Stepwise adaptations of citrate synthase to survival at
life’s extremes
From psychrophile to hyperthermophile
Graeme S. Bell
1
, Rupert J. M. Russell
2
, Helen Connaris
2
, David W. Hough
1
, Michael J. Danson
1
and Garry L. Taylor
1,2
1
Centre for Extremophile Research, Department of Biology and Biochemistry, University of Bath, UK;
2
Centre for Biomolecular
Sciences, University of St Andrews, St. Andrews, UK
The crystal structure of citrate synthase from the thermo-
philic Archaeon Sulfolobus solfataricus (optimum growth
temperature ¼ 85 °C) has been determined, extending the
number of crystal structures of citrate synthase from differ-
ent organisms to a total of five that span the temperature
range over which life exists (from psychrophile to hyper-
thermophile). Detailed structural analysis has revealed
possible molecular mechanisms that determine the different
stabilities of the five proteins. The key to these mechanisms is
the precise structural location of the additional interactions.
As one ascends the temperature ladder, the subunit interface

that optimally grows at 85 °C. The gene for Sulfolobus
solfataricus CS has been cloned and sequenced [19], and
over-expressed in E. coli. The purified recombinant protein
exists as a homodimer of M
r
¼ 81,000, with each monomer
comprising 379 amino acids. The following abbreviations
will be used for the CSs, including their optimal growth
temperatures: Arthrobacter: ArCS(31), pig: PigCS(37),
T. acidolphilum: TpCS(55), S. solfataricus:ScCS(85),and
P. furiosus: PfCS(100).
The structure of unliganded SsCS(85) reported in this
paper can now be entered into the temperature ladder of CS
structures, and fills in the gap between the 55 °C and 100 °C
enzymes. Six CS crystal structures from five host organisms
(Table 1) can now be used for comparative analysis in order
to identify some of the structural features that could confer
(hyper)thermostability in this enzyme ÔfamilyÕ.Ascanbe
seen from Table 1, the organisms span the range of
temperatures at which life is known to exist, and the
inherent stability of each CS, from in vitro measured half-
lives of thermal inactivation [19–21], increases with the
optimum growth temperature of the host cells. The structure
of the SsCS(85) is thus discussed in comparison with the
other CS structures, and trends in structural changes are
correlated with the increasing thermal stabilities across the
homologous series of enzymes. In terms of thermostability,
the enzymes fall into two broad classes based on the
temperature at which the half-life equals 8 min: the psychro-
phile and pig enzymes at the lower end with temperatures of

detector. Diffraction extended to 2.7 A
˚
resolution. The
crystal was translated stepwise perpendicular to the beam to
maximize the completeness of the data and to overcome
radiation damage of the crystal. The data were reduced and
scaled using
DENZO
/
SCALEPACK
[22] (Table 2). The asym-
metric unit of the P2
1
unit cell contains two dimers with a
solvent content of 51%. The structure of SsCS(85) was
solved by molecular replacement using the program
AMORE
[23]. Because the crystallization solutions contained both
citrate and CoA, it was assumed that the closed form of
SsCS(85) had crystallized; therefore, initial attempts were
made to solve the structure using the closed structures of
Pf CS(100) or ArCS(31) as the search model, but this did not
produce any clear solutions. Attempts were subsequently
made using the open structure of the TaCS(55) dimer as the
search model. Using data in the resolution range of 15–6 A
˚
and a Patterson integration radius of 25 A
˚
, 50 solutions
from the rotation function were calculated. Using the same

˚
1,2
. Tight non crystallographic symmetry (NCS)
restraints for both main-chain and side-chain were used
initially and six cycles of refinement carried after which the
R-factor was 36.3% (R
free
¼ 40.5%). Keeping the tight
NCS restraints, individual isotropic B-factor refinement was
then carried out, bringing the R-factor down to 24.7% (R
free
31.2%), after which the NCS restraints were gradually
loosened and the four monomers were built independently.
NCS restraints were controlled in
PROTIN
and, during the
refinement procedure, side-chain followed by main-chain
restraints were gradually loosened, with a final round
removing the NCS restraints continuing to lower the R
free
value.
The first two residues at the N-terminus and last seven
residues of the C-terminal arm were not seen in the poorly
defined electron density of these parts of the structure in all
four monomers. One conflict with the sequence data was
residue 57, which had been assigned as arginine and was
found from analysis of the electron density map to be a
proline (this is a totally conserved proline in all the other
known CSs). The position of the small domain with respect
to the large domain in SsCS(85) is the same as previously

