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The crystal structure of citrate synthase from the thermophilic Archaeon, Thermoplasma acidophilum
The crystal structure of citrate synthase from the thermophilic Archaeon, Thermoplasma acidophilum Rupert JM Russell, David W Hough, Michael J Danson and Garry LTaylor* School of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK Background: The Archaea constitute a phylogenetically distinct, evolutionary domain and comprise organisms that live under environmental extremes of temperature, salinity and/or anaerobicity. Different members of the thermophilic Archaea tolerate temperatures in the range 55-110 0C, and the comparison of the structures of their enzymes with the structurally homogolous enzymes of mesophilic organisms (optimum growth temperature range 15-45 0 C) may provide important information on the structural basis of protein thermostability. We have chosen citrate synthase, the first enzyme of the citric acid cycle, as a model enzyme for such studies.
Results: We have determined the crystal structure of Thermoplasma acidophilum citrate synthase to 2.5 A and have compared it with the citrate synthase from pig heart, with which it shares a high degree of structural homology, but little sequence identity (20%). Conclusions: The three-dimensional structural comparison of thermophilic and mesophilic citrate synthases has permitted catalytic and substrate-binding residues to be tentatively assigned in the archaeal, thermophilic enzyme, and has identified structural features that may be responsible for its thermostability.
Structure 15 December 1994, 2:1157-1167 Key words: Archaea, citrate synthase, crystal structure, thermophile, Thermoplasma acidophilum
Introduction It has been proposed that all living organisms belong to one of three phylogenetically distinct domains: the Eukarya, the Bacteria and the Archaea [1]. The Archaea inhabit a diverse range of extreme environments and phenotypically can be divided into three major groups: thermophiles, halophiles and methanogens, with some members possessing more than one of these phenotypes. Structural studies on archaeal enzymes may therefore reveal features necessary for stability under the extreme growth conditions of the organism, and in addition, possibly, features unique to archaeal proteins. In this respect, the study of homologous proteins from mesophilic and extremophilic hosts is one important approach. However, although sequence analysis can highlight specific amino acid changes between extremophilic and mesophilic proteins, the structural consequence of these changes with respect to additional specific interactions can only be confidently gained from comparisons of the threedimensional structures of the two homologous proteins followed by site-directed mutagenesis experiments to test the predictions. For our comparative studies we have chosen the enzyme citrate synthase [2], which catalyzes the entry of carbon into the citric acid cycle according to the scheme: oxaloacetate+acetyl-coenzyme A+H20--citrate+coenzyme A In the present paper we report the crystal structure of citrate synthase from the thermophilic Archaeon Thermoplasma acidophilum, which grows optimally at 55-60°C and pH 1-3.
T acidophilum citrate synthase exists as a dimer of Mr-90000, with each monomer comprising 384 amino acids. The gene for the enzyme has previously been cloned and sequenced and expressed in Escherichia coli [3,4]. A purification scheme and a preliminary crystallographic study for the recombinant enzyme have been reported [5]. Optimal activity is observed at 55°C although decreased activity is seen at room temperature, with kinetic parameters recorded at the elevated temperature being similar to those for pig heart citrate synthase at mesophilic temperatures. The enzyme is stable and active after incubation for 10 min at 78C, and no appreciable change in a-helical content is observed in circular dichroism (CD) spectra recorded at either 55 0 C or. 80 0C. In contrast, pig heart citrate synthase exhibits no activity after incubation for 10 min at 45 0 C [6]. The T acidophilum citrate synthase exhibits only 20% sequence identity with the dimeric pig heart citrate synthase, but a sequence alignment study reveals conservation of catalytically important residues and predictions from CD spectra suggest similar levels of secondary structural elements for these two phylogenetically diverse citrate synthases. The crystal structures of citrate synthase from pig and chicken heart muscle have been elucidated for 13 different crystalline complexes, including the unliganded enzyme and substrate/analogue complexes [7]. Each monomer, which is >60% ot-helical, can be divided into a small and a large domain. The active site of each monomer is situated between the two domains, comprising residues from both monomers. Upon binding of oxaloacetate to the 'open' (substrate entry/product release)
*Corresponding author. © Current Biology Ltd ISSN 0969-2126
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Structure 1994, Vol 2 No 12 conformation, the enzyme undergoes a large conformational change equivalent to a 190 rigid-body rotation of the small domain with respect to the large domain, thus forming the 'closed' (catalytically active) conformation of
the enzyme [8]. In the present paper, the crystal structure of the 'open' form of T acidophilum citrate synthase at 2.5 A resolution was determined by the method of molecular replacement using the pig enzyme as the search model. The asymmetric unit of the T acidophilum crystals contains two dimers. The structure of the thermophilic archaeal enzyme exhibits a high degree of structural homology with the
eukaryotic enzyme, and putative catalytic and substratebinding residues have been assigned. In addition, a number of structural differences have been observed that may contribute to the thermostability of T acidophilum citrate synthase.
Results and discussion The nomenclature proposed by Remington et al. [8] in naming the secondary structural elements has been adopted to describe the conformation of T acidophilum citrate synthase. Primed helices refer to the second monomer of the dimer.
Fig. 1. The T. acidophilum citrate synthase monomer. (a) Stereoview of the Ca backbone of the T. acidophilum citrate synthase monomer. (b) Schematic representation of the T. acidophilum citrate synthase monomer drawn with MOLSCRIPT [40] and Raster 3D (E Merritt, unpublished program). The secondary structure was assigned using DSSP [411. The large domain is coloured orange at the amino terminus and yellow at the carboxyl terminus, and the small domain is coloured blue. Strand 1, 21-24; strand 2, 29-32; strand 3, 35-36; helix C, 37-43; helix D, 47-56; helix E, 62-74; helix F, 80-87; helix G, 95-109; helix 1, 120-142; helix J, 156-165; helix K, 171-183; helix L, 191-200; helix M, 206-217; helix N, 225-236; helix 0, 242-249; helix P, 270-283; helix Q, 287-307; helix R, 317-327; helix S, 335-359.
