Báo cáo khoa học: Interactions of the peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase component in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus pot

Interactions of the peripheral subunit-binding domain of the
dihydrolipoyl acetyltransferase component in the assembly
of the pyruvate dehydrogenase multienzyme complex
of
Bacillus stearothermophilus
Hyo-Il Jung
1
, Alan Cooper
2
and Richard N. Perham
1
1
Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, UK;
2
Department of
Chemistry, University of Glasgow, UK
The enzymes pyruvate decarboxylase (E1) and dihydro-
lipoyl dehydrogenase (E3) bind tightly but in a mutually
exclusive manner to the peripheral subunit-binding domain
(PSBD) of dihydrolipoyl acetyltransferase in the pyruvate
dehydrogenase multienzyme complex of Bacillus stearo-
thermophilus. The use of directed mutagenesis, surface
plasmon resonance detection and isothermal titration
microcalorimetry revealed that several positively charged
residues of the PSBD, most notably Arg135, play an
important part in the interaction with both E1 and E3,
whereas Met131 makes a significant contribution to the
binding of E1 only. This indicates that the binding sites for
E1 and E3 on the PSBD are overlapping but probably
significantly different, and that additional hydrophobic
interactions may be involved in binding E1 compared with

E3. Arg135 of the PSBD was also replaced with cysteine
(R135C), which was then modified chemically by alkylation
with increasingly large aliphatic groups (R135C -methyl,
-ethyl, -propyl and -butyl). The pattern of changes in the
values of DG°, DH° and TDS° that were found to accom-
pany the interaction with the variant PSBDs differed
between E1 and E3 despite the similarities in the free ener-
gies of their binding to the wild-type. The importance of a
positive charge on the side-chain at position 135 for the
interaction of the PSBD with E3 and E1 was apparent,
although lysine was found to be an imperfect substitute for
arginine. The results offer further evidence of entropy–
enthalpy compensation (Ôthermodynamic homeostasisÕ) ) a
feature of systems involving a multiplicity of weak inter-
actions.
Keywords: pyruvate dehydrogenase multienzyme complex;
surface plasmon resonance; isothermal titration micro-
calorimetry; protein–protein interaction; thermodynamics.
The oxidative decarboxylation of pyruvate in most cells and
organisms is carried out by enzymes of the pyruvate
dehydrogenase (PDH) multienzyme complex, which con-
sists of pyruvate decarboxylase (E1; EC 1.2.4.1),
dihydrolipoyl acetyltransferase (E2; EC 2.3.1.12) and
dihydrolipoyl dehydrogenase (E3; EC 1.8.1.4). The three
enzymes are noncovalently, but tightly, assembled into a
highly organized multifunctional catalytic machine ([1–4]
and references therein).
The E2 chain of the PDH complex of Bacillus stearo-
thermophilus is composed of three major folding units: a
lipoyl domain (LD,  80 residues), a peripheral subunit-

binding domain (PSBD,  35 residues) and an acetyltrans-
ferase inner-core catalytic domain (CD,  250 residues),
which are joined together by long and flexible linker
segments ( 25–40 residues) rich in alanine, proline and
charged amino acids [2,5]. It is the acetyltransferase CD that
aggregates to form an inner core of icosahedral (60-mer)
symmetry [4,6]. The PSBD, located between the LD and
CD, is one of the smallest known globular protein domains
that lacks disulfide bridges or stabilizing metal ions.
According to the three-dimensional solution [7] and crystal
[8] structures of the binding domain, it has a compact fold
stabilized mainly by hydrophobic interactions, and consists
of two almost parallel short a-helices (H1 and H2), a turn of
distorted 3
10
-helix, and loops L1 and L2 joining these
structural elements.
The main function of the PSBD is to attach both E1 and
E3 to the icosahedral E2 core [9,10]. The binding stoichio-
metry and the kinetic and thermodynamic parameters of
the interactions have been investigated by means of non-
denaturing gel electrophoresis, surface plasmon resonance
Correspondence to 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 333667, Tel.: + 44 1223 333663,
E-mail:
Abbreviations: CD, catalytic domain; E1, pyruvate decarboxylase;
E2, dihydrolipoyl acetyltransferase; E3, dihydrolipoyl dehydrogenase;
ITC, isothermal titration microcalorimetry; LD, lipoyl domain; PDH,

pyruvate dehydrogenase; PSBD, peripheral subunit-binding domain;
SPR, surface plasmon resonance; ThDD, thrombin-cleavable
di-domain.
Enzymes: pyruvate decarboxylase (EC 1.2.4.1); dihydrolipoyl acetyl-
transferase (EC 2.3.1.12); dihydrolipoyl dehydrogenase (EC 1.8.1.4).
(Received 18 July 2003, revised 11 September 2003,
accepted 19 September 2003)
Eur. J. Biochem. 270, 4488–4496 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03842.x
(SPR) analysis and isothermal titration microcalorimetry
(ITC) [11–13]. One B. stearothermophilus PSBD (i.e. in
effect one E2 chain) is capable of binding either one E3
dimer or one E1 (a
2
b
2
) heterotetramer, but not both
simultaneously. The dissociation constant (K
d
)forthe
complex formed with E3 (5.8 · 10
)10
M
) was found to be
almost twofold higher than that for the complex with E1
(3.2 · 10
)10
M
). Although the PSBD has such a strong
affinity, formation of the complex does not appear to cause
any major conformational change near the active site of

