
8-acetyl-dihydrolipoamide product (about 4 A/mM·cm) cannot be determined (shuffling of acetyl groups in acetylated dihydrolipoamide leads to formation of a 3 to 1 mixture of the 8- and
6-acetyl-dihydrolipoamide and significant amounts of the biacetyl
derivative at high levels of acetylation; see Refs. 46 and 47).
[1-14C]Acetyl-dihydrolipoamide formation was assayed by procedure of Butterworth et al. (48). Reactions were initiated at 25 °C by addition of [1-14C]acetyl-CoA (8 × 105 cpm/µmol) to yield 0.5 mM concentration in a reaction mixture containing 25 mM potassium phosphate (pH 7.5), 0.5 mM dihydrolipoamide, 0.05 mM EDTA, 4 µg of E2 in a 0.5-ml reaction mixture. After 120 s, 1 ml of benzene was added and vortexed for 20 s to stop the reaction and to extract the radiolabeled acetyl-dihydrolipoamide formed. Two 0.2-ml aliquots of the benzene layer were withdrawn and counted; duplicate assays were within ±6%.
Acetylation CapacityTo acetylate all lipoyl groups with
[1-14C]acetyl-CoA, reductive acetylation by the reverse
E2 and E3 reactions was coupled to conversion of CoA formed to
succinyl-CoA as described previously (31, 49, 50). Reactions were
carried out at 30 °C in 20 mM potassium phosphate (pH
7.5) containing 0.2 mM EDTA, 0.25 mM NADH and
NAD+, 0.4 mM -ketoglutarate, 50 µM Ca2+, 2 µg of
-ketoglutarate
dehydrogenase complex, and the indicated level of E2 source. Reactions
were initiated by addition of acetyl-CoA (8 cpm/pmol), and after
60 s reactions were terminated and protein-bound acetyl groups
determined.
The capacity of an E2 source (4-6 µg)
to support the overall PDC reaction was evaluated by incubating E1 (2 µg/µg of E2) and variable levels of E3 for 60 s at 30 ° in
15-21 µl and then adding two-thirds of the volume to 1 ml of
standard assay mixture (51) and measuring the rate of NADH production
at 340 nm. E3 activity was measured by the cyclic E3 reduction of
lipoates and their rapid chemical reoxidation (42).3 The E3
content of a E2·E3BP subcomplex preparation was reduced to 0.6 E3
dimers/subcomplex by a second passage through the standard resolution
procedure (41) that removes E1 and most E3, followed by pelleting of
the subcomplex through a sucrose step gradient containing 2.5 M urea and 0.35 M NaCl. The lipoyl domain
sources (E2·E3BP, recombinant E2, or bilipoyl domain construct) were
added at 80-320 pmol to a 200-µl reaction mixture containing 50 mM sodium phosphate (pH 7.4), 0.3 mM NADH, 0.1 mM NAD+, 0.2 mM
5,5-dithiobis-(2-nitrobenzoate), and 1 mM EDTA. Reactions were initiated by addition of 0.1 µg of E3. 5-Thio-2-nitrobenzoate production was monitored with a UVmax microplate reader at 405 nm.
Purified recombinant human E2 with or without bovine E1 was dialyzed against Buffer B to remove Pluronic F-68. Samples were prepared and negatively stained with 0.5% uranyl acetate (52), and micrographs acquired with a Philips EM420T transmission electron microscope at a nominal magnification ×50,000 using 1000 e/nm2 (53).
