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Thecrystalstructureofahyperthermostablesubfamily II
isocitrate dehydrogenasefromThermotoga maritima
Mikael Karlstro
¨
m
1
, Ida H. Steen
2
, Dominique Madern
3
, Anita-Elin Fedo
¨
y
2
, Nils-Ka
˚
re Birkeland
2
and Rudolf Ladenstein
1
1 Center for Structural Biochemistry, Karolinska Institutet, NOVUM, Huddinge, Sweden
2 Department of Biology, University of Bergen, Norway
3 Institut de Biologie Structurale CEA-CNRS-UJF, Grenoble, France
The enzymes of hyperthermophilic organisms are
remarkably stable and can resist denaturation at tem-
peratures ranging from 80 °C to above 130 °C [1,2],
whereas their counterparts from mesophilic organisms
are usually denatured at around 50 °C. However, they
are often homologous and their catalytic mechanisms
are usually identical [3]. Moreover, the gain in free sta-
bilization energy in hyperthermostable proteins, com-
pared with their mesophilic homologues, is generally
small, especially at the growth temperature of the
organisms [4]. Typically, the net free energy of stabil-
ization in both mesophilic and hyperthermophilic pro-
teins, is 5–20 kcalÆmol
)1
, which is equivalent to only
a small number of weak intermolecular interactions
[3,5,6]. There is no single mechanism or structural fea-
ture that is responsible for the high thermotolerance of
hyperthermostable proteins [4,7]. The most common
determinants of hyperthermostability that have been
Keywords
ionic networks; isocitrate dehydrogenase;
thermostability; Thermotoga maritima
Correspondence
M. Karlstro
¨
m, Karolinska Institutet, NOVUM,
Centre for Structural Biochemistry,
S-141 57 Huddinge, Sweden
Fax: +46 8 608 9290
Tel: +46 8 608 9178
E-mail: mikael.karlstrom@biosci.ki.se
(Received 28 February 2006, revised
26 April 2006, accepted 2 May 2006)
doi:10.1111/j.1742-4658.2006.05298.x
Isocitrate dehydrogenase (IDH) fromthe hyperthermophile Thermotoga
maritima (TmIDH) catalyses NADP
+
- and metal-dependent oxidative
decarboxylation ofisocitrate to a-ketoglutarate. It belongs to the b-decarb-
oxylating dehydrogenase family and is the only hyperthermostable IDH
identified within subfamily II. Furthermore, it is the only IDH that has
been characterized as both dimeric and tetrameric in solution. We solved
the crystalstructureofthe dimeric apo form of TmIDH at 2.2 A
˚
. The
R-factor ofthe refined model was 18.5% (R
free
22.4%). The conformation
of the TmIDH structure was open and showed a domain rotation of 25–
30° compared with closed IDHs. The separate domains were found to be
homologous to those ofthe mesophilic mammalian IDHs ofsubfamily II
and were subjected to a comparative analysis in order to find differences
that could explain the large difference in thermostability. Mutational stud-
ies revealed that stabilization ofthe N- and C-termini via long-range elec-
trostatic interactions were important for the higher thermostability of
TmIDH. Moreover, the number of intra- and intersubunit ion pairs was
higher and the ionic networks were larger compared with the mesophilic
IDHs. Other factors likely to confer higher stability in TmIDH were a less
hydrophobic and more charged accessible surface, a more hydrophobic
subunit interface, more hydrogen bonds per residue and a few loop dele-
tions. The residues responsible for the binding ofisocitrate and NADP
+
were found to be highly conserved between TmIDH and the mammalian
IDHs and it is likely that the reaction mechanism is the same.
Abbreviations
AfIDH, Archaeoglobus fulgidus IDH; ApIDH, Aeropyrum pernix IDH; AUC, analytical ultracentrifugation; BsIDH, Bacillus subtilis IDH; EcIDH,
Escherichia coli IDH; HcIDH, human cytosolic IDH; HDH, homoisocitrate dehydrogenase; IDH, isocitrate dehydrogenase; PcIDH, porcine
heart mitochondrial IDH; PfIDH, Pyrococcus furiosus IDH; TmIDH, Thermotogamaritima IDH.
FEBS Journal 273 (2006) 2851–2868 ª 2006 Karolinska Institutet 2851
observed are more ionic interactions at the protein sur-
face and increased formation of large ionic networks
[8–10]. Electrostatic optimization and the reduction in
repulsive charge–charge interactions are crucial [11,12].
However, in many cases, the combined effects of
multiple, modestly stabilizing interactions seem to be
responsible for enhanced thermostability. Examples
include a reduction in the hydrophobic accessible sur-
face area, increased hydrogen bonding, stronger inter-
subunit interactions, loop deletions and structural
compactness [13–15].
In order to analyse thermotolerance comparatively
within one protein family, we chose isocitrate dehydrog-
enase (IDH), a metal-dependent (Mg
2+
or Mn
2+
)
enzyme in the tricarboxylic acid cycle which catalyses
the subsequent dehydrogenation and decarboxylation
of isocitrate to a-ketoglutarate using NAD
+
or
NADP
+
as a cofactor [16]. Owing to its central role in
metabolism, IDH is present in organisms from all three
domains of life, Archaea, Bacteria and Eukarya. Conse-
quently, IDH is also present in organisms that have
adapted to a wide range of growth temperatures making
it an attractive model enzyme for studying heat-adap-
tive traits. In a previous publication, we characterized
hyperthermostable IDHs fromThermotoga maritima
(TmIDH), Aeropyrum pernix (ApIDH), Pyrococcus
furiosus (PfIDH) and Archaeoglobus fulgidus (AfIDH)
with respect to phylogenetic affiliation, cofactor
specificity, thermostability and oligomeric state, and
identified three different subfamilies of IDH [17].
ApIDH, AfIDH and PfIDH showed high sequence
identity to IDH fromthe mesophile Escherichia coli
(EcIDH) and they formed, together with all known
archaeal IDHs as well as most bacterial IDHs,
subfamily I. Within subfamily II, the bacterial and
eukaryotic NADP
+
-IDHs were grouped into different
branches. However, TmIDH was separated from both
and represented the deepest branch of this subfamily.