either E1 or E3 [8,10].
The association of the PSBD with E3 at 25 °Cis
characterized by a small, unfavourable enthalpy change
(DH° ¼ +2.2 kcalÆmol
)1
) and a large, positive entropy
change (TDS° ¼ +14.8 kcalÆmol
)1
), whereas that with
E1 is accompanied by a favourable enthalpy change
(DH° ¼ )8.4 kcalÆmol
)1
) and a less positive entropy change
(TDS° ¼ +4.5 kcalÆmol
)1
), in both instances with marked
DC
p
effects, as described in detail elsewhere [12]. The
replacement of Arg135 in the PSBD with alanine (R135A)
causes a significant decrease in the binding affinity of PSBD
for E3 [13]. Indeed, Arg135 plays a central role in the
binding energetics; the R135A mutation is associated with
more favourable enthalpy changes and less positive entropy
changes in E3 binding. Such detailed information on the
interaction with E1 has thus far been unavailable.
The R135A mutation results in the loss of both the
charged guanidino group and most of the hydrophobic
aliphatic side-chain. To date our investigations have quan-
tified the overall effects of the R135A mutation on the

kinetics and energetics of the binding to E3 [13] but had
provided no evidence as to the relative parts played
by the ionized guanidino group and the aliphatic side-chain
of the arginine residue. In this paper we extend our studies
to the interaction of the PSBD with E1. The binding sites on
the PSBD for E1 and E3 may overlap but the interactions
are clearly distinguishable. We have also been able to
separate the thermodynamic contributions made by the
guanidino group and the aliphatic side-chain of the arginine
residue, using a technique of cysteine engineering [14].
Materials and methods
Materials
All reagents used were of analytical grade unless otherwise
stated. The sources of all restriction endonucleases, fine
chemicals, bacterial strains and media, plasmids and
antibiotics have been listed elsewhere [12,15].
Design of mutagenic oligonucleotides
The following mutagenic oligonucleotides, designed to
convert the indicated wild-type amino acid residues into
alanine, leucine, methionine and cysteine respectively,
were used: 5¢-end primer (forward), 5¢-GATAACAATTCC
CCTCTAGAAA-3¢;3¢-end primer (reverse), 5¢-GCGGG
ATATCCGGATATAGT-3¢; R135L (forward), 5¢-ATG
CCGTCCGTG
CTCAAGTATGC-3¢; R135L (reverse), 5¢-
CGCGCATACTT
GAGCACGGACGGCAT-3¢; R135M
(forward), 5¢-ATGCCGTCCGTG
ATGAAGTATGC-3¢;
R135M (reverse), 5¢-CGCGCATACTT

CATCACGGAC
GGCAT-3¢; R135K (forward), 5¢-GCCATGCCGTCCGT
G
AAGAAGTATGCGCGCGAAAAA-3¢; R135K (reverse),
5¢-TTTCGCGCGCATACTT
CTTCACGGACGGCA-3¢;
R135C (forward), 5¢-ATGCCGTCCGTG
TGCAAGTA
TGC-3¢; R135C (reverse), 5¢-CGCGCATACTT
GCACAC
GGACGGCAT-3¢. The altered codons are underlined. The
mutants were constructed in plasmid pET11ThDD as
described elsewhere [12]. The various forms of plasmid
pET11ThDD encoding the M131A, R135A, K136A,
R139A, R146A, K153A and R156A mutants have been
described previously [13].
Expression of genes and purification of proteins
Plasmid pET11ThDD carries a subgene encoding residues
1-170 of the B. stearothermophilus E2chainwithathrom-
bin-cleavage site in the linker region between the LD and
PSBD [16]. The mutant plasmids were overexpressed
in Escherichia coli BL21(DE3) cells grown at 37 °Cin
Luria–Bertani medium supplemented with ampicillin. After
induction with isopropyl thio-b-
D
-galactosidase (final
concentration, 1 m
M
) for 2 h, mutant forms of the
thrombin-cleavable di-domain (ThDD) were purified as

described by Jung et al. [13]. The LD in the ThDD was
lipoylated (on Lys42) by treatment with E. coli lipoate
protein ligase in the presence of ATP and lipoic acid [13].
Recombinant B. stearothermophilus E1 and E3 were puri-
fied from genes overexpressed in E. coli cells as described
elsewhere [10,17].
Chemical modification of the R135C ThDD
The newly introduced cysteine residue at position 135 in the
purified mutant R135C ThDD was chemically modified by
exposure to alkyl iodides, essentially as described by Hasan
and Leatherbarrow [14]. Iodomethane, iodoethane, iodo-
propane and iodobutane [2
M
solutions in 96% (v/v) ethanol]
were added to the mutant R135C protein ( 25 mg) in 0.1
M
Tris/HCl (pH 8.2), containing 10 m
M
dithiothreitol under
nitrogen, to a final concentration of 70 m
M
,andthemixture
was incubated for 6 h at 25 °C in the dark. Each reaction
tube was shaken several times at regular intervals. The extent
of reaction with each iodoalkane was estimated by subjecting
samples of the protein to electrospray mass spectrometry
(ESI-MS) intermittently in a Micromass Quattro-LC mass
spectrometer. When full alkylation was achieved, the reac-
tion mixture was loaded onto a Mono Q
TM

high-perform-
ance anion-exchange column equilibrated with buffer
solution A [20 m
M
potassium phosphate, pH 7.0, and
0.02% (w/v) sodium azide] to remove excess reagent. The
modified protein was subsequently eluted by applying a
linear gradient (15–75%) of buffer B [20 m
M
potassium
phosphate, pH 7.0, 1
M
NaCl and 0.02% (w/v) sodium
azide]. Fractions containing the chemically modified R135C
protein were pooled, dialysed exhaustively against water and
concentrated by Centriprep
TM
filtration.
Analysis of the binding affinity of mutant PSBDs
with E1 and E3
Interaction of the PSBD in each ThDD with E1 and E3
was analysed by means of SPR detection in a BIAcore
Ó FEBS 2003 Thermodynamics of protein complex assembly (Eur. J. Biochem. 270) 4489
instrument, as described elsewhere [11,13], and by ITC
measurements carried out in a Microcal calorimeter, as
described by Jung et al. [12,13].
General protein chemical techniques
Non-denaturing PAGE, SDS/PAGE and amino acid
analysis were carried out as described elsewhere [10,11].
Results