7.2% (22.9%)
I/rI 9.37 (3.25)
Total No. of reflections 148169
Unique reflections 46758
R-factor 20.8%
Free R-factor 28.5%
No. protein atoms 11 742
Rmsd bond lengths (A
˚
) 0.009
Rmsd bond angles (°) 0.032
Table 1. CS structures used for analysis.
Source
organism
Optimum growth
temperature
(°C)
CS
Temperature (°C) at which
the half-life equals 8 min
Substrates in
crystal structure
Data resolution
(A
˚
)
Arthrobacter DS23R 31
a
45 Citrate and CoA 2.1
Pig 37 58 Citrate only

equivalent in pigCS (helices A, B, H, and T are not present)
(Figs 2 and 3). Of the 16 equivalent helices, the large domain
comprises 11 helices (C-M and S) and the small domain five
helices (N-R). The small domain has been classed as
residues 217–321 inclusive for SsCS(85).
The active sites of CSs comprise residues from both
monomers and therefore CS is only active as a dimer,
stressing the importance of maintaining dimeric integrity as a
prerequisite for activity. Binding of citrate and CoA to the
active site has been discussed in detail for PfCS(100) and
ArCS(31), and the differences with respect to the pig enzyme
noted [15,18]. The SsCS(85) structure has no substrate
bound, but the location of active site residues can be
identified by comparison with the liganded PfCS(100)
structure. The citrate-binding residues comprising three
arginine residues, R267 (helix P), R338 (helix S) and R358¢
(where the prime denotes the residue of the second mono-
mer), and three histidine residues, H183 (loop K-L), H218
(loop M-N) and H258 (loop O-P), are equivalent to those
found in PfCS(100). The binding residues for the three
Fig. 2. Structurally based sequence alignment of the five CSs discussed.
Helices A to T are shaded, and the location of the small domain is
indicated by lowercase sequence letters. The three catalytic residues are
indicated by fl. The three arginines and histidines involved in binding
citrate are marked with a C. The residues involved in binding the three
phosphates of CoA are marked with an A. The sequence numbering is
shownatthestartofeachline.
Fig. 1. Stereo-diagram showing a typical region of the final 2Fo-Fc electron density map contoured at 1 r.
6252 G. S. Bell et al. (Eur. J. Biochem. 269) Ó FEBS 2002
phosphate groups of CoA are likely to be K250 (loop O-P)

sequence analysis
package, and superposition was carried out using the least
squares fit in
O
[25] for fitting of alpha-carbon atoms
(starting from three conserved atoms). These statistics are
listed in Table 3.
Sequence identities between the various CSs for which
3D-structures have been determined range from 20%
[eukaryotic
5
vs. bacterial or archaeal) to 60% (SsCS(85)
and TaCS(55)]. These identities are reflected in the root
mean square (RMS) deviations between the alpha-carbons
of the structures, with the most similar structures being the
TaCS(55) and SsCS(85), and with the PfCS(100) and
ArCS(31) pair also showing a very low RMS deviation. As
some structures are in the open conformation and some
have substrates bound, the large and small domains of each
enzyme were compared separately (Table 3); in general,
such an analysis shows the same trend as that for the whole
dimer but the small domains tend to be more highly
conserved. As is suggested later, this may correlate with
differences particularly relating to the dimer interface, to
which the small domain does not contribute, and may reflect
the fact that the majority of the substrate-binding and
catalytic residues are from the small domain.
The molecular mechanisms underlying protein thermal
stability
In our comparison of CS atomic structures from organisms