ApIDH in subfamily I was described as the most
thermostable IDH, with an apparent melting tempera-
ture of 109.9 °C and TmIDH was described as the
only hyperthermostable IDH known within sub-
family II with an apparent melting temperature of
98.3 °C. Moreover, we identified a heterogeneous mix-
ture of tetrameric and dimeric species of TmIDH in
solution, in which the tetrameric form of TmIDH repre-
sented a unique oligomeric state of NADP-IDH. Here,
we report thecrystalstructureof TmIDH, representing
the first bacterial structureof an IDH from
subfamily II. The crystallographic structure is presented
in the dimeric form as crystallization trials of the
tetrameric form of TmIDH have been unsuccessful to
date.
In order to reveal possible determinants of the
increased thermotolerance of TmIDH, we compared
the structureofthe dimeric form with the mesophilic
mammalian homologues porcine mitochondrial IDH
(PcIDH) and human cytosolic IDH (HcIDH) from the
same subfamily. The observed differences were then
compared with differences between ApIDH and its
mesophilic homologue EcIDH in subfamily I. Further-
more, the analysis was used as a guideline to design
specific mutants of TmIDH. Their properties are dis-
cussed with respect to the main mechanisms involved
in protein thermostabilization.
Results and Discussion
Purification ofthe dimeric form of TmIDH
We previously described TmIDH as a heterogenous
mixture of dimeric and tetrameric species [17].
Recently, thecrystalstructureof tetrameric homoisoci-
trate dehydrogenasefromthe hyperthermophilic bac-
terium Thermus thermophilus was solved and it was
suggested that formation ofthe tetramer was involved
in thermostabilization [18]. New analytical ultracentrif-
ugation (AUC) data on TmIDH confirmed that two
oligomeric species with S
20,W
values corresponding to
a tetramer ( 7.9 S) and a dimer ( 5.0 S) are present
in the preparation using the previously described puri-
fication protocol (data not shown) [17].
In order to separate the two oligomeric forms, a gel-
filtration step was added and the separate fractions
were analysed using AUC. The AUC data demonstra-
ted that dimeric and tetrameric species do not re-
equilibrate to a heterogenous mixture (Fig. 1). Thus,
the dimeric and tetrameric forms were not in equilib-
rium and could be separated. The two forms were con-
centrated and subjected to various crystallization trials.
To date, crystals ofthe tetrameric species of TmIDH
0
0,1
0,2
0,3
0,4
0,5
0,6
051015
Sedimentation coefficient (S)
c(s) arbit. units
Fig. 1. Sedimentation velocity analysis of dimeric Thermotoga mari-
tima IDH, (TmIDH) at 20 °C. The data recorded at 0.1 mgÆmL
)1
,in
50 m
M NaCl buffered with 50 mM Tris ⁄ HCl pH 8 were fitted using
SEDFIT software [62]. The single peak is centered on 5,4 S. This
value corresponds to the one expected for a pure dimer TmIDH.
Structure and thermal stability of T. maritima M. Karlstro
¨
m et al.
2852 FEBS Journal 273 (2006) 2851–2868 ª 2006 Karolinska Institutet
have not been obtained, whereas the dimeric form pro-
duced good quality crystals that were used to solve the
structure.
Quality and description ofthe model
The crystalstructureof TmIDH was solved by molecu-
lar replacement at 2.2 A
˚
and represents the apo form
of the enzyme. The atomic coordinates and structure
factors were deposited in the Protein Data Bank (entry
1ZOR). The model (Fig. 2A) was refined to a crystal-
lographic R-value of 18.4% and a free R-value of
22.3%. It was crystallized as a dimer in the asymmetric
unit with a solvent content of 54.8% corresponding to
a Matthews’ coefficient of 2.7 A
˚
3
ÆDa
)1
. The two sub-
units were related by a twofold noncrystallographic
rotation axis and are referred to as subunits A and B.
The space group was P2
1
2
1
2
1
and the unit cell parame-
ters a ¼ 62.5 A
˚
,b¼ 88.1 A
˚
,c¼ 180.9 A
˚
. The sym-
metry-related molecules in thecrystal did not produce
the tetrameric form. Electron-density maps of subunit
A were of very high quality throughout the structure
determination, whereas density maps ofthe large
domain of subunit B lacked density for many side
chains. This was probably caused by a local disorder
in thecrystal resulting fromthe flexibility ofthe large
domains in combination with the particular crystal
packing. However, these side chains were maintained
in the model with ideal conformations (as defined by
the rotamers ofthe o database) except for residues
B1–3, which were omitted because of dubious main
chain density. In subunit A, only three side chains
were lacking density and more solvent molecules were
built compared with subunit B. In total, 428 water
molecules were built. In both subunits, some residues
were modelled with two conformations with 50%
occupancy each (Lys A48, Lys A62, Glu A81, Lys
A84, Arg A109, Lys A183, Lys A220, Asn A240, Glu
A300, Arg A307, Arg A308, Arg A335, Glu A348, Glu
A
B
C
Fig. 2. (A) Ribbon representation oftheThermotogamaritima IDH
(TmIDH) dimer. The dimer forms through the association of the
small domains (subunit A: orange and subunit B: light blue) and the
formation ofthe clasp domain (A: pink and B: purple). Each large
domain (A: green and B: blue) is connected to the small domain via
a flexible hinge region. Both subunits were found in an open con-
formation. (B) Overlay ofthe large domains of TmIDH (green),
HcIDH (blue) and PcIDH (pink), showing the N- and C-termini and
the loop deletion of five residues between helices l and m in
TmIDH. The domains were superimposed separately because of
different conformations in the subunit. (C) Overlay ofthe small
domains including the clasp domains of TmIDH (green), Hc IDH
(blue) and PcIDH (pink) showing the loop deletion of three and four
residues in the clasp domain of TmIDH.