Possible overlap between the binding sites
on the PSBD for E1 and E3
The small but highly compact PSBD ( 35 amino acid
residues) of the B. stearothermophilus E2 chain binds either
E1 or E3 but not both simultaneously. Although the small
size of the PSBD is sufficient to explain the mutual
exclusivity, there is no evidence as to how the E1 binding
site on the PSBD relates to that for E3. The crystal structure
of B. stearothermophilus E3 complexed with the PSBD has
been determined to 2.6 A
˚
resolution [8]. Residues Arg135,
Arg139 and Arg156 of the PSBD are involved in an
Ôelectrostatic zipperÕ with AspB344 and GluB431 in the E3
dimer, and residues Ser133 and Lys136 of the PSBD make
interactions with monomer A of E3 (Fig. 1A). Alanine-
scanning mutagenesis confirmed that the positively charged
residues at positions 135, 136, 139 and 156 in the PSBD play
a vital part in the interaction with E3 and highlighted the
particular importance of Arg135 [13].
To assess their contribution, if any, to the binding of
E1, these four positively charged residues (Arg135, Lys136,
Arg139 and Arg156) plus two neutral residues (Met131 and
Ser133) and two other positively charged residues
(Arg146 and Lys153) were targeted for alanine-scanning
mutagenesis. As shown in Fig. 1B, all these residues are
located on the helix 1 and loop 2 regions of the PSBD and
are fully solvent-exposed.
Binding of mutant PSBDs to E1
All the mutant forms of the ThDD (a di-domain comprising

theLDplusPSBDofB. stearothermophilus E2 [8,15])
generated from subgenes overexpressed in E. coli (BL21)
cells, were purified as described elsewhere [13]. They are
known to be folded correctly [13]. The ThDD binds to E1
and E3 by virtue of the PSBD without interference from the
N-terminal LD [11].
The capacity of mutant PSBDs to bind to wild-type E1
was investigated by means of nondenaturing polyacryl-
amide gel electrophoresis. The mutant ThDDs were incu-
bated with wild-type E1 in 20 m
M
potassium phosphate
buffer, pH 7.0, for 5 min at room temperature, and the
mixtures were then submitted to polyacrylamide gel
electrophoresis under nondenaturing conditions (Fig. 2A).
In every case, the E1 band was found to be retarded,
indicating that all of the mutant PSBDs bound tightly to E1.
However, such gel electrophoresis, while serving to show
that none of the mutations in the PSBD prevented tight
binding to E1, gives no quantitative information on the
strength of the interaction. SPR analysis was therefore
carried out to assess rate and dissociation constants for the
interaction of mutant PSBDs with the wild-type E1 (a
2
b
2
)
tetramer.
The mutant ThDDs were immobilized on a BIAcore
CM5 sensor chip by attachment of the lipoylated LD in

each instance, leaving the PSBD free to interact with E1, as
described elsewhere [13]. The BIAcore sensor chip contained
four flow cells in each of which a different ThDD could be
immobilized independently. The wild-type ThDD was
Fig. 1. Structure of the PSBD of B. stearothermophilus E2 and its interaction with E3. (A) Details of the interaction of the PSBD with E3 [8]. (B)
Structure of the PSBD in a ribbon representation. The amino acid residues chosen for replacement by mutagenesis and chemical modification are
highlighted, and portrayed in the ball-and-stick representation. The figure was produced using
MOLSCRIPT
[22].
4490 H I. Jung et al.(Eur. J. Biochem. 270) Ó FEBS 2003
immobilized in the second flow cell as an internal reference,
and the first flow cell was used as a blank. Representative
SPR profiles for association of the wild-type E1 to the
PSBD of immobilized wild-type and mutant ThDDs are
shown in Fig. 2B. SPR response is measured in resonance
units (RU). Experimental variation was minimized by
expressing all the kinetic data as relative ratios in which the
SPR response for a mutant domain immobilized in a given
chip was divided by the response for the wild-type domain
immobilized in the same chip.
For the mutants M131A and R135A, the association of
E1 reached a steady-state immediately and generated
rectangular-shaped binding curves, which is a good indica-
tor of weak binding (k
off
is high). In such circumstances, a
large error may be incurred by the simultaneous measure-
ment of k
on
and k

off
values with the typical fitting method in
the
BIAEVALUATION
TM
software (Pharmacia). Therefore, the
equilibrium measurement method (Pharmacia Biosensor
AB, Application note 301) was used, as described elsewhere
[13,18]. The kinetic parameters for other mutants (S133A,
K136A, R139A, R146A, K153A and R156A) were meas-
ured using the
BIAEVALUATION
TM
software, also as described
elsewhere [11]. The results are summarized in Table 1.
Alanine substitution at positions Met131 and Arg135 of
the PSBD was found to lower the binding affinity for E1 by
almost 140-fold, indicating that these residues are of major
importance in the formation of the complex (Table 1).
Given that the nonpolar residue, Met131, is not involved in
the interaction with E3 [13], this observation strongly
suggests that the E1-PSBD interaction differs significantly
from the E3-PSBD interaction. In addition to Arg135, three
positively charged residues (Lys136, Arg139 and Arg156)
arealsoinvolvedintheinteractionwithE3[13].However,
only the R156A mutation, and to a much lesser extent the
R139A mutation, displayed major effects on the binding to
E1 (Table 1).
Fig. 2. Interaction of the PSBD and E1 ana-
lysed by means of nondenaturing PAGE and