Compactness and surface characteristics
The accessible surface area was calculated using the program
GRASP
[26] and the volume and cavity detection were
determined using the program
VOIDOO
[27] with a probe
radius 1.4 A
˚
and grid spacing of 0.75 A
˚
. All calculations for
closed structures were carried out in the absence of substrate,
and the results are summarized in Table 4.
ArCS(31), TaCS(55) and PfCS(100) have very similar
surface areas, with that of SsCS(85) being slightly higher;
however, all four enzymes have a considerably smaller
surface area and volume than the pigCS(37), even when
deleting the first 35 residues from the pig enzyme (these 35
amino acids comprise helices A and B, which are absent in
the other CSs being considered). A similar pattern to the
total accessible surface area (ASA)
6
is found when compar-
ing the overall volume, with pigCS(37) having a consider-
ably larger volume than the other CSs (again, even when
calculated with the N-terminally deleted structure).
However, it is also notable that the smallest volume is
exhibited by the psychrophilic CS (8.36 · 10
4

˚
2
) of hydrophobic surface area shows a
decrease as the thermostability of the protein increases, a
trend observed in other structural comparisons [4]. SsCS(85)
also follows the trend observed in PfCS(100), with the
elimination of all cavities capable of accommodating a
solvent molecule, indicating that this is a prerequisite for
maintaining integrity at high temperatures. The number of
internal cavities (and their total volumes in A
˚
2
calculated by
VOIDOO
) are 1 (104), 6 (476), 3 (218), 3 (184), 0 (0) and 0 (0)
Table 3. Overall comparison of primary and 3D structures of CSs. In the top half of the table the RMS deviations between Ca atoms (in A
˚
)aregiven
for complete dimers, the large domain and the small domain, with the number of contributing pairs of Ca atoms in parantheses. In the bottom half
of the table, the percentage sequence identities and similarities are shown, the latter in parentheses.
Enzyme
(open)
ArCS(31)
(closed) PigCS(37) PigCS(37) TaCS(55) SsCS(85) PfCS(100)
ArCS(31) – 2.27 (560) 2.12 (630) 1.97 (604) 1.94 (610) 1.32 (719)
1.74 (242) 1.53 (252) 1.57 (259) 1.07 (262)
1.79 (96) 1.59 (90) 1.51 (90) 1.27 (92)
PigCS(37) (open) – – 1.19 (730) 1.95 (651) 1.88 (646) 2.15 (550)
2.16 (533) 2.08 (519) 2.04 (631)
PigCS(37) (closed) 27% (50%) – – 1.81 (233) 1.79 (232) 1.84 (245)

2
) 2.72 3.34 3.20 2.99 2.72 2.82 2.72
No. of atoms calculated for 5784 6888 6884 6344 5722 5879 5961
No. of atoms buried 3044 3469 3601 3307 2955 3014 3248
Atoms buried (%) 52.6 50.4 52.3 52.1 51.6 51.3 54.5
Volume ( · 10
4
A
˚
3
) 8.36 9.96 9.98 9.18 8.71 8.51 8.65
Total area hydrophobic exposed (A
˚
2
) 7854 6654 6246 – 6001 5513 4942
% Hydrophobic of total ASA 29 20 20 – 22 20 18
6254 G. S. Bell et al. (Eur. J. Biochem. 269) Ó FEBS 2002
for ArCS(31), PigCS(37) open, PigCS(37) closed, TaCS(55),
SsCS(85) and PfCS(100), respectively.
The subunit interface: ion pairs, hydrophobicity
and complementarity
Ion pairs were classed as residues of opposite charge
situated 4.0 A
˚
or less apart [28]. Looking simply at the total
numbers of ion pairs, it can be seen that all the thermophilic
CSs have a greater total number of ion pairs than the pig
enzyme, but that the psychrophilic enzyme actually has the
greatest number of all (Table 5). Looking then at the trends
towards inter/intrasubunit ion pairs, PfCS(100) has the

