Báo cáo khoa học: Interaction of the E2 and E3 components of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus ppt

Interaction of the E2 and E3 components of the pyruvate
dehydrogenase multienzyme complex of Bacillus
stearothermophilus
Use of a truncated protein domain in NMR spectroscopy
Mark D. Allen, R. William Broadhurst, Robert G. Solomon and Richard N. Perham
Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, UK
The 2-oxo acid dehydrogenase complexes consist of
multiple copies of three distinct enzymes that together
catalyse the oxidative decarboxylation of 2-oxo acids,
in the presence of thiamine diphosphate (ThDP), coen-
zyme A (CoA), Mg
2+
and NAD
+
, to generate CO
2
and the corresponding acyl-CoA. The complexes are
assembled around an oligomeric [octahedral (24-mer)
or icosahedral (60-mer)] dihydrolipoyl acyltransferase
(E2) core to which multiple copies of the relevant
2-oxo acid decarboxylase (E1) and dihydrolipoyl dehy-
drogenase (E3) bind tightly, but noncovalently, to
form the intact multienzyme complexes [1]. The pyru-
vate dehydrogenase (PDH) complex has a pivotal role
in most organisms, catalysing the irreversible reaction
that links the glycolytic pathway and the tricarboxylic
acid cycle. Pyruvate is oxidatively decarboxylated to
acetyl-CoA, which can be either broken down further
in the tricarboxylic acid cycle or used as an important
Keywords
pyruvate dehydrogenase; protein–protein

interaction; NMR spectroscopy;
multienzyme complex; protein domains
Correspondence
R.N. Perham, Department of Biochemistry,
University of Cambridge, Sanger Building,
Old Addenbrooke’s Site, 80 Tennis Court
Road, Cambridge CB2 1GA, UK
Fax: +44 1223 338707
Tel: +44 1223 338635
E-mail:
(Received 19 July 2004, revised 27 September
2004, accepted 28 September 2004)
doi:10.1111/j.1432-1033.2004.04405.x
A
15
N-labelled peripheral-subunit binding domain (PSBD) of the dihydro-
lipoyl acetyltransferase (E2p) and the dimer of a solubilized interface
domain (E3int) derived from the dihydrolipoyl dehydrogenase (E3) were
used to investigate the basis of the interaction of E2p with E3 in the assem-
bly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearo-
thermophilus. Thirteen of the 55 amino acids in the PSBD show significant
changes in either or both of the
15
N and
1
H amide chemical shifts when
the PSBD forms a 1 : 1 complex with E3int. All of the 13 amino acids
reside near the N-terminus of helix I of PSBD or in the loop region
between helix II and helix III.
15

N backbone dynamics experiments on
PSBD indicate that the structured region extends from Val129 to Ala168,
with limited structure present in residues Asn126 to Arg128. The presence
of structure in the region before helix I was confirmed by a refinement of
the NMR structure of uncomplexed PSBD. Comparison of the crystal
structure of the PSBD bound to E3 [Mande SS, Sarfaty S, Allen MD,
Perham RN & Hol WGJ (1996) Structure 4, 277–286] with the solution
structure of uncomplexed PSBD described here indicates that the PSBD
undergoes almost no conformational change upon binding to E3. These
studies exemplify and validate the novel use of a solubilized, truncated pro-
tein domain in overcoming the limitations of high molecular mass on
NMR spectroscopy.
Abbreviations
E1, pyruvate decarboxylase; E2, dihydrolipoyl acetyltransferase; E3, dihydrolipoyl dehydrogenase; E3int, dimer of a solubilized interface
domain; PDH, pyruvate dehydrogenase; PSBD, peripheral subunit-binding domain; ThDD, thrombin-cleavable di-domain; ThDP, thiamine
diphosphate.
FEBS Journal 272 (2005) 259–268 ª 2004 FEBS 259
metabolite in the synthesis of fatty acids, cholesterol,
steroids in eukaryotes and N-acetyl-derived carbo-
hydrates.
The structural and mechanistic core of the 2-oxo
acid dehydrogenase complexes is provided by the E2
component, each chain of which is composed of three
independent domains. At the N-terminus are 1–3 tan-
demly repeated lipoyl domains, followed by a peri-
pheral subunit-binding domain (PSBD) responsible for
binding E3 in the majority of organisms. In icosahe-
dral complexes, the PSBD is also involved in the bind-
ing of E1 [1,2]. The catalytic (acyltransferase) core
domain, which assembles to form the octahedral or

