Download Molecular architecture of the pyruvate dehydrogenase complex

Information Processing and Molecular Signalling
Molecular architecture of the pyruvate
dehydrogenase complex: bridging the gap
M. Smolle*†1 and J.G. Lindsay*
*Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, U.K., and †Division of
Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8TA, U.K.
Abstract
The PDC (pyruvate dehydrogenase complex) is a high-molecular-mass (4–11 MDa) complex of critical
importance for glucose homoeostasis in mammals. Its multi-enzyme structure allows for substrate
channelling and active-site coupling: sequential catalytic reactions proceed through the rapid transfer of
intermediates between individual components and without diffusion into the bulk medium due to its
‘swinging arm’ that is able to visit all PDC active sites. Optimal positioning of individual components
within this multi-subunit complex further affects the efficiency of the overall reaction and stability of
its intermediates. Mammalian PDC comprises a 60-meric pentagonal dodecahedral dihydrolipoamide (E2)
core attached to which are 30 pyruvate decarboxylase (E1) heterotetramers and six dihydrolipoamide (E3)
homodimers at maximal occupancy. Stable E3 integration is mediated by an accessory E3-binding protein
associated with the E2 core. Association of the peripheral E1 and E3 enzymes with the PDC core has been
studied intensively in recent years and has yielded some interesting and substantial differences when
compared with prokaryotic PDCs.
Introduction
The mitochondrial 2-oxoacid dehydrogenase complexes are
a family of large, multi-enzyme assemblies that serve as
models for the study of protein–protein interactions and molecular recognition phenomena. They catalyse the oxidative
decarboxylation of a vital group of 2-oxoacid intermediates in
carbohydrate and amino acid metabolism. Principal members
include the PDC (pyruvate dehydrogenase complex), OGDC
(2-oxoglutarate dehydrogenase complex) and BCOADC
(branched-chain 2-oxoacid dehydrogenase complex).
PDC controls the key committed step in carbohydrate utilization, namely the conversion of pyruvate into acetyl-CoA
and NADH, thus linking glycolysis to the citric acid cycle
in a three-step reaction. Initially, pyruvate is decarboxylated by the ThDP (thiamin diphosphate)-dependent E1
enzyme that also catalyses the subsequent transfer of the
resulting acetyl group from ThDP on to the lipoamide moiety
of E2 in a reductive acetylation step, generating a stable
intermediate. In the presence of CoASH, E2 catalyses the
transacetylation reaction to form acetyl-CoA. Finally, the E2
dihydrolipoamide is reoxidized by E3. E3 transfers electrons
via a redox-active pair of cysteine residues to FAD and on to
the terminal electron acceptor NAD+ . The overall reaction
Key words: active-site coupling, energy metabolism, mammal, molecular architecture, 2-oxoacid
dehydrogenase, pyruvate dehydrogenase.
Abbreviations used: BCOADC, branched-chain 2-oxoacid dehydrogenase complex; cryo-EM,
cryo-electron microscopy; E3BP, E3-binding protein; LD, lipoyl domain; OGDC, 2-oxoglutarate
dehydrogenase complex; PDC, pyruvate dehydrogenase complex; PDK, PDC kinase; PDP, PDC
phosphatase; SAXS, small angle X-ray scattering; SBD, subunit-binding domain; ThDP, thiamin
diphosphate.
1
To whom correspondence should be addressed (email [email protected]).
can be summarized as:
pyruvate + CoASH + NAD+ → acetyl-CoA
+ CO2 + NADH + H+ .
Genetic and physiological defects in PDC have been
implicated (i) in a wide range of diseases including various
types of metabolic acidosis and mitochondrial myopathies;
(ii) as major autoantigens in the autoimmune disease, primary
biliary cirrhosis; (iii) in neurodegenerative disorders,
e.g. Alzheimer’s disease; and (iv) as primary targets
for modification by environmental toxins and oxidative
damage.
Basic core organization
All three 2-oxoacid complexes display a similar mode of
quasi-symmetric organization of their subunits E1, E2 and
E3. Apart from playing a role in the catalytic mechanism, E2
also forms a central 24-meric or 60-meric core, consistent with
octahedral (432) or icosahedral (532) symmetry respectively
[1,2]. For OGDC and BCOADC, as well as PDC from
Gram-negative bacteria, the E2 core is assembled as an
octahedron. Only in Gram-positive bacteria and eukaryotes
does the PDC core assume an icosahedral morphology.