SPR detection. (A) Wild-type and mutant
ThDDs (each 100 pmol) were incubated with
E1 (100 pmol of a
2
b
2
heterotetramer) in
20 m
M
potassium phosphate buffer, pH 7.0,
at 25 °C, and samples of the mixtures were
subjected to nondenaturing PAGE. Lane 1,
Arg135 wild-type; lane 2, M131A; lane 3,
S133A; lane 4, R135A; lane 5, K136A; lane 6,
R139A; lane 7, R146A; lane 8, K153A; lane 9,
R156A. (B) SPR sensorgrams of E1 binding to
wild-type and mutant ThDDs. Wild-type E1
(a
2
b
2
) was injected onto the sensor surface
using a series of increasing concentrations
(12.5, 25, 50, 100 and 200 n
M
) and represen-
tative sensorgrams at 50 n
M
are shown. The
start of the association and dissociation phases

are indicated by the arrows a and d, respect-
ively. Wt, wild-type.
Table 1. Kinetic and thermodynamic parameters for the interaction of
PSBD mutants with E1. Kinetic data were determined by SPR analysis.
All measurements were made in HBS buffer (10 m
M
Hepes, 150 m
M
NaCl, 3.4 m
M
EDTA), pH 7.4 at 25 °C, as in Materials and methods.
The kinetic parameters for the binding of the wild-type PSBD to E1
are k
on
¼ 3.27 · 10
6
M
)1
Æs
)1
, k
off
¼ 1.06 · 10
)3
s
)1
, K
d
¼ 3.24 ·
10

)10
M
)1
[11]. The standard errors on kinetic parameters were less
than 5% except for mutants R135A and M131A (£ 10%). DG° ¼
–RTlnK
d
,whereR¼ 1.987 calÆmol
)1
ÆK
)1
and T ¼ 298K. NM, not
measurable by ITC; mut, mutant; wt, wild-type.
PSBD
k
on
(mut)/
k
on
(wt)
k
off
(mut)/
k
off
(wt)
K
d
(mut)/
K

d
(wt)
DG°
(kcalÆmol
)1
)
Arg135 (wt) 1.0 1.0 1.0 )12.9
M131A NM NM 142
a
)10.0
S133A 1.0 1.0 1.0 )12.9
R135A NM NM 140
a
)10.0
K136A 1.0 3.2 3.2 )12.2
R139A 1.0 6.1 6.1 )11.8
R146A 1.0 1.9 1.9 )12.6
K153A 1.0 2.1 2.1 )12.5
R156A 1.0 18.7 18.7 )11.2
a
Obtained by equilibrium measurement method.
Ó FEBS 2003 Thermodynamics of protein complex assembly (Eur. J. Biochem. 270) 4491
For the other amino acid residues tested (Ser133, Arg146
and Lys153), the sensorgrams for the mutants were
essentially identical to that of the wild-type PSBD and no
significant effect on the binding to E1 was detected
(Table 1). In summary, values of the dissociation constant
(K
d
) for the mutant PSBDs interacting with E1 were in the

order M131A ‡ R135A  R156A > R139A > (K136A,
K153A, R146A, S133A). Indeed, the last four residues in
this list contributed virtually nothing to the interaction.
Chemical modifications of Arg135 in the ThDD
In order to dissect the particular contributions of the
positively charged guanidino group and the aliphatic side-
chain of Arg135 of the PSBD, this Arg residue in a
recombinant di-domain (i.e. LD plus PSBD), was first
replaced with cysteine by site-directed mutagenesis. The
newly introduced thiol group was then chemically modified
by alkylation (i.e. R135C -methyl, -ethyl, -propyl and
-butyl), as described in Materials and methods. The thiol
group of cysteine residues reacts rapidly with alkyl halides,
such as methyl, ethyl, propyl and butyl iodides, to give the
corresponding stable alkyl derivatives:
As the PSBD contains no cysteine residue in its primary
structure, Cys135 created by mutagenesis (R135C) is a
unique site for the subsequent chemical modification. The
LD has one cysteine residue at position 37, but this residue is
completely buried inside the protein [19]. Thus, when the
wild-type ThDD was treated with alkyl iodides, no chemical
modification could be detected by mass spectrometry (data
not shown). Likewise, when the alkylated forms of the
mutant ThDD (R135C) were examined, only one alkyl
group was found to have been added in each instance (data
not shown).
Binding constants for Arg135 variants of the PSBD
From Tables 2 and 3, the dissociation constants for the
interaction of the PSBD with E3 and E1, determined by
means of ITC or SPR, do not seem to be influenced greatly

by the incremental changes in the length of the aliphatic
side-chain of the Arg135 variants (especially bearing in mind
the experimental uncertainties in determining K
d
values in
this region by ITC). The R135K mutant behaved most like
the wild-type. This suggests that electrostatic interactions
are the dominant factor in the binding affinity. In each
instance, when the dissociation constants for each mutant at
25 °Cand37°C were compared, no major difference was
observed. Taken together, these data support the view that a
Table 2. Thermodynamic parameters for the interaction of E3 with PSBD variants having different side-chain lengths at position 135. The numbers in
parentheses are the standard errors from repeated measurements. NM, not measurable by ITC; ND, not determined; wt, wild-type.
Variant Side-chain
K
d
a
(25 °C)
( · 10
)8
M
)
K
d
a
(37 °C)
( · 10
)8
M
)

DH(25 °C)
(kcalÆmol
)1
)
DH(37 °C)
(kcalÆmol
)1
)
DC
p
(calÆmol
)1
ÆK
)1
)
R135A (–CH
3
) 2.0 (7.0
b
) 1.6 )2.6 (0.1) )8.4 (0.1) )483
R135C-methyl (–CH
2
SCH
3
) 1.2 2.0 )2.2 (0.4) )7.4 (0.5) )433
R135C-ethyl (–CH
2
SCH
2
CH