icosahedral inner core of the complexes, is proposed to
bind E1 in the octahedral complexes [2,3], and is loca-
ted at the C-terminus. Each of the individual domains
is separated by long and flexible linker regions, which
make possible active site coupling by allowing for large
movements of the lipoyl domain(s) [1,4–6].
To date it has proved impossible to obtain crystals
of an intact PDH complex. However, several three-
dimensional structures have been determined for the
individual domains of E2 chains from both icosahedral
and octahedral complexes: lipoyl domain structures
from Bacillus stearothermophilus, Escherichia coli and
Azotobacter vinelandii PDH complexes [7–9], and
E. coli and A. vinelandii 2-oxoglutarate dehydrogenase
complexes [10,11]; PSBD structures from E. coli 2-oxo-
glutarate dehydrogenase and B. stearothermophilus
PDH complexes [12,13]; the octahedral acyltransferase
core from A. vinelandii PDH [14] and E. coli 2-oxo-
glutarate dehydrogenase [15] complexes and the icosa-
hedral core from the B. stearothermophilus PDH
complex [16]. Additionally, E3 and E1 structures from
a number of sources have been solved by X-ray crys-
tallography [17–24], and it has been possible to obtain
a crystal structure of a B. stearothermophilus E3–PSBD
complex formed between a lipoyl domain-PSBD
di-domain and an E3 dimer [24]. Most recently, mod-
els for the overall structures of the assembled PDH
complexes, of some 10 MDa in molecular mass, have
been proposed based on cryoelectron microscopy data
[25,26].

An E3 dimer can bind only one PSBD domain of
E2 [1,27]. This is due to steric hindrance, the associ-
ation with one PSBD close to the twofold axis of E3
preventing the association of a second PSBD [28,29].
The interaction occurs at the C
2
-axis of symmetry in
the E3 dimer, chiefly with the interface domain of E3,
which is highly conserved and generates the majority
of contacts across the dimer interface. A surface loop
region of the PSBD appears to undergo a conforma-
tional change when PSBD binds to the E3 dimer, as
judged by the observed differences between the struc-
tures obtained by NMR spectroscopy for the free
PSBD [13] and X-ray crystallography for the
E3–PSBD complex [28].
This paper describes the interaction of a
15
N-labelled
PSBD (residues 119–171) of the B. stearothermophilus
E2p polypeptide chain with the dimer of a protein
domain (E3int) representing the interface domain (resi-
dues 343–470) of B. stearothermophilus E3. The use of
the engineered E3int domain (27 kDa as the dimer)
was introduced because the high molecular mass
(112 kDa) of the intact E3 dimer limited the applica-
tion of NMR spectroscopy. Chemical shift differences
between the backbone resonances of the uncomplexed
PSBD and PSBD bound to E3int indicate that several
amino acids near the N-terminus of helix I of the

PSBD are at or near the E3-binding site, confirming
and extending the earlier crystal structure [28]. The
backbone dynamics of PSBD were also investigated, as
the rigidity or otherwise of the proposed E3-binding
site is crucial to a proper understanding of protein–
protein interactions within the multienzyme complex.
Further, an improved solution structure of PSBD was
subsequently determined and compared with that of
the PSBD in the crystal structure of the PSBD–E3
complex [28], thereby allowing a better estimate of the
extent of molecular rearrangement that accompanies
binding to E3.
Results and Discussion
Interaction of PSBD with the intact E3 dimer
E3 (0.1 nmol of dimer) was mixed with various
amounts of PSBD before being subjected to nondena-
turing polyacrylamide gel electrophoresis, as described
elsewhere [30]. Saturation of binding, as evidenced by
the appearance of free PSBD in the Coomassie-stained
gel, was found to occur when 0.1 nmol of E3 dimer
was mixed with 0.1 nmol of PSBD. As expected, there-
fore, free PSBD interacts with B. stearothermophilus
E3 in a 1 : 1 stoichiometry identical to that observed
previously with the PSBD as part of the lipoyl-PSBD
di-domain [30].
The changes in backbone amide
15
N and
1
H chem-