loop G-I, in addition to the above-mentioned Asp-Lys
pair.
Interestingly, in the psychrophilic ArCS(31) the first
Asp-Lys ion pair is part of a four-residue network (in
conjunction with D95 and R98, both in helix G). The four
residue network in ArCS(31) only comprises two single
interactions directly across the interface, whereas the
PfCS(100) five-residue network has four such interactions.
This would suggest that the PfCS(100) networks contri-
bute considerably more to the intermolecular interactions
than they do in the psychrophilic enzyme. Even so, the
psychrophilic enzyme does have an intersubunit ionic
network, which may be related to cold-stability in the face
of diminished hydrophobic interactions at very low
temperatures [15]. The ionic interactions at the central
helices G and M of the five CS structures are shown in
Fig. 5.
The nature of the dimer interface of SsCS(85) differs from
PfCS(100) in that there is a higher degree of hydrophobic
interactions (Table 6). Similar levels to those in SsCS(85)
are also observed in TaCS(55). All the thermophilic CSs
show a low value for the Ôgap volume indexÕ,theratioofthe
gap volume to the accessible surface area of the interface, an
indication that these proteins exhibit greater surface com-
plementarity at the interface compared with ArCS(31) and
pigCS(37).
Finally, examining the part of the dimer interface near the
active site, all the archaeal CSs and the ArCS(31) have ionic
interactions that also tend to stabilize the N-terminus;
however, PfCS(100) certainly has the most extensive ionic

have additional relevance to the strength of the subunit
interactions. The lengths of the C-terminal arms vary, with
ArCS(31) being six residues shorter than those of PfCS(100)
and TaCS(55), and five shorter than SsCS(85). ArCS(31)
has fewer interactions of the C-terminal arm with the other
monomer than the three thermophilic CSs (including one
ion pair that appears to anchor the end of the arm: R375-
E48¢ in PfCS(100)). R375 and E48 are conserved in
TaCS(55) and SsCS(85) suggesting the likelihood of this
ion pair being present at their C-termini. ArCS(31) also has
an arginine residue (R375) which interacts with E56¢ but, as
this residue is four residues from the end of the C-terminus,
there may be more chance of fraying of this terminal arm in
the psychrophile. Both N-termini in PfCS(100) also have an
interconnecting ion pair (K8-D16¢) but this is a three residue
interaction in ArCS(31) (K7, D15¢ and D359¢). SsCS(85)
also has several terminal interactions (E9,R259 and R355¢)
but TaCS(55) does not.
Loop regions: length and ionic interactions
It has previously been suggested that loop regions tend to be
the most flexible regions within a protein, and are therefore
often the first areas to be subject to proteolytic cleavage or
heat denaturation [29]. It is possible therefore that increased
thermostability may be achieved by shortening loops or by
additional interactions stabilizing these regions.
The equivalent loop regions of the five CSs have
therefore been compared (several extra loops are present in
the pig enzyme). Although some of the differences in loop
conformations (particularly near the active site) may be
due to the open or closed nature of the structures, it is

has a four residue intramolecular network and SsCS(85) has
a five residue network that involves a residue at the
N-terminal end of the loop.
Loop I-J. All the enzymes have similarly large loops but
there are no ion pairs in pigCS(37) with one ion pair in
TaCS(55). ArCS(31), SsCS(85) and PfCS(100) all have
multiple ionic interactions linking loops I-J and J-K.
Loop J-K. The loop in pigCS(37) is slightly shorter than the
others and contains no interactions, whilst the TaCS(55)
loop has an ion pair. ArCS(31), SsCS(85) and PfCS(100)
have interactions linking this loop with the previous loop I-J.
Loop N-O. This appears to be a long and flexible loop in
ArCS(31) and contains six charged residues, but no ion
pairs. PigCS(37) also has a longer and more extended loop
than SsCS(85) and TaCS(55) (which both contain ion pairs)
and PfCS(100) has the shortest loop.
Loop O-P. Although not obvious from the length of loops
as designated by
PROMOTIF
[30], the loop in the pig enzyme
is considerably more extended than the others. ArCS(31),
pigCS(37) and PfCS(100) all have single ion pairs
stabilizing this loop (the interaction in the pigCS(37) loop
links it to loop B-C), with TaCS(55) and SsCS(85) loops
having multiple ionic interactions that link loops O-P and
K-L.
Loop P-Q. TaCS(55) and SsCS(85) loops both contain
ionic interactions. In the case of SsCS(85), this is in the form
of a three residue network linking it with loop J-K. This
loop is absent in PfCS(100).