M. Karlstro
¨
m et al. Structure and thermal stability of T. maritima
FEBS Journal 273 (2006) 2851–2868 ª 2006 Karolinska Institutet 2853
B81, Glu B170, Asn B240, Glu B257, Arg B308 and
Glu B385). Four cis-peptides were identified, two of
which are cis-prolines (Pro376 in both subunits). The
rmsd values from ideal geometry were within reason-
able limits and the Ramachandran plot showed that
90.8 and 89.6% ofthe residues in subunits A and B,
respectively, fall within the most favourable region.
Asn159 in subunit B was found in a disallowed confor-
mation, which may be explained by unclear density
and its location in a sharp turn between strand M2
and M3. Table 1 summarizes the quality ofthe data
and the model.
Several crystal structures of different IDHs have
been reported: E. coli IDH [19] [EcIDH, PDB codes
3ICD (closed) and 1SJS (open)], Aeropyrum pernix
IDH [20] (ApIDH, PDB codes 1XGV, 1TYO and
1XKD) and Bacillus subtilis IDH [21] (BsIDH, PDB
code 1HQS) representing subfamily I and PcIDH [22]
(PcIDH, PDB code 1LWD) and HcIDH [23] [HcIDH,
PDB code 1T0L (closed) and 1T09 (open)] from sub-
family II. They are all homodimeric NADP
+
-IDHs
and their common folds are shared also by the crystal
structures of isopropylmalate dehydrogenase which
uses a substrate containing the malate moiety in com-
mon with isocitrate and belongs to the same family of
b-decarboxylating dehydrogenases [24–26]. TmIDH
showed highest structural similarity to PcIDH and
HcIDH, the two other members ofsubfamilyII IDHs
for which thestructure are known.
Hyperthermostable enzymes are often smaller than
their homologues from mesophiles. The amino acid
sequence of TmIDH is shorter than PcIDH and HcIDH.
TmIDH has 399 amino acid residues, whereas PcIDH
and HcIDH contain 413 and 414 residues, respectively.
However, all ofthe sequenced bacterial IDHs in this
subfamily (i.e. also those from mesophiles and psycro-
philes) have shorter sequences than the eukaryotic
IDHs. Therefore, the shorter sequence of TmIDH is
most likely a phylogenetic characteristic and not an
adaptation to higher temperature. All IDH subunits
consist of three domains: a large domain, a small
domain and a clasp domain (Fig. 2A). In TmIDH, resi-
dues 1–119 and 281–399 belong to the large domain, res-
idues 120–140 and 182–280 constitute the small domain
and residues 141–181 form the clasp domain. The large
domain is connected to the small domain by a flexible
hinge region. TmIDH was found in an open conforma-
tion with relative differences in the rotation ofthe large
domain of 30° compared with the closed ternary iso-
citrate–NADP
+
–Ca
2+
–HcIDH complex (PDB code:
1T0L) and of 25° compared with the binary iso-
citrate–PcIDH complex. Compared with the most open
subunit ofthe binary NADP
+
–HcIDH complex (PDB
code: 1T09), TmIDH was 6° more open. The clasp
domain in TmIDH was typical for subfamily II, with
two stacked four-stranded antiparallel b sheets instead
of the two antiparallel a helices beneath a single four-
stranded antiparallel b sheet characteristic for sub-
family I IDHs.
Because of different conformations, the large and
the small domains of each homologue were compared
separately. The rmsd values between the C
a
-carbons of
the large domain of TmIDH and that of PcIDH and
HcIDH were 0.92 A
˚
(using 231 C
a
-atoms) and 0.97 A
˚
(230 C
a
-atoms), respectively, when the nonconserved
loops in PcIDH and HcIDH were excluded. Superposi-
tion ofthe small domains, together with the clasp
domain, exhibited rmsd values between TmIDH and
PcIDH and HcIDH of 0.84 A
˚
(161 C
a
-atoms) and
0.88 A
˚
(159 C
a
-atoms), respectively. Overlays of the
different domains of TmIDH, HcIDH and PcIDH are
shown in Fig. 2B,C. Because ofa transformation of
the secondary structure in the open form of HcIDH,
Table 1. Data collection and refinement statistics. Values in paren-
theses refer to data in the highest-resolution shell.
TmIDH
PDB code 1ZOR
Wavelength (A
˚
) 0.996
Resolution limits (A
˚
) 2.24–35.56
(2.24–2.29)
Mosaicity 0.5
Unit cell parameters a ¼ 62,5
b ¼ 88,1
c ¼ 180,a
a ¼ b ¼ c ¼ 90°
No. of unique reflections 49143(2500)
Redundancy 4.8
Completeness (%) 99.7 (100)
I ⁄ sigma (I) 15.84(3.32)
R
merge
(%) 6.6 (37.9)
Wilson B-factor 40.94
Space group P2
1
2
1
2
1
Refinement
No of non hydrogen atoms 6536
No of ions 2
Missing residues B1-3
Solvent molecules 428
Resolution range 2.24–35.56
(2.236–2.294)
R
cryst
(overall)% 18.4 (22.3)
R
free
(%) 22.3 (25.5)
Ramachandran plot (excl Gly and Pro)
Most favourable region subunit A ⁄ B (%) 90.8 ⁄ 89.6
Allowed regions A ⁄ B (%) 9.2 ⁄ 10.1
Disallowed regions A ⁄ B(%) 0⁄ 0.3
rmsd from ideal values
Bond lengths (A
˚
) 0.012
Bond angles (°) 1.251
Structure and thermal stability of T. maritima M. Karlstro
¨
m et al.
2854 FEBS Journal 273 (2006) 2851–2868 ª 2006 Karolinska Institutet
which is discussed more in detail below, the separate
domains ofthe closed form of HcIDH were used in
the superposition.
All secondary structural elements were conserved in
TmIDH, PcIDH and HcIDH. In total there were 16
a helices (44.9% ofthe residues in TmIDH) and 16
b strands (19.5% ofthe residues in TmIDH). In addi-
tion, one 3
10
helix was identified in TmIDH. The total
secondary structure content of 65.2% was only slightly
different from that of PcIDH (64.4%) and HcIDH
(66.2%). A structure-based sequence alignment is
shown in Fig. 3.