Sucrose Gradient AnalysesTo evaluate removal of residual
non-E2 bands and test E1 binding to E2, fractionation of E2 and E2 + E1
was evaluated using a modification of the step gradient procedure
of Powers-Greenwood (35). 25 µg samples of purified E2 with or
without 20 µg of purified bovine E1 were incubated for 60 min at
4 °C in 70 µl of 50 mM sodium phosphate (pH 7.2)
containing 0.2 mM EDTA, 0.5 mM dithiothreitol,
1 µg/ml leupeptin and aprotinin. These samples were layered over a
three-step gradient (50 µl each of 7.5, 10, and 15% sucrose in the
same buffer) in 5 × 20-mm ultraclear tubes (Beckman) and
centrifuged at 130,000 × g for 150 min at 4 °C. Fractions were withdrawn as upper 120 µl (S1), middle 50 µl (S2), and bottom 50 µl (S3); and the pellets were dissolved in 50 µl of
the same buffer. SDS-PAGE analysis (43) with silver staining (44) was
conducted as described in Fig. 4.
E1a Kinase and E1b Phosphatase Activities
[32P]Phosphate incorporation into E1 was
measured by standard procedures using E2-free resolved E1 as a
substrate (44, 54, 55). Kinase assays were conducted at 30 °C in 20 mM potassium phosphate (pH 7.4), 1 mM
MgCl2, 1 mM dithiothreitol, 0.05 mM
EDTA, 0.2% Pluronic F-68, and 0.2% Triton X-100 using the level of
components indicated in the figure legend. Reactions were initiated by
addition of [-32P]ATP (~3 × 105
cpm/nmol) to 0.1 mM and terminated at the indicated time by
spotting to trichloroacetic acid-containing disks; protein-bound
[32P]phosphate was determined as described previously
(54).
[32P]Phosphate release from PDCb or E1b was measured at 30 °C in 30-µl reaction mixtures containing 20 mM Mops-K buffer (pH 7.3), 0.4 mM dithiothreitol, 0.6 mM EDTA, 1.0 mM EGTA, 1.2 mM CaCl2, and the indicated level of E2 oligomer (56, 57). E1b phosphatase was added for 120 s and then activity initiated by addition of MgCl2 to 10 mM. After 120 s, reactions were terminated by addition of 50 µl of 20% trichloroacetic acid and precipitated protein was pelleted after at least 30 min of incubation at 4 °C by centrifugation for 4 min in a microcentrifuge at 8000 × g. Aliquots (28 µl) were withdrawn and radioactivity determined by counting in scintillation fluid. All kinase and phosphatase assays were conducted in duplicate or triplicate; absolute deviations are shown.
The initial
expression vector that was provided coded for all of mature E2, 16 amino acids of the presequence (location of a convenient
EcoRI restriction site), and a small terminal portion of
-galactosidase (58). The expression product accumulated only in
inclusion bodies and lacked E2 activity before and after a variety of
solubilization procedures but was fully antigenic in Western blots
using polyclonal and monoclonal
antibodies.5 The construction of the
expression vector, as outlined in Fig. 1, removed the
leader sequence and adjoined a start codon at the front of the coding
region for mature E2. The pSE420 vector was chosen for its
bacteriophage T7 gene 10 element that enhances translation of eukaryotic genes. Cell lysates from pShE2 transformed colonies gave strong responses in dot blots using lipoyl
domain-specific monoclonal antibodies and contained soluble E2 activity
well beyond that of E. coli PDC (data not shown).
The pShE2 plasmid was sequenced not only at the ligated 5-end but
throughout most of the E2-encoding insert, including all regions in
which the sequences of Coppel et al. (19) and of Thekkumkara
et al. (20) differed. Sequencing confirmed that the desired
construct was obtained and established a sequence for this
placental-cDNA derived construct was identical to that reported for
the human liver E2 (20), establishing that previously reported
differences were due to sequencing errors.