Furthermore, eukaryotic PDC contains an additional protein,
E3BP (E3-binding protein; previously protein X), which is
associated with the E2 core [3,4] and responsible for binding
E3.
Both E2 and E3BP have similar, modular domain structures: human E2 and E3BP consist of two (E2) or one (E3BP)
N-terminal LD(s) [lipoyl domain(s)] of approx. 80 amino
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Figure 1 Subcomplex formation in bacterial and human PDCs
(A) Complex of G. stearothermophilus E1 (α, yellow/pink; β, green/purple) with E2-SBD (blue) (PDB code 1W85).
(B) Complex of G. stearothermophilus E3 (green/red) with E2-SBD (blue) (PDB code 1EBD). (C) Low-resolution structure
(blue) superimposed on to a model of a 2:1 subcomplex of E3BP-didomain (green/magenta, lime/yellow) and E3 (orange).
acids. Each LD carries a lipoic acid moiety covalently linked
to a lysine residue situated at the tip of a type I β-turn.
The LD is followed by an SBD (subunit-binding domain)
of approx. 35 residues, responsible for interaction with E1
and/or E3, and a C-terminal domain of approx. 250 residues
that is essential for core formation. In E2, the C-terminal
domain is also the catalytic domain, while the active-site motif
is mutated in E3BP, rendering it incapable of catalysing the
acetyltransferase reaction. All domains are interconnected by
alanine- and proline-rich linker regions of approx. 30 amino
acids in length that impart the flexibility necessary for the LD
to visit all three active sites during catalysis.
Two models for the eukaryotic PDC core have been
proposed: in the ‘addition model’ based on cryo-EM (cryoelectron microscopy) of recombinant PDC cores from
Saccharomyces cerevisiae, 60 molecules of E2 are thought to
form the edges of the icosahedron, while 12 copies of E3BP
are located on the inside of the E2 core, apparently associated
with the tips of E2 trimers [5]. More recently, Hiromasa
et al. [6] proposed an alternative ‘substitution’ model based
on results from analytical ultracentrifugation and SAXS
(small angle X-ray scattering) where 12 E3BP molecules are
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thought to replace an equal number of E2 enzymes within the
core.
Active-site coupling
The ability of PDC to sustain substrate channelling and
active-site coupling is the basis for its efficient and tightly
controlled function (see [7] for an excellent review).
Fluorescence energy transfer studies revealed that the three
different active sites have to be at least 40 Å (1 Å = 0.1 nm)
apart, since no energy transfer could be detected for
Azotobacter vinelandii PDC [8,9]. Cryo-EM reconstructions
of Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) and bovine PDC indicate even larger distances.
Milne et al. [10] have shown the existence of an annular
gap of approx. 90 Å between the E2 core and deposited
E1, while it is somewhat smaller (∼75 Å) between E2 and
E3 [11]. The distances reported for bovine PDC are shorter
with a space approx. 50 Å in length between the core and
both E1 and E3 respectively [12]. The difference in spacing
observed in Geobacillus and bovine PDC remains unclear.
However, taking the LD and its linker into account, bridging
Information Processing and Molecular Signalling
the distances between active sites becomes straightforward,
as gaps as large as 200 Å or more could be traversed [7,13].
Figure 2 A model for ‘cross-bridge’ formation in eukaryotic PDC
E3BP/E3 cross-bridges are shown in magenta/cyan and E2/E1
cross-bridges in green/blue. PDK and PDP/PRP are shown in purple.
The E2/E3BP core is based on a cryo-EM structure [37].
PDC architecture
The PDC core provides the structural and mechanistic framework for the tight but non-covalent association of E1 and E3.
In eukaryotes, tight protein–protein interactions are formed
specifically between E2-SBD and E1, and E3BP-SBD and
E3 respectively. However, in Gram-positive bacteria such
as G. stearothermophilus, E1 and E3 both associate with
E2, as E3BP features only in eukaryotes. Consequently,
E1 and E3 have to compete for overlapping binding sites
on E2; interaction of E2 with either E1 or E3 prevents
complex formation with the other [14]. Interestingly, PDCdeficient patients who totally lack E3BP possess partial
complex activity (10–20% of controls) [15], apparently
because the SBD of E2 has retained a limited ability to mediate
low-affinity E3 binding (S.D. Richards and J.G. Lindsay,
unpublished work).