ical shifts upon mixing PSBD with E3 were slight
(maximum chemical shift changes are 0.213 p.p.m. and
0.029 p.p.m. for
15
N and
1
H shifts, respectively; results
not shown). Significant chemical shift changes (greater
than 0.10 and 0.02 p.p.m. in the
15
N and
1
H dimen-
sions, respectively) were observed for Asn126, Arg127,
Ala131, Gly156 and Glu161. However, for all but the
eight and two residues in the N- and C-terminal
Assembly of pyruvate dehydrogenase complex M. D. Allen et al.
260 FEBS Journal 272 (2005) 259–268 ª 2004 FEBS
regions, respectively, linewidths in the PSBD–E3 dimer
(10 : 1) mixture were much broader than those found
for PSBD alone, greatly diminishing the information
that could be derived from the spectrum. This is pre-
sumably due to the high molecular mass (115 kDa)
and correspondingly long rotational correlation time
of the PSBD–E3 complex.
Interaction of PSBD with E3int
To overcome these problems connected with the high
molecular mass of the PSBD–E3 complex, the inter-
action of PSBD with a solubilized interface domain
(E3int) of B. stearothermophilus E3 was studied. The

E3int domain comprises the C-terminal portion, resi-
dues 343–470, of the B. stearothermophilus E3 chain
and, based on the crystal structure of E3, contains a
majority of the intersubunit contacts in the E3 dimer
[1,18,28]. It has proved possible to generate a dimer-
ic form of the corresponding interface domain of the
homologous glutathione reductase, a flavoprotein like
E3, from E. coli. To achieve this, hydrophobic pat-
ches on the surface of the domain were altered to
display charged or hydrophilic residues in key posi-
tions, thereby rendering the domain soluble but not
prone to aggregation beyond the desired dimer stage
[31]. A similar programme of mutation was therefore
undertaken on a subgene encoding the interface
domain of the B. stearothermophilus E3. The final
version of the solubilized E3int domain contains
seven mutations of surface hydrophobic residues
(I384D, V352N, L391A, I465G, I384D, V352N and
D443N) chosen to render the domain more soluble
than its wild-type counterpart, but not to interfere
with dimerization. The E3int domain forms a dimer
in the same way as E3, as determined by gel filtra-
tion (data not shown), and can be expected to inter-
act with PSBD in essentially the same way as the
intact E3 dimer [29,30]. However, the E3int dimer
has a molecular mass of only 27 kDa compared with
110 kDa for E3.
Linewidths for all but the 12 and terminal residues
in the N- and C-terminal regions of PSBD (which do
not form part of the structured region) were found to

be narrower than those observed on complexation
with E3. This reflects the smaller size and shorter
rotational correlation time of the PSBD–E3int com-
plex (molecular mass 32 kDa). However, linewidths
were still broader than those in the spectra of PSBD
alone.
The changes in backbone amide
15
N and
1
H chem-
ical shifts upon mixing PSBD with E3int are shown in
Fig. 1A,B, respectively. The chemical shift changes
between the free PSBD and PSBD–E3int spectra are
more pronounced (maximum changes are 0.500 p.p.m.
and 0.065 p.p.m. for
15
N and
1
H shifts, respectively)
than those observed between free PSBD and PSBD–E3
spectra. Significant changes in chemical shift were
observed for Ala123, Asn126, Arg127, Arg128, Val129,
Ile130, Ala131, Met132, Val135, Arg136, Lys137,
Lys154 and Arg157. The location of residues undergo-
ing large chemical shift changes upon interaction with
E3 and E3int are mapped on to the three-dimensional
structure of PSBD in Fig. 1C (where changes greater
than 0.10 p.p.m. or 0.02 p.p.m. in the
15