Immunoblot analysis revealed substantial full-sized E2 (as expected with a mobility in SDS-PAGE somewhat faster than bovine E2; Ref. 59) along with degradation products, which were not reduced by inclusion of a variety of protease inhibitors in cell lysates. Furthermore, there was no time-dependent change in the pattern of intact versus partially cleaved E2 when cell lysates were incubated at 25 °C in buffer A and the same pattern of E2 bands was observed when cells were disrupted in 2% SDS-sample buffer by immediate insertion in boiling water (data not shown). Co-expression of groELS chaperonin proteins did not reduce E2 cleavage or increase the portion of E2 fractionating into the supernatant fraction. Thus, if chaperonins are required to aid the folding or assembly of E2, the endogenous levels in E. coli are sufficient. Using pShE2 transformed into the protease-deficient BL21 strain, a greater proportion of full-sized E2 was found in the soluble fraction from cell lysates than with the Top10. Using the BL21 host, changing the IPTG induction from stationary to mid-log phase, and lowering the temperature of expression to 27 °C gave a higher level of soluble E2, significantly increased the proportion of intact E2, and reduced the number of E2-derived bands arising from the limited proteolysis. With a correction for the low level of E. coli PDC E2, the transacetylase activity indicated that recombinant E2 constituted about 3% of the soluble protein after 8 h of post-induction growth.
Purification of Recombinant E2Using the steps detailed under
"Experimental Procedures," the recombinant E2 was purified to near
homogeneity. The initial PEG fractionation step left >99% of the
contaminating E. coli PDC in solution, but a significant
portion of the bacterial -ketoglutarate dehydrogenase complex
co-precipitated with the E2 in the 8-16% cut. Human E2 (intact
E260 has calculated Mr of 3.57 × 106) eluted prior to the similarly sized
-ketoglutarate dehydrogenase complex in gel-filtration
chromatography, demonstrating that E2 subunits assembled into a large
aggregate.6 The most prominent protein
(Mr
42,000 in SDS-PAGE) not separated from
E2 by the gel filtration, as well as the E. coli
-ketoglutarate dehydrogenase complex, were removed by fractionation
with ammonium sulfate prior to the size exclusion step; both
contaminants stayed in solution when E2 was precipitated.
Gel-filtration chromatography then removed low molecular weight
contaminants, followed by a clarifying centrifugation step, which
reduced or removed additional contaminants. A variety of other
procedures either failed to allow E2 to be recovered (ion exchange or
hydroxylapatite chromatography) or failed to significantly improve the
purification (additional PEG fractionations, sucrose gradient
centrifugation).
Fig. 2 shows the change in SDS-PAGE profile, and Table
I presents information on the recovery and change in
specific activity at each stage in the purification. Intact E2 is the
major band in the SDS-PAGE profile of the purified product. Two other
bands (labeled in the SDS-PAGE pattern of Fig. 3 as E2
and E2I) were identified by Western blotting as cleavage
products of E2 retaining the oligomer forming inner domain. The other
minor contaminants all changed in their ratio to mature E2 during the
purification process. NH2-terminal sequencing gave
SLPPHQKV, AKILVAEG, and VVPPTGPG for bands identified as E2, E2
, and
E2I, respectively, establishing that the upper band is
mature E2 with start codon Met removed and that E2
and E2I
resulted from cleavage following Met-59 (located in L1 domain) and
Leu-318 (located in the third linker region just prior to the inner
domain of E2), respectively.
|
Densitometric area scanning of silver-stained bands of SDS-PAGE patterns of E2 loaded over a 6-fold range (see "Experimental Procedures") gave estimates that intact E2 constituted about 70 ± 5% of the protein and that the two E2 cleavage products contributed about 15 ± 5% of the protein. These estimates were made assuming that staining is proportional to protein. On a molar basis, this suggests that about 75% of the E2 protein consists of full-sized E2 subunits. Somewhat higher relative staining intensity of the ~38-kDa contaminant was observed when Coomassie Brilliant Blue-stained patterns were similarly analyzed, but that analysis gave roughly the same relative molar proportion of intact versus cleaved E2. Corrected specific activities and acetylation levels (below) would be about 9% higher if the proportion of E2 protein was estimated based on Coomassie staining rather than silver staining.