3
) 1.6 1.2 )3.7 (0.2) )8.9 (0.5) )433
R135C-propyl (–CH
2
SCH
2
CH
2
CH
3
) 1.5 1.3 )2.8 (0.4) )8.2 (0.5) )450
R135C-butyl (–CH
2
SCH
2
CH
2
CH
2
CH
3
) 1.2 1.5 )2.8 (0.2) )8.0 (0.4) )433
R135M (–CH
2
CH
2
SCH
3
) 2.3 1.3 )2.7 (0.4) )7.2 (0.5) )375
R135L (–CH

2
CH(CH
3
)(CH
3
)) NM 1.9 0 )4.6 (0.6) )383
R135K (–CH
2
CH
2
CH
2
CH
2
NH
3
+
) 0.6
b
ND 0 )4.0 (0.7) )333
Arg135 (wt) (–CH
2
CH
2
CH
2
NHC(¼ NH)NH
3
+
) 0.06

b
ND +2.2 (0.1) )1.8 (0.3) )316
c
a
Determined by means of ITC.
b
Determined by means of SPR.
c
Taken from previous results [12,13].
Table 3. Thermodynamic parameters for the interaction of E1 with PSBD variants having different side-chain lengths at position 135. The numbers in
parentheses are the standard errors from repeated measurements. NM, not measurable by ITC; ND, not determined; wt, wild-type.
Variant Side-chain
K
d
a
(25 °C)
( · 10
)8
M
)
K
d
a
(37 °C)
( · 10
)8
M
)
DH(25 °C)
(kcalÆmol

)1
)
DH(37 °C)
(kcalÆmol
)1
)
DC
p
(calÆmol
)1
ÆK
)1
)
R135A (–CH
3
) 1.1 (4.5
b
)ND )6.6 (0.2) )13.1 )542
R135C-methyl (–CH
2
SCH
3
) 1.5 1.3 )5.9 (0.5) )11.1 (0.4) )433
R135C-ethyl (–CH
2
SCH
2
CH
3
) 1.9 1.0 )4.5 (0.5) )8.0 (0.6) )292

R135C-propyl (–CH
2
SCH
2
CH
2
CH
3
) 2.2 3.1 )3.1 (0.3) )8.1 (0.2) )417
R135C-butyl (–CH
2
SCH
2
CH
2
CH
2
CH
3
) 1.8 7.1 )3.9 (0.6) )8.3 (0.1) )367
R135M (–CH
2
CH
2
SCH
3
) 4.5 ND )2.6 (0.2) ) 7.5 )408
R135L (–CH
2
CH(CH

3
)(CH
3
)) 2.5 2.2 )3.8 (0.0) )8.6 (0.0) )400
R135K (–CH
2
CH
2
CH
2
CH
2
NH
3
+
) 0.4
b
NM )2.6 (0.1) )7.7 (0.1) )425
Arg135 (wt) (–CH
2
CH
2
CH
2
NHC(¼ NH)NH
3
+
) 0.03
b
NM )8.4 (0.1) )14.3 (0.4) )470

c
a
Determined by means of ITC.
b
Determined by means of SPR.
c
Taken from previous results [12,13].
4492 H I. Jung et al.(Eur. J. Biochem. 270) Ó FEBS 2003
specific electrostatic interaction between Arg135 and negat-
ively charged side-chains contributed by E3 [13] and E1 (see
above) has a major influence on the binding affinity.
Thermodynamic changes on binding Arg135 variants
of the PSBD
ITC experiments were performed at 25 °Cand37°Cto
study the interaction of the variant ThDDs described above
with E3 or E1, and the associated thermodynamic changes
(DH°, DS° and DC
p
) are compared in Tables 2,3 and 4. In
all cases, the enthalpies and entropies of binding showed
significant temperature-dependence (large negative DC
p
), as
described previously for the E3-PSBD interaction [12], but
with enthalpy–entropy compensation resulting in relatively
smaller changes in free energies of binding with temperature.
Consequently, the absolute values of DH° and DS° under
specific conditions are difficult to interpret, but the relative
changes in these parameters observed for the variants under
otherwise similar experimental conditions might be more

tractable.
For the binding of the PSBD variants to E3, the major
changes in both standard Gibbs free energy of binding (DG°)
and its component parts (DH° and TDS°) compared with the
wild-type are associated with the loss of the positive charge
on Arg135 (Table 2). In all cases, binding is predominantly
entropy-driven (positive TDS°) with only a relatively small
enthalpic component, which may be exothermic, endother-
mic or even athermal (Table 4), depending on conditions.
With the exception of the R135L mutant (for which ITC data
could not be obtained), all the PSBD variants with an
uncharged side-chain at position 135 displayed similar DH°
and TDS° values regardless of the side-chain length. Com-
pared with the wild-type PSBD, the reduction in binding free
energy (DDG°) of around 2 kcalÆmol
)1
can be seen to arise
from a more favourable DH° (more exothermic by
 5kcalÆmol
)1
) offset partly by less favourable reductions
in TDS° of around 7 kcalÆmol
)1
. The R135K mutant, which
carries a positive charge at position 135, although
slightly different from Arg135, shows roughly inter-
mediate changes.
With E1 the changes appear to follow a different pattern.
Although the binding free energies are very similar to those
seen with E3, again with a DDG° of  +2 kcalÆmol