N and
1
H
dimensions, respectively, are indicated by grey
spheres). The major locations include residues 126–137
straddling the start of helix I (residues 134–142) and
residues Lys154, Gly156 and Arg157 in the loop (L2)
between helix III (residues 145–149) and helix II (resi-
dues 159–168). As shown in Fig. 1C, all the affected
residues lie relatively close in space, with both the
region near the N-terminus of helix I of the domain
and the three residues in loop L2 contributing to the
E3-binding site.
Backbone dynamics of PSBD
The region (Met132 to Ser134) before the start of
helix I of the PSBD structure was reported previously
[13] to be largely unstructured owing to an absence of
detectable long-range NOEs in the NMR spectrum.
The availability of uniformly
15
N-labelled PSBD
enabled us now to analyse the backbone dynamics
using a steady-state [
1
H]-
15
N NOE experiment [32,33]
in 20 mm potassium phosphate buffer, pH 6.5, at 298
K. The calculated values of g for PSBD plotted
against residue number are shown in Fig. 2. The plot

indicates that the structured region extends from
Val129 to Ala168, but residues Asn126, Arg127 and
Arg128 do appear to be partly mobile.
Structure determination of PSBD
Because the backbone dynamics experiment revealed
that residues 117–125 and 171 of the PSBD are highly
flexible in solution, only residues 126–170 were inclu-
ded in the structure calculations. The final set of struc-
tures of PSBD contained 841 unambiguous
conformational constraints (summarized in Table 1).
The final ensemble comprised 25 structures, all of
which had no distance violations > 0.25 A
˚
, no dihed-
ral angle violations > 5 °, and very small deviations
from ideal covalent geometry. Most of the residues are
restricted to favoured or additionally allowed regions
of (/,w) space with, of the nonglycine residues, only
M. D. Allen et al. Assembly of pyruvate dehydrogenase complex
FEBS Journal 272 (2005) 259–268 ª 2004 FEBS 261
Lys154 consistently falling into disallowed regions.
Figure 3 illustrates the ensemble and secondary struc-
ture regions of PSBD.
All statistical calculations were carried out on the
structured region from Val129 to Ala168. Outside this
region the torsion angles of / and w deviated rapidly
away from their mean values. The average root mean
square (rms) deviation from the mean coordinates for
backbone nuclei over the structured region from
Val129 to Ala168 is 0.35 A

˚
(Table 1).
Backbone fractional H- N-
NOE enhancement
1
15
-4.0
-3.6
-3.2
-2.8
-2.4
-2.0
-1.6
-1.2
-0.8
-0.4
0.0
120 125 130 135 140 145 150 155 160 165 170
Residue number
Fig. 2. Backbone dynamics of PSBD. Plot of
the backbone steady state {
1
H}-
15
N NOE
enhancement, g, for PSBD as a function of
residue number.
-0.06
-0.04
-0.02

0.00
0.02
0.04
0.06
K154
R127 V129
I130
A131
M132
V135
K137
B
Residue Number
H- chemical shift change (ppm)
1
120 125 130 135 140 145 150 155 160 165 170
-0.5
-0.4
-0.3
-0.2
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
Residue Number
N126
V129

I130
R136
R157
A
C
A123
N- chemical shift change (ppm)
15
120 125 130 135 140 145 150 155 160 165 170
Lys137
Arg136
Val135
Met132
Ala131
Ile130
Val129
Arg 128
Arg127
Asn126
Lys154
Gly156
Arg157
Glu161
N
C
Fig. 1. NMR spectroscopy of the interaction of PSBD and E3int. Plots of the (A)
15
N and (B)
1
H