Binding of Bovine E1 and Morphology of Human E2Fig. 3 shows the SDS-PAGE patterns for three supernatant fractions and one pellet fraction derived from microsucrose-gradient fractionation of E1, human E2, and the combination. Alone, E1 is concentrated at the top and E2 toward the bottom of the gradient, consistent with E1 existing as a tetramer and E2 as a large oligomer. In combination, virtually all the E1 fractionated with the recombinant E2, establishing the B domain is fully functional in binding E1. The most prominent contaminant in the E2 preparation (band position between E1 subunits) must be in a large aggregate that does not interact with E2, since it sedimented more rapidly whether or not E1 was present.
The upper panel of Fig. 4 shows a portion of a field of negatively stained human E2. As previously presented and modeled (53, 60), only the inner core is observable and presents images characteristic of an icosahedrally symmetric pentagonal dodecahedron. Typical views at or almost down the 5-fold axis are common (left center) but examples of images viewed approximately along the 2-fold (lower image; upper right) and 3-fold axis (top upper right) are apparent. The lower panel shows images with a high (left) and a lower (right) level of E1 bound around the periphery of the inner core, further supporting effective E1 binding.
Lipoyl Content and Catalytic ActivityTo evaluate the lipoate
content, we compared the extents of acetylation of our preparations of
human and bovine E2. Using [1-14C]acetyl-CoA and CoA
removal to drive the acetylation reaction, 75% as high an extent of
acetylation of the recombinant E2 as the E2·E3BP subcomplex was
observed (Table II, third column). This falls within
experimental error of the value of 80 ± 7% as high of
acetylation, calculated using the proportion of bands estimated from
gel scanning (silver stain) and accounting for single site acetylation
of E3BP in bovine E2·E3BP and E2 (truncated in the first lipoyl
domain) in the recombinant E2 oligomer. Acetylation of E2
was
confirmed by autoradiography (54) after SDS-PAGE separation of subunits
(data not shown). This agreement indicates that all or nearly all
lipoyl domains are lipoylated.
|
The specific activity
(A232 nm·min
1·mg
1)
of the purified E2 oligomer was 58% higher than that of a recent
bovine E2·E3BP subcomplex (Table II). The other transacetylase assay
measuring [14C]acetyl-dihydrolipoamide, which uses a
20-fold higher acetyl-CoA concentration, also gave a higher activity
for the recombinant E2 than for E2·E3BP subcomplex (Table II, second
column). We estimate the E2 specific activity of the E2 oligomer
(following corrections due to non-E2 protein (decrease) and due to the
presence of active, truncated forms of cleaved E2 (increase)) to be
about 5.0 µmol·min
1·mg
1.
The capacity to support the overall PDC reaction was compared for E2 oligomer relative to E2·E3BP subcomplex when both were reconstituted with excess E1 and the E3 components (Table II, last column). The E2 oligomer supported a low but significant rate of the overall reaction in the absence of E3BP. Even when a 4-fold higher level of E3 was added to the E2 oligomer, there was no increase in reconstituted PDC activity (data not shown). We have not been able to prepare an active form of E3BP in the absence of E2. Retention of about 4% activity is consistent with previous studies with bovine E2 (35) from which E3BP was removed, whereas with S. cerevisiae E2 no activity in the overall reaction was observed in the absence of E3BP (61). Although not as extreme as in the case of the yeast PDC, the retention of such a low activity indicates that the overall PDC reaction is facilitated by molecular mechanisms that efficiently channel reducing equivalents to E3BP-bound E3 (cf. "Discussion").