)1
compared with the wild-type PSBD, the separate DH° and
TDS° contributions show much greater variability with
respect to changes in the side-chain at position 135. In
particular, and in contrast with E3, removal of the positive
charge results in a less exothermic interaction, albeit to a
lesser extent as the side-chain length is reduced. These
unfavourable enthalpy changes are offset partially by more
favourable (more positive) TDS° contributions, with a
similar general trend to smaller effects associated with
shorter side-chains.
This difference in pattern is also seen in the heat capacity
data. For the binding of PSBD variants to E3, the DC
p
values are consistently more negative than that found with
the wild-type, and the effect is generally larger the shorter
the length of the side-chain at position 135. In contrast, with
the possible exception of the R135A mutant, DC
p
for
binding of the PSBD to E1 is consistently less negative than
for the wild-type. (Absolute values of DC
p
should be treated
with caution because they are derived from measurements
at only two temperatures.)
Discussion
The PSBD of the E2 chain in the B. stearothermophilus PDH
complex is responsible for binding both the E1 and E3
components to the multimeric (60-mer) E2 core. One PSBD

is capable of binding one E1 tetramer (a
2
b
2
) or one E3 dimer
but not both simultaneously [9–11]. The crystal structure of
E3 bound to the PSBD [8], together with a combination of
thermodynamic analyses and site-directed mutagenesis stud-
ies [12,13], has provided detailed information about the
binding interface in the E3-PSBD complex. Electrostatic
attractions constitute the driving force for complex forma-
tion and Arg135 of the PSBD is a key residue. Important
subsidiary roles are played by Arg139 and Arg156.
Although we lack a structure for the E1-PSBD complex,
it is now clear from the results described above (Table 1)
that the binding site on the PSBD for E1 has something in
common with that for E3, not least in the major importance
of Arg135 and the lesser importance of Arg156. However,
Met131 is also exceptionally important in the interaction
with E1, the M131A mutation causing a decrease in binding
Table 4. Comparison of thermodynamic parameters for the interaction of E3 and E1 with PSBD variants having different side-chain lengths at position
135. All the values are at 25 °C. The units of DG°, DH° and TDS° are kcalÆmol
)1
. DG° wascalculatedas-RTlnK
d
,whereR ¼ 1.987 calÆmol
)1
ÆK
)1
,

T ¼ 298K and all K
d
values are from Tables 2 and 3. TDS° was calculated as DH°–DG°.ValuesofDH° were determined by means of ITC. ND, not
determined (Table 2); wt, wild-type.
Variant Side-chain
E3 E1
DG° DH° TDS° DG° DH° TDS°
R135A (–CH
3
) )10.5 )2.6 +7.9 )10.9 )6.6 +4.3
R135C-methyl (–CH
2
SCH
3
) )10.8 )2.2 +8.6 )10.7 )5.9 +4.8
R135C-ethyl (–CH
2
SCH
2
CH
3
) )10.6 )3.7 +6.9 )10.5 )4.5 +6.0
R135C-propyl (–CH
2
SCH
2
CH
2
CH
3

) )10.7 )2.8 +7.9 )10.4 )3.1 +7.3
R135C-butyl (–CH
2
SCH
2
CH
2
CH
2
CH
3
) )10.8 )2.8 +8.0 )10.6 )3.9 +6.7
R135M (–CH
2
CH
2
SCH
3
) )10.4 )2.7 +7.7 )10.0 )2.6 +7.4
R135L (–CH
2
CH(CH
3
)(CH
3
)) ND 0 ND )10.4 )3.8 +6.6
R135K (–CH
2
CH
2

CH
2
CH
2
NH
3
+
) )11.2 0 +11.2 )11.5 )2.6 +8.9
Arg135 (wt) (–CH
2
CH
2
CH
2
NH
2
C(¼ NH)NH
3
+
) )12.6 +2.2 +14.8 )12.9 )8.4 +4.5
Ó FEBS 2003 Thermodynamics of protein complex assembly (Eur. J. Biochem. 270) 4493
affinity similar to that observed for R135A. The M131A
mutation has no effect on the binding of E3 [13]. Thus, the
binding sites for E1 and E3, although at least overlapping,
are also likely to be significantly different. Moreover, the
involvement of Met131 suggests that hydrophobic inter-
actions make a large contribution to the binding of E1, in
contrast with the electrostatic interactions that dominate E3
binding. These results are consistent with the earlier
speculation, based on the favourable enthalpy change and

modest entropy change, that a mixture of electrostatic and
hydrophobic interactions drives the E1-PSBD complex
formation [12].
With the exception of Met131, the residues identified as
important for the binding to E1 (Arg135, Arg156) and to E3
(Arg135, Arg139, Arg156) are located in helix 1 and loop 2
of the PSBD (Fig. 1B). The residue Met131 is located at the
C-terminal end of the linker region between the LD and the
PSBD, just before the beginning of helix 1 in the PSBD [6].
Preceding Met131 in the linker sequence, there is a series of
hydrophobic residues (e.g. Val128, Ile129 and Ala130) as
well as charged residues (Arg126 and Arg127). To explore
their participation, Arg126, Arg127, Val128 and Ile129 were
replaced by alanine and the effects on the affinity of PSBD
for E1 were monitored by SPR analysis (data not shown).
None of these mutations changed the binding constants
from that of the wild-type PSBD. Thus, we can rule out the
possibility that the linker region in general interacts
significantly with E1; Met131 appears to be the residue
principally involved.
The differences between the binding sites for E1 and E3
on the PSBD are further highlighted by the changes in the
thermodynamic parameters in response to mutation and
chemical modification at position Arg135. As listed in
Tables 2 and 3, and replotted for convenience in Fig. 3
in terms of DH° values, the enthalpy changes varied
comparatively little with the different side-chains introduced
at this position until a positive charge was included. In the
case of E3, the value changed to zero (R135K) or became
positive (Arg135, wild-type). With E1, the R135K mutation