N
chemical shift changes between the
[
15
N,
1
H]-HSQC spectra of free PSBD and the PSBD–E3int dimer (10 : 1) mixture as a function of residue number. (C) Schematic MOLSCRIPT
[46] drawing of the three-dimensional structure of PSBD; residues undergoing significant chemical shift changes (> 0.10 p.p.m. or
> 0.02 p.p.m. in the
15
Nor
1
H dimensions, respectively) are illustrated as grey spheres. The molecule is coloured from the N- to the C-termi-
nus following the colours of the visible spectrum (violet for N-terminus and red for the C-terminus).
Assembly of pyruvate dehydrogenase complex M. D. Allen et al.
262 FEBS Journal 272 (2005) 259–268 ª 2004 FEBS
Description of the three-dimensional structure
of PSBD
The refined NMR structure of PSBD is consistent with
the two previously reported E3-binding domain struc-
tures: that of synthetic peptides representing the
PSBD of the dihydrosuccinyltransferase chain of the
2-oxoglutarate dehydrogenase complex of E. coli [12]
and of the dihydroacetyltransferase chain of the PDH
complex of B. stearothermophilus [13]. The domain
consists of two a-helices comprising residues Ser134 to
Lys142 (helix I) and Leu159 to Leu168 (helix II), and
a short 3
10
-helix Asp145 to Val149 (helix III), with

loops connecting the helices.
The N-terminal region (Val129 to Pro133) is relatively
well-defined and possesses a hydrogen bond between
Val158 H
N
and Ile130 C¢. This hydrogen bond was not
observed for the construct used by Kalia et al. [13] and
undoubtedly the inclusion of this restraint in our struc-
ture calculations permitted this region to adopt a more
rigid conformation. The region is further stabilized by a
number of hydrophobic contacts between Val129,
Ala131, Val135 and Ile146. The loop L2 between the
3
10
-helix and helix II contains several residues with
amide protons exhibiting reduced rates of exchange with
the solvent. Analysis of the initial structures allowed
hydrogen bond acceptors for each of these residues to
be identified. The structure also appears to be stabilized
by hydrogen bonds between Tyr138 O
g
H
g
and Asp164
O
d2
(although this was not included as a restraint in the
structure calculations).
Structural comparisons
The three-dimensional structure of the PSBD from the

E2 chain of the B. stearothermophilus PDH complex
has been determined before, by NMR spectroscopy of
the free PSBD [13] and X-ray crystallography of the
E3–PSBD complex [28]. All three structures now avail-
able are very similar with respect to the arrangement
of structural motifs and loops, with the exception of
loop L2 where differences exist. Previously it was
noted that upon superposition of the NMR structure
and crystal structure, the tip of loop L2 appeared to
Table 1. Summary of constraints and statistics for the 20 accepted
structures of B. stearothermphils PSBD domain.
Structural constraints
Intra-residue 341
Sequential 178
Medium-range ( 2 £ |i-j| £ 4 ) 119
Long-range ( |i-j| > 4 ) 137
Dihedral angle constraints 22
Distance constraints for 22 hydrogen bonds 44
Total 841
Statistics for accepted structures
Statistics parameter (± SD)
Rms deviation for distance constraints 0.006 A
˚
± 0.001A
˚
Rms deviation for dihedral constraints 0.02 ° ± 0.01 °
Mean X-PLOR energy term (kcalÆmol
)1
± SD)
E (overall) 40.0 ± 3.0

E (van der Waals) 11.3 ± 1.4
E (distance constraints) 2.5 ± 0.5
E (dihedral and TALOS constraints) 0.003 ± 0.002
Rms deviations from the ideal geometry (± SD)
Bond lengths 0.0011 A
˚
± 0.0001 A
˚
Bond angles 0.35 ° ± 0.01 °
Improper angles 0.16 ° ± 0.01 °
Average atomic rmsd from the mean structure (± SD)
Residues 129–168 (N, Ca, C atoms) 0.33 A
˚
±0.09A
˚
Residues 129–168 (all heavy atoms) 0.33 A
˚
±0.09A
˚
Ser134
Lys142
Asp145
Ala168
Leu159
Val149
Fig. 3. Solution structure of PSBD from
NMR spectroscopy. Superposition of back-
bone traces of the 25 accepted structures
over residues 129–168, and a
MOLSCRIPT [46]