Liu et al. (42) found that the individual lipoyl domains of E2, prepared by recombinant techniques, were effectively utilized by the E3 component in a cyclic form of the reverse reaction involving NADH-dependent reduction by E3, followed by rapid chemical reoxidation to the disulfide by reaction with DTNB. Table III shows E3 reaction rates using 0.4-1.6 µM intact E2 subunits of the E2·E3BP subcomplex or recombinant oligomer and of the bilipoyl domain portion of E2 (L1·H1·L2). With the recombinant sources (lines 1 and 2, Table III), concentration-dependent increases in rates fit E3 using these lipoyl domain sources as a substrate with a Km above this concentration range. Indeed, increasing the level of a free lipoyl domain yields a maximal specific activity that is much higher.3 The somewhat higher rates with the free bilipoyl domain structure than with the E2 oligomer may reflect more efficient delivery of lipoyl domains to the E3 active site with the freely diffusing structure than when lipoyl domains are grouped in close proximity within an E2 structure that E3 cannot bind.
|
Results from varying the E2·E3BP subcomplex were very different (Table III). The subcomplex contained a low level of endogenous E3 even after additional resolution steps to reduce the E3 level. Both this endogenous E3 (line 3) and added E3 (line 4) gave a fixed and nearly identical specific activity as the subcomplex level was increased. This implies that E3 bound to E3BP is not responding to variation in the E2 concentration but only to the fixed stoichiometry of lipoyl domains surrounding it within the E2·E3BP subcomplex. Addition of E3 to the E2·E3BP to levels exceeding 30% of its E3 binding capacity lowered E3 specific activities (data not shown). This outcome suggests an increased competition for localized lipoates as more E3 is bound in the subcomplex. The results are consistent with E3, localized by binding to E3BP, encountering a fixed availability of lipoyl domains to introduce reducing equivalents.
Capacity of the Recombinant E2 Oligomer to Support Kinase and Phosphatase FunctionBovine kidney E1a kinase binds to the inner
(L2) domain of E2 (30); binding increases the rate of phosphorylation
of E1a severalfold by lowering the Km for E1a
20-fold and increasing the Vm of the kinase
2-fold (51). In the resolution of bovine kidney PDC that produces the
E2·E3BP subcomplex and E1, a major portion of the kinase fractionates
with the subcomplex and a small portion with E1. The activation state
of the dilute kinase that fractionates with E1 is enhanced with the
level of E2. With 2.9 µM E1, 6 µg of the recombinant E2
oligomer gave somewhat more than a 2-fold increase and 19 µg provided
>4-fold increase in this kinase activity (Fig. 5). In
contrast, no increase in kinase specific activity was found when
E2·E3BP-kinase subcomplex was varied to provide a similar range of E2
levels.7 The activation of the kinase
associated with E1 but not that associated with E2·E3BP as the
concentration of recombinant or subcomplex E2 was varied in the same
range may reflect enrichment of different kinase isozymes (63-65) with
E1 or with E2·E3BP during the resolution of bovine PDC.
The Ca2+-dependent E2 enhancement of the
Mg2+-requiring E1b phosphatase activity is pronounced, with
activation in the presence of saturating Mg2+ primarily
resulting from a decrease in the Km of the phosphatase for E1b (66). Fig. 6 shows that, in 25-µl
reaction mixtures, the recombinant E2 oligomer markedly increased
phosphatase activity with as little as 2 µg of E2 giving a 10-fold
increase in activity and 8 µg of E2 facilitating up to a 16-fold
increase in phosphatase. Relatively low E1b (1.52 µM) was
included, which maximizes E2 activation in these assays. From a
reciprocal plot of activity to E2 subunit concentration, half-maximal
activation of the phosphatase is estimated to occur at 0.74 ± 0.1 µ E2 subunit concentration, consistent with tight binding
of the phosphatase to E2.
When 8 µg of phosphorylated PDCb and 4 µg of E1b (3.8 µg of bovine E2, 1.88 µM E1b in total) were present, phosphatase activity was increased by further addition of 8 µg of recombinant E2 by 2-fold over the enhancement by bovine E2 (right series, Fig. 6). Comparing the absolute activities for a change from 3.8 µg of bovine E2 to a total of 7.8 µg of E2 (3.8 µg of bovine plus 4 µg of human E2) to that for a change from 4 µg to 8 µg of human E2, the initial activity is 2-fold higher with 4 µg of human E2 than with the bovine E2 and the final activity is still 41% higher with 8 µg of human E2 than with the combination of human and bovine. These data suggest human E2 is more effective than bovine E2 in activating the phosphatase. It remains to be determined whether that is due to interference of other components (E3BP or E3), damage to bovine E2 during preparation of the complex, or an innately greater capacity of the human E2 to activate the bovine phosphatase in dephosphorylating bovine E1b. Regardless, these data further support high functionality of the recombinant human E2.