again led to a less negative DH°, but the interaction with the
wild-type domain was substantially the most exothermic.
The importance of the positive charge at Arg135 for the
interaction with E3 (Table 2) and E1 (Tables 1 and 3) is
abundantly clear.
The thermodynamic parameters for the interaction of E3
([12,13] and above) and E1 ([12] and above) with the various
forms of the PSBD are summarized in Table 4. For E3 these
can be interpreted in detail, in terms of the origin of the
entropy-driven interaction with the PSBD, and from a
comparison with the X-ray crystal structure of the
E3-PSBD complex [12,13]. The discrepancy between the
measured value of DC
p
for this interaction and that
calculated from the buried polar and nonpolar surfaces in
the interface has been discussed previously [12]. Once again,
the particular importance of the positive charge on the side-
chain at position 135 in the PSBD is evident from the data in
Table 2. We are not in a position to carry out an
equivalently detailed analysis for the E1-PSBD interaction,
as we lack a crystal structure on which to base the
calculations of buried surface area. However, it is clear
from the data in Table 3 that including a positive charge on
the side-chain at position 135 is not associated with a large
change in DC
p
; the one major curiosity is associated with
R135C-ethyl and R135A, which cause DC
p

to change
significantly in positive and negative directions, respectively.
This remains to be explained.
In mutagenesis studies, lysine is usually regarded as the
best substitute for arginine in conserving the positively
charged side-chain and many binding proteins appear to be
unaffected by interchanging these residues. However, the
dissociation equilibrium constant (K
d
) of the R135K mutant
for binding E3 (Table 2) and E1 (Table 3) was observed to
Fig. 3. Effect of side-chain length and charge
on the binding enthalpy for the interaction of
PSBD variants with E3 and E1. All measure-
ments were made in HBS buffer (10 m
M
Hepes, 150 m
M
NaCl, 3.4 m
M
EDTA), pH 7.4
at 25 °C. The side-chain lengths at position
135 were calculated from the sum of the indi-
vidual bond lengths using values of 1.54 A
˚
and 1.82 A
˚
for C-C and C-S bonds, respect-
ively. Terminal C-H and S-H bond lengths are
not included. Enthalpy changes for the inter-

actions with R135K and wild-type Arg135
PSBDs are highlighted. Solid line, interaction
with E3; broken line, interaction with E1.
4494 H I. Jung et al.(Eur. J. Biochem. 270) Ó FEBS 2003
be about 10-fold higher than that of the wild-type PSBD. In
fact, the side-chains of lysine and arginine are significantly
different, both in length and in the point (lysine) and
delocalized (arginine) positive charge. Unlike lysine, argi-
nine is capable of multiple types of interaction; it has the
ability to form both simple and bidentate ionic interactions
with carboxyl groups and a hydrogen bond network with up
to five hydrogen bonds. These properties account for the
favoured occurrence of arginine residues at binding inter-
faces. Indeed, in a database analysis of the contributions of
individual amino acid residues to protein–protein inter-
actions [20], arginine appears with a frequency of more than
10% (21% for tryptophan, 13.3% for arginine and 12.3%
for tyrosine residues). This is borne out in the importance of
arginine residues in the PSBD for the interactions with E1
andE3detailedabove.
A striking feature of the thermodynamic data described
here is that, although mutations and other side-chain
replacements at Arg135 of the PSBD give rise to very similar
changes in the free energies of binding to E1 and E3, the
underlying pattern of enthalpy, entropy and heat capacity
changes is significantly different in each case. This is in line
with a general pattern of entropy–enthalpy compensation or
Ôthermodynamic homeostasisÕ that is coming to be seen as
commonplace in (macro)molecular interactions involving a
multiplicity of weak interactions [21]. The structural or

molecular basis for these differences is harder to understand.
Electrostatic interactions appear to dominate the binding of
E3 [8], whereas hydrophobic interactions may make a more
substantial contribution to the binding of E1 (see above).
Nonetheless, the positive charge of the side-chain of one
particular residue, Arg135, in the PSBD of E2 assumes a
particular significance in the thermodynamics, although not
the kinetics, of both these competitive bimolecular inter-
actions. There are useful lessons to be learned here for the
rational design of protein–protein binding interfaces, which
will be amplified when a crystal structure of the E1-PSBD
complex is determined.
Acknowledgements
This work was supported in part by a research grant (to R.N.P.) from
the Biotechnology and Biological Sciences Research Council. We are
grateful to the BBSRC and the Wellcome Trust for their support of the
core facilities in the Cambridge Centre for Molecular Recognition, and
to the BBSRC and the Engineering and Physical Sciences Research
Council for funding the biological microcalorimetry facilities in the
University of Glasgow. We thank the Cambridge Overseas Trust, the
Department of Biochemistry and St John’s College, Cambridge for
financial support to H.I.J.
References
1. deKok,A.,Hengveld,A.F.,Martin,A.&Westphal,A.H.(1998)
The pyruvate dehydrogenase multi-enzyme complex from Gram-
negative bacteria. Biochim. Biophys. Acta 1385, 353–366.
2. Perham, R.N. (2000) Swinging arms and swinging domains in
multifunctional enzymes: catalytic machines for multistep reac-
tions. Annu.Rev.Biochem.69, 961–1004.
3. Zhou, H.Z., McCarthy, D.B., O’Connor, C.M., Reed, L.J. &