representation of the PSBD structure in the
same orientation. The residues defining the
secondary structural elements are labeled.
M. D. Allen et al. Assembly of pyruvate dehydrogenase complex
FEBS Journal 272 (2005) 259–268 ª 2004 FEBS 263
move by 9.2 A
˚
[28]. It was suggested that the differ-
ence might be due to changes taking place in loop L2
upon binding to the E3 dimer or might reflect an
intrinsic flexibility of loop L2. The results from the
backbone dynamics experiment described above, how-
ever, reveal that the loop is inherently rigid on the sub-
nanosecond time scale. Together with the absence of
significant chemical shift changes for more residues in
loop L2 when the PSBD binds to E3int, this suggests
that the loop does not undergo significant structural
rearrangement upon binding to E3. Moreover, super-
positioning shows that the crystal [28] and present
NMR (see above) structures of PSBD are virtually
identical. Comparative Ramachandran plots of the
PSBD NMR and crystal structures reveal only minor
differences in loop L2, which are confined to residues
Gly153, Lys154 and Asn155.
The differences observed between the previous
NMR structure [13] and that determined here (see
above) are probably due to the use of uniformly
15
N-labelled PSBD and the inclusion of additional resi-
dues in the construct at the N-terminus of the domain.

In particular, the absence of residues Arg127 and
Arg128 from the earlier construct [13] may have affec-
ted the stability of the N-terminal region, thereby pre-
venting it from adopting the defined conformation
observed in the crystal and new NMR structures. The
different conformations of loop L2 between the two
NMR structures can also be attributed to the different
N-terminal regions, as the lack of the observed hydro-
gen bond constraint between Val158 H
N
and Ile130 C¢
would have significantly affected the earlier calcula-
tions [13]. The remaining slight differences between the
crystal structure and the new NMR structure are prob-
ably due to the relatively low number of NOEs
observed between Lys154 and Asn155 in the loop L2.
The site of interaction between E3 and PSBD
The chemical shift changes observed on formation of a
complex between PSBD and E3int are likely to be due
principally to direct contact between the two proteins,
together with some changes in the conformation of
PSBD upon binding. Figure 1 shows large chemical shift
changes for Asn126, Arg127, Val129, Ile130, Ala131,
Met132, Val135, Arg136, Lys137, Lys154, Gly156 and
Arg157, all of which are located close in three-dimen-
sional space. The crystal structure of the E3-lipoyl-
PSBD di-domain complex has already been determined
[28]. The hydrophobic residues Val129, Ile130, Ala131,
Met132, Val135 and basic residues Arg127, Arg128,
Arg136, Lys137, Lys154 and Arg157 implicated by these

NMR studies are arranged in the crystal structure such
that the hydrophobic regions on PSBD and E3 form
multiple van der Waals contacts, while the acidic resi-
dues of E3 and basic residues of PSBD generate multiple
salt-bridges. The results obtained by means of NMR
spectroscopy are now wholly consistent with the struc-
ture of the PSBD–E3 complex determined by X-ray
crystallography. This rules out any doubt that the crys-
tal structure might not be a valid representation of the
interaction in solution and indicates that the interaction
between PSBD and E3, though very tight (K
d
 10
)9
m
[1,30]), is of the direct ‘lock-and-key’ kind rather than
an induced fit. As a corollary, it is clear that the
approach we have been developing, of creating a soluble
construct containing the interface region of the E3 dimer
[31,34], has made it possible to overcome the molecular
mass limitation in using NMR spectroscopy to study
protein structure and protein–protein interaction. This
augurs well for future studies, for example by transverse
relaxation compensated NMR spectroscopy.
Experimental procedures
Materials
Bacteriological media were from Difco (Detroit, MI, USA).
The pBSTNAVDD vector carrying the dihydrolipoyl ace-
tyltransferase gene that encodes residues 1–171 of B. stearo-
thermophilus E2p was generated earlier [35]. Plasmid

pET11d and E. coli host strain BL21(DE3) [(F
–
, ompT,
hsdS
B
(r
b
–
,m
b
–
), gal, dcm (DE3)] were obtained from Nov-
agen Inc (Madison, WI, USA).
Construction of expression vector pET11ThDD
Construction of plasmid pET11ThDD encoding residues
1–171 of B. stearothermophilus E2p with a thrombin-cleavage
site (LVPRGS) in place of Ala118 proceeded via double
overlapping PCR mutagenesis using standard techniques
[36]. Plasmid pBSTNAVDD [35] was used as the template
DNA. The PCR product was digested with NcoI and
BamH1, purified by means of agarose gel electrophoresis and
ligated into vector pET11d previously digested with NcoI
and BamH1 and treated with calf intestinal alkaline phospha-
tase. The resulting vector encodes the sequence of a
di-domain with a thrombin-cleavable linker region (ThDD).
The DNA was fully sequenced to ensure its fidelity.
Expression and purification of
15
N-labelled PSBD
E. coli strain BL21(DE3) cells transformed with