E2 components have many roles in the organization, operation, and
regulation of -keto acid dehydrogenase complexes. Because E2
subunits invariably have modular structures with independently folded
domains set off from each other by mobile linker regions, the
domain-specific roles can be fruitfully diagnosed by recombinant DNA
approaches as exemplified by using individual domains or groups of
domains in native or mutated forms in studies of E. coli
(e.g. Refs. 37 and 67-69), B. stearothermophilus
(e.g. Refs. 21, 22, and 24-26), A vinelandii
(16, 17, 70), and S. cerevisiae PDC-E2s (e.g.
Refs. 13 and 61). However, as noted in the Introduction, some roles
require assembled E2 structures. Guest and colleagues have
characterized a variety of assembled E. coli PDC-E2
structures for lipoyl domain roles and found the three lipoyl domains
have equivalent capacities in supporting the overall PDC reaction
(67-69). As in the case of S. cerevisiae PDC (27),
production of mammalian E2 free of E3BP is important for distinguishing
the contribution of these two lipoyl-bearing components in catalytic
and regulatory processes. Mammalian PDC is regulated by phosphorylation
and dephosphorylation, but that is not the case in the well
characterized bacterial PDCs, and yeast PDC has only recently been
shown to produce a kinase and phosphatase under specialized growth
conditions (71).
We have successfully expressed and prepared in a highly purified state
the assembled E2 core of human PDC without coexpression of other
components of the complex. Beyond deleting the upstream coding region
(including a partial leader sequence), key conditions were expression
of E2 in a protease-deficient host (E. coli BL21), lipoate
supplementation, and induction of expression in mid-log growth phase at
a reduced temperature of 27 °C. The latter two conditions minimized
but did not completely eliminate proteolytic cleavage and allowed
recovery of an assembled structure with about 75% of the subunits
intact. Sequencing of NH2 termini identified a truncated
structure, labeled E2, whose sequence began after Met-59 located in
the first domain; E2
may have been produced by the Met-59 codon
serving as an internal start site. There is a good Shine-Dalgarno
sequence (GGAGGAG) located 6 nucleotides upstream of the Met codon.
This Met residue aligns with Leu, Val, and Ile in other PDC-E2 lipoyl
domains and corresponds to a buried amino acid in the established
three-dimensional structures of bacterial domains (22, 23), suggesting
that production of E2
by proteolysis might require cleavage to occur
prior to folding of the lipoyl domain. A second site of cleavage is
located near the end of the third linker region. By whatever means, the
E2
and E2I structures were created prior to rupture of
E. coli cells since they were observed in immunoblots when
cells were extracted with immediate heating in SDS-sample buffer.
Residual contaminants in E2 preparations are large structures that do
fractionate somewhat differently in sucrose gradients (e.g.
Fig. 3), and there is no indication that these E. coli
proteins interact with human E2.
Although not fully intact, the recombinant E2 exhibited high activity
in the acetyltransferase reaction, bound the E1 component, and
supported the activated function of the kinase and phosphatase. The
observable inner core has the expected dodecahedron structure. Supporting the independent folding of lipoyl domains, internal disruption of the terminal lipoyl domain in E2 did not prevent folding
and lipoylation of the inner lipoyl domain since it could be
effectively acetylated by the E1 (data not shown) or E2 reactions.