TmIDH has a loop deletion of three and four resi-
dues, compared with PcIDH and HcIDH, respectively,
between strands M2 and M3 in the clasp domain
(Fig. 2C). Other loop deletions were found between
helices g1 and g2 (one residue), strands E and D (one
residue) and helices l and m (five and four residues,
respectively, Fig. 2B). The two latter loop deletions
were also identified in several bacterial IDHs of meso-
philes from this subfamily and must be considered as
phylogenetic characteristics. The two first-loop dele-
tions, however, were not found in any mesophilic IDH.
This finding is in agreement with earlier observations
that proteins of hyperthermohiles often have shorter
loops [15,27]. In this way, fraying elements where the
structure might begin to unfold, are reduced.
Differences between the subunits
Subunits A and B of TmIDH were very similar. Super-
position ofthe small domains showed an rmsd of
0.27 A
˚
for the C
a
-atoms, whereas the rmsd between
the large domains was 0.32 A
˚
. The relative difference
in the rotation ofthe large domain between the two
subunits was 2.6°.
Dimer association
The interface in TmIDH was found to be similar to
that of PcIDH and HcIDH. The dimer associates
through the formation ofthe clasp domain and via
hydrophobic interactions between helices h and i in
both subunits to form a stable four-helix bundle.
The active site
The active site ofthe IDHs is formed in the cleft
between the small and the large domains and contains
residues from both domains and both subunits. The
substrate-binding residues of TmIDH were investigated
by superposition ofthe active site residues from each
domain of TmIDH and the closed substrate-bound
HcIDH and PcIDH separately. The putative isocitrate-
binding residues fromthe large domain, Thr77, Ser94
and Asn96, were very well aligned with rmsd values of
0.362 and 0.257 A
˚
between TmIDH vs. PcIDH and
HcIDH, respectively, for all 21 atoms. Arg100 and
Arg109 are also putative isocitrate-binding residues
but showed slightly different conformations in
TmIDH, most likely due to the absence ofisocitrate in
TmIDH. However, the isocitrate-binding residues from
the small domain, Arg132, Tyr139 and Lys208¢ (the
prime indicates the neighbouring subunit ofthe dimer)
were still well aligned with rmsd values of 0.525 and
0.950 A
˚
between TmIDH vs. PcIDH and HcIDH,
respectively, for all 32 atoms. The metal-coordinating
residues Asp247¢, Asp270 and Asp274 were also struc-
turally similar with rmsd values of 0.745 and 1.056 A
˚
for all 24 atoms between TmIDH vs. PcIDH and
HcIDH, respectively. (The equivalent of Asp247¢
shows quite a different conformation in HcIDH which
is likely due to the absence ofisocitrate in TmIDH.)
In the metal (Mg
2+
or Mn
2+
)-binding site, a Na
+
ion was built as it was the only positively charged ion
present in the crystallization buffer and no remaining
|F
o
| ) |F
c
| electron density could be observed at 2.0
sigma after it was built. The Na
+
ion was coordinated
by six oxygen ligands in both subunits. Two waters
(waters 185 and 427 in subunit A and waters 284 and
289 in subunit B), the carbonyl oxygen of Asp270 and
Asp247¢ represented the equatorial ligands, whereas
Asp270 and Asp274 were the axial ligands.
Cofactor binding
The b-decarboxylating dehydrogenases share a unique
cofactor-binding site that differs fromthe well-known
Rossmann fold found in many other dehydrogenases
[19,24,28]. Only a few amino acid residues appear to
be responsible for the discrimination between NAD
+
and NADP
+
[29–32]. The residues which interact with
the 2¢-phosphate of NADP
+
are responsible for the
discrimination between NAD
+
⁄ NADP
+
.
All ofthe residues involved in binding of the
NADP
+
in the ternary HcIDH complex (PDB code
1T0L) were found to be conserved in TmIDH except a
lysine (Lys260 in HcIDH) which interacts with the
2¢-phosphate of NADP
+
. This lysine was replaced by
Arg255¢ in TmIDH and might still be important for
the discrimination between NAD
+
and NADP
+
. Pre-
sumably, the other residues interacting with the 2¢-
phosphate in TmIDH are Arg308, His309 and
Gln252¢. However, close to Arg308 and His309 there
was another arginine (Arg312) in TmIDH directed
towards the supposed 2¢-phosphate of NADP
+
.In
M. Karlstro
¨
m et al. Structure and thermal stability of T. maritima
FEBS Journal 273 (2006) 2851–2868 ª 2006 Karolinska Institutet 2855
Structure and thermal stability of T. maritima M. Karlstro
¨
m et al.
2856 FEBS Journal 273 (2006) 2851–2868 ª 2006 Karolinska Institutet
PcIDH and HcIDH, this arginine is replaced by a Glu
(318) and Met (318), respectively. It is therefore poss-
ible that the cofactor binding is slightly different in
TmIDH compared with PcIDH and HcIDH. However,
all other residues putatively involved in binding of
NADP
+
were conserved structurally. The adenine
ring-binding residues correspond to His303, Val306,
Thr321 and Asn322, whereas Gly304, Thr305 and
Val306 should interact with the 5¢-phosphate of the
adenine. Thr77 and Arg82 are equivalent to the resi-
dues which bind the hydroxyl groups ofthe nicotina-
mide ribose. Thr75, Lys72 and Asn96 most likely bind
the amide group ofthe nicotinamide. The rmsd
between TmIDH and the NADP
+
–isocitrate–HcIDH–
complex (PDB code: 1T0L) was only 0.359 A
˚
for all
92 atoms of these residues which do not bind the
2¢-phosphate. Between TmIDH and PcIDH without
NADP
+
, the rmsd was 0.615 A
˚
for all 92 atoms.