pET11ThDD were grown to an A
600
of 1.5 in K-Mops
minimal medium [37] containing 10 mm
15
N-NH
4
Cl before
being induced by the addition of isopropyl thio-b-d-gal-
Assembly of pyruvate dehydrogenase complex M. D. Allen et al.
264 FEBS Journal 272 (2005) 259–268 ª 2004 FEBS
actoside (1 mm final concentration). The cells were harves-
ted after 3 h of induction, resuspended in 50 mm Tris ⁄ HCl
buffer, pH 7.5, containing 1 mm EDTA, 1 mm phenyl-
methanesulfonyl fluoride and 0.02% (w ⁄ v) sodium azide,
and disrupted in a French press. Cell debris was removed
by centrifugation and the supernatant was fractionally pre-
cipitated with ammonium sulphate. The protein that was
precipitated between 35 and 80% saturation was dialysed
overnight into 50 mm potassium phosphate, pH 7.0. ThDD
was purified by consecutive cation-exchange and anion-
exchange chromatography using Hi-load
TM
-S and Mono
TM
-
Q columns, respectively [38]. Purified ThDD (10 mgÆmL
)1
)
was treated with thrombin, 40 UÆmL

)1
final concentration,
in 20 mm potassium phosphate buffer, pH 7.0, at 37 °C for
6 h, to cleave the linker between the lipoyl domain and
PSBD. PSBD released in this way was purified by cation-
exchange chromatography and its purity checked by
SDS ⁄ PAGE [38].
Generation of the E3int dimer
B. stearothermophilus E3 was purified from an over-expres-
sion system in E. coli described previously [39]. The dimeric
interface domain, comprising residues 343–470 of B. stearo-
thermophilus E3 [34] was prepared in essentially the same
way as the dimeric interface domain was excised from the
homologous E. coli glutathione reductase and rendered
more soluble by appropriate mutations on its freshly
exposed hydrophobic surfaces [31]. To achieve this for the
B. stearothermophilus E3 interface domain, the following
amino acid exchanges were made to its freshly exposed
hydrophobic surfaces: four residues making hydrophobic
contacts with the FAD domain (L389D, A390S, L391A
and I465G) and three residues abutting the NAD or central
domains (I348D, V352N and I443N). The interface domain
with these seven changes was designed to retain one of the
two C
2
axes of symmetry present in the intact wild-type E3
dimer.
NMR spectroscopy
For 2D NMR spectroscopy, samples of
15

N-labelled PSBD
(5 mm) and unlabelled PSBD (8 mm)in20mm potassium
phosphate buffer, pH 6.5 (90% H
2
O ⁄ 10% D
2
O, v ⁄ v) were
used. Two-dimensional [
1
H]
15
N-HSQC spectra, used to
detect backbone and side-chain amide resonances which
slowly exchange with D
2
O, were recorded in 20 mm potas-
sium phosphate buffer, pH 6.5, in 99.996% (v ⁄ v) D
2
O.
NMR spectra were recorded on a Bruker AM-500 spectro-
meter (500.13 MHz for
1
H and 50.68 MHz for
15
N) at
298 K. Mixing times were 60 ms and 150 ms for the TOCSY
and NOESY experiments, respectively. Proton and nitrogen
chemical shifts were determined relative to sodium 2,2-di-
methyl-2-silapentane-5-sulfonate and liquid ammonium,
respectively. Sequential assignment of the cross-peaks was