comparing the thermophilic archaeal CSs with the pig-
CS(37), and the tendency towards fewer cavities should
also correlate with the improved hydrophobic packing of
these proteins. The increased complementarity of the dimer
interface, as measured by the gap volume index, in the
thermophilic enzymes may also be a significant feature.
Although the total percentage of atoms buried is similar
for all the CSs, the decreased burial of hydrophobic groups
of ArCS(31) compared with the other CSs probably reflects
the decreased entropic penalty of exposure of hydrophobic
side-chains at psychrophilic temperatures (reviewed by
[32,33]).
Loop regions
There is a tendency towards shorter (even absent) loop
regions in the thermophilic CSs, correlating with the
compactness of these proteins when compared with pig-
CS(37). This trend has also been highlighted by analysis of
mesophilic and thermophilic genome sequences, and was
suggested to be a general strategy for thermostabilization
[34]. However, many of the shorter loops in the thermophilic
CS are similarly short in ArCS(31) (apart from loop N-O).
A more dramatic difference in the loops is seen in the
Fig. 5. Diagram showing ionic interactions in the central helices (G and
M) of the dimer interface of ArCS, PigCS, TaCS, SsCS and PfCS.
Helices from different monomers are coloured blue and orange.
Table 6. The dimer interface of CS. Statistics are calculated using the
protein–protein interactions server (Jones and Thornton, 1995) for the
CS crystal structures with the C-terminal arm removed.
ArCS
PigCS

disrupted, resulted in a less stable protein [36].
Ionic interactions and hydrophobicity
at the subunit interface
The archaeal and bacterial CSs have a higher total number
of ionic interactions than the pigCS(37), which in fact
exhibits the lowest percentage participation of charged
residues in ion pairs or networks of the five enzymes in the
comparison. The psychrophilic ArCS(31) actually has the
most ionic interactions, which we have suggested may be
related to cold stability [15], but with respect to subunit
association, PfCS(100) has the most extensive interactions
across the dimer interface whilst ArCS(31) has more than
either TaCS(55) or SsCS(85).
The eight-helical sandwich part of the dimer interface
shows a definite trend towards increasing hydrophobicity
going from ArCS(31) and pigCS(37) to TaCS(55) and
SsCS(85), and this may be indicative of the increasing
strength of the hydrophobic interaction with temperature,
at least to temperatures approaching 100 °C [37]. PfCS(100)
also has a greater degree of hydrophobicity in this region
than ArCS(31) and pigCS(37) but lower than the other two
thermophilic CSs, and this may be compensated by the
more extensive ionic interactions in the hyperthermophilic
protein. That is, the ionic interactions in the central helices
(G and M) of the eight-helical sandwich also show an
increase from none in pigCS(37), two single ion-pairs in
TaCS(55), four single ion-pairs in SsCS(85) and the two
five-residue networks in PfCS(100). ArCS(31) also has two
four-residue networks here, but these seem to be less
extensive than those in PfCS(100) (with fewer interactions

The importance of electrostatic interactions and their
precise location to stabilizing proteins has been shown in
other crystal structures of (hyper)thermostable proteins, as
discussed in the recent review by Karshikoff and Ladenstein
[10]. The most striking examples include glutamate dehy-
drogenase [39–41], glyceraldehyde 3-phosphate dehydro-
genase [42,43] and lumazine synthase [44]. Again, the
electrostatic strengthening of the intersubunit contacts is a
common theme in these proteins. Finally, computational
analyses [7,45,46] and genomic comparisons [8,9,47] add
further support to these findings.
Concluding remarks
The importance of the determination of the structure of the
SsCS(85) is principally that it ÔcompletesÕ aseriesofCS
structures from which we are now able to identify trends in
the structures of CSs that appear to be correlated with the
different degrees of thermostability. Our findings correlate
well with the growing number of studies that conclude that
ionic interactions stabilizing crucial areas of structure are
perhaps the most common
7
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studies on a mesophilic and thermophilic pair of Rnase H
proteins [49]. These studies suggest that it may be difficult to
dissect the contributions of individual interactions to

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