The structural conservation ofthe isocitrate- and
NADP
+
-binding residues suggests that isocitrate and
NADP
+
binding is highly conserved between TmIDH
and the mammalian IDHs and that these enzymes
most likely share the same catalytic mechanism. For a
description ofthe mechanism, see Karlstro
¨
m et al. [20]
and Hurley et al. [16].
Putative phosphorylation site
In EcIDH, phosporylation of Ser113 in the so-called
‘phosphorylation loop’ by IDH kinase ⁄ phosphatase is
proposed to inactivate the enzyme by blocking the
binding ofisocitrate to the active site both sterically
and by electrostatic repulsion ofthe c–carboxyl group
of isocitrate [33–36]. Whether this regulatory mechan-
ism exists in other IDHs is not clear. In the closed
ternary complex of HcIDH (PDB code: 1T0L), the
conserved helix i in the small domain is similar to that
observed in all other NADP
+
–IDH structures and is
part ofthe four-helix bundle at the dimer interface.
However, in the absence of isocitrate, the enzyme has
adopted an open conformation (PDB code: 1T09) and
helix i is found unwound into a loop conformation
where Asp279 interacts with Ser94 ofthe large domain
in the active site. This interaction mimics the phos-
phorylation ofthe equivalent serine in EcIDH which
inhibits the binding ofisocitrate and makes the enzyme
inactive. It has been postulated that the new interac-
tion in HcIDH is competing with isocitrate in binding
to the active site and a self-regulatory mechanism of
activity is thereby provided [23]. In the open form of
TmIDH without isocitrate that we report here, this
helix is maintained and the self-regulating mechanism
is therefore not supported. The serine in the ‘phos-
phorylation loop’ is, however, conserved in TmIDH
and PcIDH (Ser94 and Ser95, respectively).
Thermostability
By sequence comparison, PcIDH and HcIDH are 51.3
and 52.2% identical to TmIDH, respectively. Neverthe-
less, the structural homology makes them suitable for a
comparison in order to identify differences which can be
related to thermostability. The apparent melting tem-
perature (T
m
)ofPcIDH was determined to 59.0 °C, i.e.
39.3 °C lower than the apparent T
m
of TmIDH. Below,
we try to relate the large T
m
difference to structural
determinants that presumably cause this highly
increased thermotolerance of TmIDH. The revealed dif-
ferences between TmIDH and its mesophilic homo-
logues in subfamilyII are thereafter related to the
differences observed between ApIDH and its mesophilic
homologue EcIDH in subfamily I. The latter compar-
ison was made between the open Ap IDH (PDB code
1TYO) and the open EcIDH (PDB code 1SJS) and
might be slightly different from comparison between
ApIDH and the closed EcIDH done previously [20]. The
sequence identity between TmIDH and ApIDH is only
22.3% (a sequence alignment is shown in Fig. 3).
The major driving force in protein stability and fold-
ing is considered to be ‘the hydrophobic effect’ which
results in the burial of most ofthe hydrophobic resi-
dues in the protein core [37,38]. The hydrophobic
effect explains why many hyperthermostable proteins
show a significant increase in the number of buried
hydrophobic residues at the core or at subunit interfa-
ces, and is sometimes reflected by more hydrophobic
residues in the sequence [39–42]. This trend could also
be observed in TmIDH which has 42.9% hydrophobic
residues, whereas PcIDH and HcIDH have 39.5 and
39.4%, respectively (Table 2). However, many differ-
Fig. 3. Structure-based sequence alignment of TmIDH with porcine mitochondrial IDH (PcIDH, PDB code 1LWD), human cytosolic IDH
(HcIDH, PDB code 1T0L), Aeropyrum pernix IDH (ApIDH, PDB code 1TYO) and Escherichia coli IDH (EcIDH, PDB code 1SJS). The residues
occurring within structurally equivalent regions are boxed. Helices and strands appear as cylinders and arrows. Conserved residues are
shown in green, positions showing conservation of polar or charged character are in bold, those showing conservation of hydrophobic char-
acter are in yellow and residues showing a conservation of small size have smaller font. Sequence numbering according to TmIDH is red.
Secondary structure elements were given the nomenclature as implemented in EcIDH [19].
M. Karlstro
¨
m et al. Structure and thermal stability of T. maritima
FEBS Journal 273 (2006) 2851–2868 ª 2006 Karolinska Institutet 2857
Table 2. Characteristics of TmIDH, HcIDH, PcIDH, ApIDH and EcIDH.