Cryoelectron electron microscopy and quasielastic light scattering measurements indicate that hydrated mammalian PDC has a diameter of ~500 Å, that the E1 and E3 components are tethered with gaps between them and the inner core, that there is considerable solvent entrapped at the surface of the complex (53, 72). This solvent-rich environment can support domain and component movement, a capacity eminently important for the operation of active site coupling in catalytic processes and for regulatory interconversion of the E1 component. The high kinase and phosphatase activities supported by the recombinant E2, lacking E3BP, establish that it has the full capacity to facilitate the molecular processes whereby individual kinase and phosphatase dimers rapidly encounter many molecules of their E2-bound E1a and E1b substrates. In the case of the tightly bound kinase, this is proposed to involve a kinase dimer moving across the surface of the complex by a "hand over hand" mechanism that allows it to find and phosphorylate bound E1 without losing its grip on the complex (30). Rapid dissociation and reassociation of the less tightly bound phosphatase in a Ca2+-regulated process are thought to account for the dephosphorylation of E1b (31, 51, 66). Our results suggest that the recombinant human E2 is significantly more effective than bovine E2·E3BP in enhancing phosphatase activity. Although many explanations are possible (cf. "Results"), testing the interesting prospect that E3BP has an inhibitory role will require preparation of human E2·E3BP.
In agreement with an earlier preparation (35) in which bovine E2 was freed of E3BP (then designated as protein X), the recombinant E2 oligomer was able to support about 4% of the PDC activity obtained with the E2·E3BP subcomplex. These results differ from those obtained with yeast PDC-E2, prepared in the absence of the E3BP, which failed to support any PDC activity even in the presence of high levels of E3 (61). Nevertheless, both outcomes demonstrate that the binding of E3 by the E3BP component must greatly enhance the capacity to feed reducing equivalents through E3 in the PDC reaction. This suggests unique organizational features allow limited E3 dimers, tethered at the surface of the complex, to function efficiently. The results in Table III indicate E3BP-bound E3 has access to a fixed number of lipoyl domains based on the unchanging specific activity of low levels of E3 (endogenous or added) with variation of the level of E2·E3BP subcomplex. The lipoyl domains of E2 and E3BP are concentrated at about 2 mM at the surface of the E2 core (53, 72) and are probably anchored in ways that limit their orientations. These conditions may not only facilitate reducing equivalents being directly channeled to E3 but also aid a rapid thiol-disulfide interchange between lipoates. The latter may be coupled to electrons being fed to E3 with some degree of preference via the lipoate on E3BP's lipoyl domain (8, 73). The E3 component must operate at >280 µmol/min/mg of E3 to give the observed specific activity of 20 µmol/min/mg of bovine heart PDC in the overall reaction of the complex (bound E3 is maximally 7% of the protein; Ref. 9). Given this high rate, it seemed likely that reaction of 0.2 mM DTNB with reduced lipoates is probably limiting when E3 was confined by binding E3BP. In support of this possibility, halving the DTNB concentration to 0.1 mM reduced the E3 reaction rates of E3 associated with E2·E3BP structures by ~40% (data not shown). Thus, a rapid E3 reaction followed by a rate-limiting chemical reoxidation of lipoates is indicated.
The production of assembled recombinant E2 that functions in all the roles of mammalian PDC-E2 creates abundant opportunities for further studies in which recombinant techniques are used to dissect catalytic, binding, and regulatory roles of this central component of mammalian PDC. Furthermore, new insights and questions are raised by the differences that we have observed between bovine E2·E3BP and human E2 in supporting catalytic processes and in their relative capacities to facilitate high E1b phosphatase activity.
We are thankful to Shengjiang Liu for advice and technical assistance, to Jason Baker for preparing lipoyl domain constructs and for help in analyzing kinase activation with different E2 and E1 sources, to Sundari Ravindran for preparing bovine complex and components, to Robert Grassucci for electron microscopy, for the availability of the Wadsworth Center electron microscopy core facility and Kansas State University Biotechnology core facility, and to Amy Paulin for help in figure preparation and experiments.