In G. stearothermophilus PDC, up to 60 molecules of E1 or
E3 can be associated with the E2 core. Furthermore, binding
of both E1 and E3 to E2-SBD has been shown to occur at
stoichiometries of 1:1 in solution [16–19] as well as by crystallography (Figures 1A and 1B). The crystal structure for the
E1–E2-SBD subcomplex shows that the binding site for E2SBD is located across the E1 2-fold axis [20]. Similarly, in
the E3–E2-SBD structure, the binding site on E3 is close
to the 2-fold axis of symmetry [21]. Thus the association of
a second molecule of E2-SBD with either E1 or E3 is not
possible. Occupation of both binding sites would result in
steric clashes in one of the E2-SBD loop regions.
In contrast, it has been known for a long time that a total of
30 E1 and six E3 molecules can associate with the mammalian
PDC core [22], suggesting the possibility of formation
of 2:1 stoichiometric subcomplexes. The crystal structures
determined for human E3 complexed with its cognate SBD
from E3BP [23,24] resemble those solved for the bacterial
subcomplexes. However, when tested in solution using
various biochemical and biophysical techniques, including
non-denaturing PAGE, isothermal titration calorimetry,
analytical ultracentrifugation and SAXS, results indicate the
formation of subcomplexes at a ratio of 2:1 rather than 1:1
(E3BP/E3) [25], suggesting the existence of a network of
so-called ‘cross-bridges’ on the surface of PDC (Figure 2).
Essentially, cross-bridge formation is thought to constrain
the mobility of the E3BP (and E2) LD, especially if analogous
cross-bridges exist between E1 and E2 as suggested by
preliminary evidence from our laboratory. Consequently, this
arrangement may impair the ability of any one LD to interact
with all three different active sites. However, migration of
acetyl groups between the S-6 and S-8 atoms of lipoamide
has been documented [26], and experiments using labelled
pyruvate have confirmed that acetyl groups and reducing
equivalents can be shuttled between different LDs [27–29].
The discrepancy between crystal and solution structures is
thought to arise from differences in the conformation of E3
in solution apparent in low-resolution structures of both
E3 on its own (M. Smolle, D. Byron and J.G. Lindsay,
unpublished work) as well as a complex formed between
E3 and two molecules of E3BP-didomain (consisting of the
LD and SBD; Figure 1C). Further contributing factors could
be differences in buffer composition and/or the effects of
crystal packing. Indeed, the solution conformations of other
proteins, including yeast pyruvate decarboxylase [30], can
differ significantly from their crystal structures [31–34].
Another aspect of PDC function potentially impacted
by a network of cross-bridges is its regulation by the
tightly bound PDK (PDC kinase) and a loosely bound PDP
(PDC phosphatase). It has been reported that only one
to three molecules of PDK are bound per complex [35].
Both regulatory enzymes associate with the inner E2 LD
whereby the lipoyl cofactor provides an important part of
the recognition site. PDP and PDK both act by de-/phosphorylation of E1 on any of three specific E1α serine residues.
Phosphorylation of E1 renders the complex inactive [15].
Since only one to three PDK molecules are present per
complex, the enzyme has to migrate around the surface of
the complex in order to act on the entire population of bound
E1 molecules. A hand-over-hand mechanism has been
proposed where the dimeric PDK molecule migrates from
one inner LD to its nearest neighbour [36]. Regular spacing
of the E2 LDs linked by a system of E1 cross-bridges
could greatly facilitate this type of movement and provide
convenient access to all E1 molecules.
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Concluding remarks
Understanding PDC assembly, subunit organization and the
molecular basis for its operational efficiency and precisely
regulated activity remain at the forefront of scientific research
due to its pivotal role in energy metabolism. Furthermore,
increasing recognition of PDC involvement in a wide
spectrum of major human diseases and as a prime target
for oxidative stress and environmental toxins has served to
stimulate renewed interest in this fascinating multi-enzyme
assembly as an attractive area for further basic research and
drug development.
Research performed in our laboratory and presented here was
supported by The Wellcome Trust and the Biotechnology and
Biological Sciences Research Council.
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Received 11 July 2006