TmIDH open HcIDH open HcIDH closed PcIDH closed ApIDH open EcIDH open
PDB code 1ZOR 1T09 1T0L 1LWD 1TYO 1SJS
Apparent melting temperature (°C) 98.3 N. d. N. d. 59.0 109.9 52.6
No of amino acids per subunit 399 414 414 413 435 416
Hydrophobic residues
a
(%) 42.9 39.4 39.4 39.5 46.2 44.4
Polar residues
b
(%) 25.5 32.8 32.8 32.4 27.6 28.4
Charged residues
c
(%) 31.6 27.8 27.8 28.1 26.2 27.2
Resolution (A
˚
) 2.24 2.7 2.4 1.85 2.1 2.4
rmsd of C
a
versus TmIDH large ⁄ small & clasp domain (A
˚
) – 0.85 ⁄ nd 0.97 ⁄ 0.88 0.92 ⁄ 0.84 2.27 ⁄ 1.57 nd
No of hydrogen bonds in subunit A 391 364 382 400 361 339
No of hydrogen bonds per residue in subunit A 0.98 0.88 0.92 0.97 0.84 0.82
No of hydrogen bonds (SS)
d
per residue in subunit A 0.12 0.08 0.09 0.12 0.08 0.04
No. of hydrogen bonds (SM)
e
per residue in subunit A 0.16 0.14 0.17 0.19 0.15 0.15
No. of hydrogen bonds (MM)
f
per residue in subunit A 0.70 0.66 0.66 0.66 0.61 0.62
No. of intersubunit hydrogen bonds 30 31 40 26 27 22
No. of intrasubunit ion pairs in subunit A at a 4 ⁄ 6 ⁄ 8A
˚
cut-off 31 ⁄ 58 ⁄ 96 24 ⁄ 49 ⁄ 78 29 ⁄ 56 ⁄ 85 28 ⁄ 55 ⁄ 77 29 ⁄ 57 ⁄ 85 27 ⁄ 50 ⁄ 77
No. of intrasubunit ion pairs per residue at a 4.0 A
˚
cut-off 0.078 0.058 0.070 0.075 0.067 0.065
Total no. of ion pairs per monomer at a 4 ⁄ 6 ⁄ 8A
˚
cut-off 35 ⁄ 63.5 ⁄ 24.5 ⁄ 53 32 ⁄ 61 30 ⁄ 58 31 ⁄ 63 29 ⁄ 55
Total no. of ion pairs per residue at a 4 A
˚
cut-off 0.088 0.059 0.077 0.073 0.071 0.070
Net charge (dimer) + 6 + 5 + 5 + 20 + 1 ) 19
No. of 3 ⁄ 4 ⁄ 5-member intrasubunit networks
in the monomer at a 4.0 A
˚
cut-off
1 ⁄ 1 ⁄ 23⁄ 1 ⁄ 04⁄ 1 ⁄ 04⁄ 1 ⁄ 03⁄ 1 ⁄ 06⁄ 0 ⁄ 0
No. of 3 ⁄ 4 ⁄ 5 ⁄ 6 ⁄ 7 ⁄ 8 ⁄ 9 ⁄ 10-member intrasubunit networks
in monomer A at a 6.0 A
˚
cut-off
2 ⁄ 2 ⁄ 2 ⁄ 0 ⁄ 0 ⁄ 1 ⁄ 0 ⁄ 15⁄ 2 ⁄ 0 ⁄ 1 ⁄ 1 ⁄ 0 ⁄ 1 ⁄ 04⁄ 1 ⁄ 1 ⁄ 0 ⁄ 0 ⁄ 0 ⁄ 2 ⁄ 05⁄ 1 ⁄ 2 ⁄ 1 ⁄ 0 ⁄ 1 ⁄ 0 ⁄ 04⁄ 2 ⁄ 0 ⁄ 0 ⁄ 2 ⁄ 0 ⁄ 0 ⁄ 08⁄ 1 ⁄ 2 ⁄ 0 ⁄ 1 ⁄ 0 ⁄ 0 ⁄ 0
No. of intersubunit ion-pairs at a 4 ⁄ 6A
˚
cut-off 8 ⁄ 11 1 ⁄ 86⁄ 10 4 ⁄ 64⁄ 12 4 ⁄ 10
Intersubunit 3 ⁄ 4 ⁄ 6 ⁄ 8-member networks at a 4.0 A
˚
cut-off 2 ⁄ 2 ⁄ 0 ⁄ 00⁄ 0 ⁄ 0 ⁄ 00⁄ 2 ⁄ 0 ⁄ 02⁄ 2 ⁄ 0 ⁄ 02⁄ 1 ⁄ 0 ⁄ 02⁄ 2 ⁄ 0 ⁄ 0
Intersubunit 3 ⁄ 4 ⁄ 5 ⁄ 6 ⁄ 7-member networks at a 6.0 A
˚
cut-off 1 ⁄ 2 ⁄ 0 ⁄ 0 ⁄ 12⁄ 0 ⁄ 0 ⁄ 0 ⁄ 11⁄ 2 ⁄ 0 ⁄ 1 ⁄ 02⁄ 0 ⁄ 0 ⁄ 2 ⁄ 01⁄ 0 ⁄ 0 ⁄ 2 ⁄ 02⁄ 2 ⁄ 0 ⁄ 0 ⁄ 4
Intersubunit 8 ⁄ 9 ⁄ 10 ⁄ 15 ⁄ 23-member networks at a 6.0 A
˚
cut-off 0 ⁄ 1 ⁄ 0 ⁄ 0 ⁄ 00⁄ 0 ⁄ 0 ⁄ 0 ⁄ 01⁄ 0 ⁄ 2 ⁄ 0 ⁄ 00⁄ 0 ⁄ 2 ⁄ 0 ⁄ 00⁄ 0 ⁄ 0 ⁄ 1 ⁄ 10⁄ 0 ⁄ 0 ⁄ 0 ⁄ 0
Volume (· 10
4
A
˚
3
) 15.5 15.9 15.7 15.4 15.8 15.2
Accessible surface area of dimer (A
˚
2
) 32780 35564 32564 31820 32916 32527
Surface ⁄ volume ratio 0.211 0.224 0.207 0.207 0.208 0.214
Buried surface at dimer interface (% of dimer) 16.7 13.7 18.5 17.6 14.9 15.6
Distribution of hydrophobic ⁄ polar ⁄ charged area at accessible
surface of dimer (% of dimer)
54.4 ⁄ 19.1 ⁄ 26.5 55.7 ⁄ 23.9 ⁄ 20.4 55.4 ⁄ 24.6 ⁄ 20.0 54.6 ⁄ 24.2 ⁄ 21.1 53.2 ⁄ 23.5 ⁄ 23.3 53.7 ⁄ 21.6 ⁄ 24.7
Distribution of hydrophobic ⁄ polar ⁄ charged area
at dimer interface (% of interface)
69.5 ⁄ 18.4 ⁄ 12.1 67.1 ⁄ 22.8 ⁄ 10.1 64.5 ⁄ 22.2 ⁄ 13.3 73.1 ⁄ 18.7 ⁄ 8.2 72.4 ⁄ 16.1 ⁄ 11.5 69.2 ⁄ 18.9 ⁄ 11.9
a
Hydrophobic residues: A,V,L,I,W,F,P,M.
b
Polar residues: G,S,T,Y,N,Q,C.
c
Charged residues: R,K,H,D,E.
d
SS, side chain–side chain hydrogen bonds.
e
SM, side chain–main chain hydro-
gen bonds.
f
MM main chain–main chain hydrogen bonds.
Structure and thermal stability of T. maritima M. Karlstro
¨
m et al.