Stoops, J.K. (2001) The remarkable structural and functional
organization of the eukaryotic pyruvate dehydrogenase complex.
Proc.NatlAcad.Sci.USA98, 14802–14807.
4. Milne, J.L.S., Shi, D., Rosenthal, P.B., Sunshine, J.S., Domingo,
G.J., Wu, X., Brooks, B.R., Perham, R.N., Henderson, R. &
Subramaniam, S. (2002) Molecular architecture and mechanism of
an icosahedral pyruvate dehydrogenase complex: a multi-
functional catalytic machine. EMBO J. 21, 1–12.
5. Borges, A., Hawkins, C.F., Packman, L.C. & Perham, R.N.
(1990) Cloning and sequence analysis of the genes encoding the
dihydrolipoyl acetyltransferase and dihydrolipoamide dehydro-
genase components of the pyruvate dehydrogenase multienzyme
complex of Bacillus stearothermophilus. Eur. J. Biochem. 194,
95–102.
6. Izard, T., Ævarsson, A., Allen, M.D., Westphal, A.H., Perham,
R.N., de Kok, A. & Hol, W.G.J. (1999) Priciples of quasi-
equivalence and Euclidean geometry govern the assembly of cubic
and dodecahedral cores of pyruvate dehydrogenase complexes.
Proc.NatlAcad.Sci.USA96, 1240–1245.
7. Kalia, Y.N., Brocklehurst, S.M., Hipps, D.S., Appella, E., Sak-
aguchi, K. & Perham, R.N. (1993) The high resolution structure of
the peripheral subunit-binding domain of dihydrolipoamide
acetyltransferase from the pyruvate dehydrogenase multienzyme
complex of Bacillus stearothermophilus. J. Mol. Biol. 230, 323–341.
8. Mande, S.S., Sarfaty, S., Allen, M.D., Perham, R.N. & Hol,
W.G.J. (1996) Protein–protein interactions in the pyruvate dehy-
drogenase multienzyme complex: dihydrolipoamide dehydroge-
nase complexed with the binding domain of dihydrolipoamide
acetyltransferase. Structure 4, 277–286.
9. Hipps, D.S., Packman, L.C., Allen, M.D., Fuller, C., Sakaguchi,

K.,Appella,E.&Perham,R.N.(1994)Theperipheralsubunit-
binding domain of the dihydrolipoyl acetyltransferase component
of the pyruvate dehydrogenase complex of Bacillus stearo-
thermophilus: preparation and characterization of its binding to
the dihydrolipoyl dehydrogenase component. Biochem. J. 297,
137–143.
10. Lessard, I.A.D. & Perham, R.N. (1995) Interaction of component
enzymes with the peripheral subunit-binding domain of the
pyruvate dehydrogenase multienzyme complex of Bacillus stearo-
thermophilus: stoichiometry and specificity in self-assembly. Bio-
chem. J. 306, 727–773.
11. Lessard, I.A.D., Fuller, C. & Perham, R.N. (1996) Competitive
interaction of component enzymes with the peripheral subunit-
binding domain of the pyruvate dehydrogenase multienzyme
complex of Bacillus stearothermophilus: kinetic analysis using
surface plasmon resonance detection. Biochemistry 35, 16863–
16870.
12. Jung, H.I., Bowden, S.J., Cooper, A. & Perham, R.N. (2002)
Thermodynamic analysis of the binding of component enzymes in
the assembly of the pyruvate dehydrogenase multienzyme complex
of Bacillus stearothermophilus. Protein Sci. 11, 1091–1100.
13. Jung, H.I., Cooper, A. & Perham, R.N. (2002) Identification
of key amino acid residues in the assembly of enzyme into
the pyruvate dehydrogenase complex of Bacillus stearo-
thermophilus: a kinetic and thermodynamic analysis. Biochemistry
41, 10446–10453.
14. Hasan, Z. & Leatherbarrow, R.J. (1998) A study of the specificity
of barley chymotrypsin inhibitor 2 by cysteine engineering of the
P1 residue. Biochim. Biophys. Acta 1384, 325–334.
15. Fries, M., Chauhan, H.J., Domingo, G.J., Jung, H I. & Perham,

R.N. (2003) Site-directed mutagenesis of a loop at the active site of
E1 (a
2
b
2
) of the pyruvate dehydrogenase complex. A possible
common sequence motif. Eur. J. Biochem. 270, 861–870.
16. Wallis, N.G., Allen, M.D., Broadhurst, R.W., Lessard, I.A.D. &
Perham, R.N. (1996) Recognition of a surface loop of the lipoyl
domain underlies substrate channelling in the pyruvate dehy-
drogenase multienzyme complex. J. Mol. Biol. 263, 463–474.
17. Lessard, I.A.D., Domingo, G.J., Borges, A. & Perham, R.N.
(1998) Expression of genes encoding the E2 and E3 components of
Ó FEBS 2003 Thermodynamics of protein complex assembly (Eur. J. Biochem. 270) 4495
the Bacillus stearothermophilus pyruvate dehydrogenase complex
and the stoichiometry of subunit interaction in assembly in vitro.
Eur. J. Biochem. 258, 491–501.
18. Karlsson, R., Michaelsson, A. & Mattsson, L. (1991) Kinetic
analysis of monoclonal antibody–antigen interactions with a new
biosensor based analytical system. J. Immunol. Methods 145,
229–240.
19. Dardel, F., Davis, A.L., Laue, E.D. & Perham, R.N. (1993) Three-
dimensional structure of the lipoyl domain from Bacillus stearo-
thermophilus pyruvate dehydrogenase multienzyme complex.
J. Mol. Biol. 229, 1037–1048.
20. Bogan, A.A. & Thorn, K.S. (1998) Anatomy of hot spots in
protein interfaces. J. Mol. Biol. 280,1–9.
21. Cooper,A.,Johnson,C.M.,Lakey,J.H.&No
¨
llmann, M. (2001)

Heat does not come in different colours: entropy–enthalpy com-
pensation, free energy windows, quantum confinement, pressure
perturbation calorimetry, solvation and the multiple causes of heat
capacity effects in biomolecular interactions. Biophys. Chem. 93,
215–230.
22. Kraulis, P. (1991) MolScript, a program to produce both detailed
and schematic plots of protein structures. J. Appl. Crystallog. 24,
946–950.
4496 H I. Jung et al.(Eur. J. Biochem. 270) Ó FEBS 2003