achieved using interresidue NOE connectivities by standard
2D NMR procedures [40,41]. Stereospecific assignments of
H
b
resonances were determined by the use of cross-peak
intensities in the 2D NOESY and 2D TOCSY [40]. The back-
bone dynamics of PSBD were investigated using steady-state
{
1
H}-
15
N nuclear Overhauser enhancement (NOE) experi-
ments [32,33]. Values of the {
1
H}-
15
N NOE were determined
according to the formula g ¼ (I ¢ – I
ref
) ⁄ I
ref
, where I ¢ is the
intensity of a cross-peak in an experiment with 3 s broad-
band
1
H presaturation and I
ref
is the intensity in a reference
spectrum recorded without presaturation.
NMR spectroscopic studies of protein–protein

interaction
For 2D NMR spectroscopic studies of protein–protein
interaction, samples of
15
N-labelled PSBD (1 mm)in
20 mm potassium phosphate buffer, pH 6.5 (90%
H
2
O ⁄ 10% D
2
O, v ⁄ v), were used. B. stearothermophilus E3
dimer (final concentrations of either 0.25 mm and 0.1 mm)
was added to PSBD samples to study the interaction of
PSBD with E3. B. stearothermophilus E3int dimer (final
concentration 0.1 mm) was added to PSBD samples to
study the interaction of PSBD with E3int.
Distance constraints
A set of distance constraints was derived from NOESY
spectra recorded in H
2
O and D
2
O with mixing times of
150 ms. Each NOESY spectrum was integrated according
to the cross-peak strengths and calibrated by comparison
with NOE connectivities obtained for standard interresidue
distances within an a-helix. After calibration, the NOE con-
straints were classified into four categories: 1.8–2.8, 1.8–3.5,
1.8–4.75 and 2.5–6.0 A
˚

. The distance constraints in the ini-
tial ensemble of structure calculations were derived only
from NOESY cross-peaks that could be unambiguously
assigned on the basis of chemical shift alone.
Structure calculation and refinement
Structure refinement proceeded in an iterative manner in
which distance constraints were added or modified follow-
ing analysis of the previous ensemble of structures. The vic-
inal proton coupling constant (
3
J
aN
) was determined by the
use of a series of 2D J-modulated (
15
N-
1
H)-COSY spectra
[42]. Comparison of the coupling constant with an experi-
mentally derived Karplus curve [43,44] enabled the torsion
angle, /, to be estimated when the coupling constant was
greater than 7.1 Hz.
In the final round of structure calculations, hydrogen
bond constraints were included for a number of backbone
H
N
groups whose signals were observed to change slowly
when the sample buffer was exchanged for D
2
O. For

M. D. Allen et al. Assembly of pyruvate dehydrogenase complex
FEBS Journal 272 (2005) 259–268 ª 2004 FEBS 265
hydrogen bond partners, two distance constraints were used
where the distance
(D)
H-O
(A)
corresponded to 1.8–2.1 A
˚
and
(D)
N-O
(A)
to 2.8–3.2 A
˚
. The stereospecific assignments
of H
b
resonances determined from NOESY and TOCSY
spectra were confirmed by analysing the initial ensemble of
structures. Additional stereospecific assignments were iden-
tified for resolved resonances when the side-chain atoms
were sufficiently well-defined in the ensemble of structures.
Each round of assignment was followed by a set of struc-
ture calculations with the structural constraints including
the stereospecific assignment, and confirmed when the
resulting structures did not show any distance violations
greater than 0.25 A
˚
. The 3D structure of PSBD was calcu-

lated from 841 experimental constraints using cns version
1.1 [45]. Twenty structures were calculated from an exten-
ded conformation using torsion angle dynamics in a
standard simulated annealing protocol. Stereospecific
assignments of prochiral protons were used, where avail-
able; otherwise r
)6
averaging was used over all equivalent
protons. Structures were accepted where no distance vio-
lation was greater than 0.25 A
˚
and no dihedral angle viola-
tions > 5°. The final coordinates have been deposited in
the Protein Data Bank (PDB accession no. 1w3d).
Acknowledgements
We thank the Biotechnology and Biological Sciences
Research Council (BBSRC) for the award of a research
grant (to RNP) and a Research Studentship (to MDA).
The core facilities of the Cambridge Centre for Molecu-
lar Recognition were funded by the BBSRC and The
Wellcome Trust. We are grateful to Mr C Fuller for
skilled technical assistance and to Dr ARC Raine for
his help with the structure calculation.
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