2858 FEBS Journal 273 (2006) 2851–2868 ª 2006 Karolinska Institutet
ences between proteins from hyperthermophiles and
mesophiles are observed on the surface ofthe protein
subunits. The contribution of hydrophobic residues at
the protein surface will affect the stability ofthe pro-
tein unfavorably in aqueous solution, whereas hydro-
philic residues will help to solvate the protein and
thereby stabilize it. Accordingly, the accessible surface
area ofhyperthermostable proteins is commonly more
polar or charged and less hydrophobic [8,9,11,43–45].
Surface and interface characteristics
First, the open form of TmIDH reported here, was
compared with the open form of HcIDH. However,
one must bear in mind that two ofthe helices (i and i¢)
in the interfacial four-helix bundle are unfolded in the
open HcIDH, giving the surface and the subunit inter-
face a different character. Therefore, we show data for
the open HcIDH, the closed HcIDH and the closed
PcIDH in Table 2. It was found that TmIDH has a
6.1% increase in charged accessible surface area, a
1.3% decrease in hydrophobic surface area and 4.8%
decrease in polar surface area compared with the open
HcIDH (Fig. 4A and Table 2). This is in line with ear-
lier comparisons between enzymes from hyperthermo-
philes and mesophiles [3,42]. In this context, Ap IDH is
unusual having a less charged and more polar access-
ible surface than its mesophilic homologue EcIDH.
However, the hydrophobic part is still reduced by
0.5% compared with the open EcIDH (Table 2). The
small decrease in the hydrophobic part ofthe surfaces
might seem insignificant. However, using 25 calÆ
mol
)1
ÆA
˚
)2
for the hydrophobic contribution to the free
energy of folding applied on the difference in accessible
hydrophobic area in TmIDH (which was 2005 A
˚
2
),
gives a free energy difference of 50 kcalÆmol
)1
indica-
ting a contribution to stability in the expected order of
magnitude [46,47].
Moreover, a reduction ofthe total solvent-exposed
surface area has also been associated with increased
stability [48,49]. The solvent-exposed surface area of
TmIDH was 32 780 A
˚
2
which is considerably smaller
than the 35 564 A
˚
2
of the open form of HcIDH. How-
ever, this is partially due to the shorter sequence of
TmIDH. Therefore, the surface-to-volume ratio was
determined [50]. For TmIDH it was 0.211, whereas for
the open HcIDH it was 0.224, indicating a slight
decrease in the accessible surface. The same trend was
observed between ApIDH (0.208) and EcIDH (0.214).
In principle, a decrease in the accessible surface
might be due to increased burial ofthe molecular sur-
face at the subunit interface. However, both TmIDH
and ApIDH were found to have a smaller relative
interface area compared with their respective homo-
logues. Because ofthe unwinding of helix i belonging
to the interfacial four-helix bundle in the open HcIDH,
we preferred to compare the interface area of TmIDH
with that ofthe closed HcIDH and PcIDH. It was
found that 16.7% ofthe total molecular surface of the
two TmIDH subunits was buried at the interface,
whereas 18.5 and 17.6% ofthe closed HcIDH and
PcIDH surfaces were buried, respectively. The inter-
face of ApIDH was proportionately smaller than both
the open and the closed EcIDH (Table 2) [20].
The interface of TmIDH showed a 5% increase in
hydrophobic area and a 1.2% decrease in charged area
compared with the closed HcIDH (Fig. 4B, Table 2).
A
B
Fig. 4. (A) Distribution of hydrophobic, polar and charged accessible
surface area (ASA) ofthe different IDHs. (B) Distribution of hydropho-
bic, polar and charged area at the interface ofthe different IDHs.
M. Karlstro
¨
m et al. Structure and thermal stability of T. maritima
FEBS Journal 273 (2006) 2851–2868 ª 2006 Karolinska Institutet 2859
A similar trend was found also when the interface of
ApIDH was compared with that ofthe open homo-
logue EcIDH. However, compared with the closed
PcIDH, the interface of TmIDH was 3.5% less hydro-
phobic and 3.9% more charged (Table 2). Part of the
hydrophobic contribution at the subunit interface in
TmIDH involves a cluster of four methionines located
in the interfacial four-helix bundle (Met272 and
Met275 from both subunits). This methionine cluster
was not present in PcIDH or HcIDH. However, below
the four-helix bundle, on the top ofthe clasp domain,
there was another, almost identical, intersubunit four-
membered methionine cluster (formed by Met176 and
Met178 from both subunits), which was found to be
conserved in these three IDHs. The additional methi-
onine cluster in TmIDH might be important for the
subunit interaction. It was also found that helix h of
the interfacial four-helix bundle might be stabilized in
TmIDH by three Ala residues in a row instead of one
in PcIDH and two separate Ala residues as in HcIDH.
Alanine is considered to be the most optimal helix-
forming amino acid [51,52].
Aromatic interactions
Aromatic interactions are usually identified using a cut-
off distance of 7 A
˚
between the aromatic ring centres
[53]. The role of additional aromatic interactions in
increasing the thermostability of proteins has been dis-
cussed elsewhere [53,54]. However, Tm IDH was found
to have a decreased fraction of aromatic residues
(10.8%) compared with HcIDH (12.1%) and PcIDH
(12.7%). However, a cluster of aromatic residues invol-
ving Phe205, Phe188, Phe219, Phe223, Tyr215, Tyr241
and His133 was identified in the small domain of
TmIDH. All of these residues were conserved in PcIDH
and HcIDH except Phe205, which has a central posi-
tion in this cluster (Fig. 5C). A few other nonconserved
A
B
C
Fig. 5. (A) The mutation D389N decreased the apparent melting
temperature (T
m
)ofTmIDH by 21.8 °C. At a cut-off distance of
4A
˚
, Asp389 formed a conserved ion pair with Lys29, connecting
both termini with each other. At a 6 A
˚
cut-off, the ion pair was
involved in a nonconserved four-member ionic network with
Lys318 and Glu385. (B) Arg186 made interactions with Glu182,
Glu225 and Glu226 and was part ofa five-member ionic network.
However, mutation R186M did not affect the apparent T
m
of
TmIDH. (C) Mutation F205M reduced the apparent T
m
by 3.5 °C.
Phe205 was found to have a central position in an aromatic cluster
in the small domain involving Phe188, Phe219, Phe223, Tyr215,
Tyr241 and His133. All but Phe205 were conserved within sub-
family II. The result confirms that aromatic clustering plays a role in
increasing the apparent melting temperature of proteins.
Structure and thermal stability of T. maritima M. Karlstro
¨
m et al.
2860 FEBS Journal 273 (2006) 2851–2868 ª 2006 Karolinska Institutet
[...]... sequences TmIDH mutant Primer sequence R186M 5’-CCTGGAGAAATCGATCATGAGCTTCGCTCAGTCGTG-3’ 5’-CACGACTGAGCGAAGCTCATGATCGATTTCTCCAGG-3’ 5’-AAAAGGTCGACATCTGGATGGCGACGAAAGACACGATC-3’ 5’-GATCGTGTCTTTCGTCGCCATCCAGATGTCGACCTTTT-3’ D36N-1 5’-CATCCTTCCCTATCTCAACATCCAGCTGGTTTACT-3’ D341N-1 5’-AAGGGGAGAACTCAACGGAACACCGGAGG-3’ 5’-CACTCTCGAAGAGTTCATAAACGAAGTGAAGAAGAATCTC-3’ 5’-GAGATTCTTCTTCACTTCGTTTATGAACTCTTCGAGAGTG-3’ F205M... to the thermotolerance of TmIDH Comparison of ion pairs and hydrogen bonds was based on subunit A in all cases because many side chains lacked electron density in the large domain of subunit B in TmIDH The quality ofthe electron-density map ofthe interfacecontaining small domain of subunit B was, however, fine, which allowed safe analysis ofthe number of intersubunit ion pairs The total number of. .. pairs and larger ionic networks, more hydrophobic residues in total, a more hydrophobic interface, a less hydrophobic accessible surface area and a decreased surface-tovolume ratio However, TmIDH lacked interdomain Structure and thermal stability of T maritima ˚ ionic networks at a cut-off of 4.0 A and the size ofthe networks were not as dramatically increased at 4.2 and ˚ 6.0 A cut-offs like in ApIDH... clustering of aromatic residues are vital for the stability of TmIDH However, analysis of one mutant, R186M, demonstrated that the location ofthe ionic network and the local environment probably is essential, because the mutation did not affect the apparent Tm of TmIDH Several properties of TmIDH were shared in common with ApIDH, which is the most thermostable IDH: stabilized N-terminus, more ion pairs and... equal volume ofthe reservoir solution Crystals appeared after a few days and grew to a typical dimension of 0.2 · 0.2 · 0.5 mm Data were collected at the synchrotron beamline I711 at MAXLAB (Lund, Sweden) Thecrystal was flash-frozen with boiling nitrogen at 100 K in the presence of 20% ethylene glycol as cryoprotectant A MarCCD 165 detector and marccd v 0.5.33 software were used for data collection The. .. program truncate [64] Data collection parameters and processing statistics are given in Table 1 The Matthews’ coefficient (Vm) was ˚ 2.7 A3 ÆDa)1 suggesting a dimer in the asymmetric unit Molecular replacement Phase information was obtained in space group P212121 by molecular replacement with the program phaser [65] using ˚ data up to 2.5 AThe different domains of IDH were searched for separately because... subunit A on the same domain in subunit B, with the small domains of subunit A and B remaining in a fixed superimposed position Rotation angles were calculated fromthe O rotation matrix by convrot (W Meining, unpublished) No significant translation component was detected A structure- based sequence alignment was performed with the program stamp [71] and was based on the Ca-atom coordinates and secondary... using a water probe radius of 1.4 A Hydrogen bonds were calculated using hbplus v 3.15 [74] and the following default parameters: maximum distan˚ ˚ ces for D A, 3.9 A and for H A, 2.5 A; minimum angles for D–H A, D A AA and H A AA were 90° [75] Figure preparations Figures 2 and 5 were made using pymol [76] Figure 3 was prepared using the program alscript [77] Acknowledgements We are grateful to Marit... IH, Birkeland NK & Ladenstein R (2005) Isocitratedehydrogenasefromthe hyperthermophile Aeropyrum pernix: X-ray structure analysis ofa ternary enzyme–substrate complex and thermal stability J Mol Biol 345, 559–577 21 Singh SK, Matsuno K, LaPorte DC & Banaszak LJ (2001) Crystalstructureof Bacillus subtilis isocitrate ˚ dehydrogenase at 1.55 A Insights into the nature of substrate specificity exhibited... of different domain orientations found in other IDH crystal structures The rotation and the position ofthe dimer ofthe small domains, including the clasp domain, were located using the equivalent domains ofthe porcine mitochondrial IDH structure (PDB entry 1LWD) as search model The large domains were 2864 M Karlstrom et al ¨ located using both the porcine mitochondrial IDH (PDB entry 1LWD) and the . D36N-1
5’-CATCCTTCCCTATCTC
AACATCCAGCTGGTTTACT-3’
D341N-1
5’-AAGGGGAGAACTC
AACGGAACACCGGAGG-3’
D389N 5’-CACTCTCGAAGAGTTCATA
AACGAAGTGAAGAAGAATCTC-3’
5’-GAGATTCTTCTTCACTTCGT
TTATGAACTCTTCGAGAGTG-3’
M. Karlstro
¨
m. 5’-AAAAGGTCGACATCTGG
ATGGCGACGAAAGACACGATC-3’
5’-GATCGTGTCTTTCGTCGC
CATCCAGATGTCGACCTTTT-3’
D36N + D341N D36N-1
5’-CATCCTTCCCTATCTC
AACATCCAGCTGGTTTACT-3’
D341N-1
5’-AAGGGGAGAACTC
AACGGAACACCGGAGG-3’
D389N