Crystal structure of Thermoplasma acidophilum citrate synthase Russell et al. The overall folded conformation T acidophilum citrate synthase comprises 16 ot-helices per monomer, equivalent to 57% of residues, a value that correlates well with that predicted by CD spectra. Each monomer can be described as having two domains: a large domain containing 11 helices (residues 1-224 and 326-384; helices C-G, I-M and S), and a small domain containing five helices (residues 225-325; helices N-R). A small section of surface-accessible antiparallel -sheet, present as three small strands, is formed by residues 21-36 (Fig. 1). Upon dimerization, 20% of the accessible surface area of each monomer is calculated to be buried. The subunit interface consists of an eight ot-helix sandwich, with four pairs of antiparallel helices (FF', GG', LL' and MM') whose axes lie approximately perpendicular to the twofold axis relating the monomers (Fig. 2). Wrapped around the sandwich is a pair of antiparallel helices (I and S) both of which bend smoothly. Similar bending was observed in the equivalent helices in pig heart citrate synthase, although helix I of the pig enzyme contains a proline residue that is thought to induce the bending, whereas the archaeal citrate synthase contains a serine at the equivalent position. Comparison with pig citrate synthase The overall conformation Upon superposition onto the pig enzyme (Fig. 3), T acidophilum citrate synthase exhibits an overall root mean square deviation (rmsd) of 2.27 A for 356 Cot atoms with respect to the open form of pig heart citrate synthase, with the 16 helices in T acidophilum citrate synthase all possessing their structural counterparts in the pig enzyme, despite the lack of sequence identity between the two proteins (Fig. 4). The main areas of structural differences are found at loops connecting the ol-helices, and these are discussed in detail below in relation to the proposed features conferring thermal stability on T acidophilum citrate synthase. The subunit interface helices and helices I and S display the highest degree of structural conservation (rmsd for 96 Cot atoms 1.36 A), with most divergence occurring in surface-accessible regions.
Fig. 3. Stereoview of the superposed Ca backbone tracings of T.acidophilum (red) and pig heart (black) citrate synthases.
Fig. 2. Schematic representation of the T. acidophilum citrate synthase dimer drawn with MOLSCRIPT [40] and Raster 3D (E Merritt, unpublished program). The view is down the two-fold axis. Putative catalytic residues (His222, His262 and Asp317) are highlighted. Colour scheme as for Fig. 1b. The small domains of both citrate synthases comprise five equivalent helices, but the large domain of the T acidophilum enzyme contains four fewer helices (helices A, B, H and T) than that of pig heart; this difference may actually be two helices (A and H), as the amino and carboxyl termini of T acidophilum citrate synthase have very poorly defined electron density (i.e. in the regions corresponding to helices B and T of pig citrate synthase). This reduction in the number of helices and loops in the archaeal citrate synthase results in a markedly more compact enzyme. The orientation of the two domains with respect to each other is subtly different in the two citrate synthases. Within the small domain, helices R and N in the T
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Structure 1994, Vol 2 No 12
Fig. 4. Structure-based sequence alignment of pig heart (PigCS) and T. acidophilum (TpCS) citrate synthases. Helices are indicated by horizontal lines, conserved residues by shading and active-site residues in inverse type. The small domain of each enzyme is shown in smaller type (residues 280-394). Residues from the second monomer involved in the active site are asterisked. The numbers in parentheses indicate residue numbers. (Figure produced using ALSCRIPT [42].) acidophilum citrate synthase show only small differences to the equivalent helices in the pig enzyme, but the remaining three helices (0, P and Q) are all shifted in the direction of the small domain closure observed in pig citrate synthase upon substrate binding, resulting in a more compact enzyme. The conformational change upon substrate binding in the archaeal citrate synthase cannot be determined until the structure of the 'closed' form is elucidated; however, a conformational change probably does occur in the T acidophilum citrate synthase, as addition of the substrate, oxaloacetate, to native crystals causes the crystals to crack and disintegrate. Doubts have been raised as to whether the 'open' form of citrate synthase represents a stable conformation of the enzyme or is just an artefact of the crystallization conditions. The structure reported here supports the findings of Liao et al. [9] that the 'open' form must represent a natural conformation of citrate synthase. A main-chain temperature factor plot
(see Materials and methods section) indicates increased flexibility of the small domain with respect to the large domain. This trend is also observed in the 'open' form of pig heart citrate synthase and may therefore reflect the conformational change that the enzyme undergoes upon binding of substrates. The active site The active sites of pig citrate synthase lie between the large and small domains of each monomer, and each is composed of residues from both monomers. The activesite residues of the pig enzyme have been elucidated by the combinatorial approach of mutagenesis studies and determination of the crystal structures of substrate-bound forms (see [7,10-12] and references therein). Three residues, His274, His320 and Asp375, have been shown to act in a concerted acid-base mechanism, and all three have their structural counterpart in T acidophilum citrate
Crystal structure of Thermoplasma acidophilum citrate synthase
synthase (His222, His262 and Asp317), suggesting conservation of the catalytic mechanism (Fig. 5). A number of positively charged residues have been shown to be essential in the binding of both substrates in pig citrate synthase, and all are maintained except for three putative modifications in the archaeal enzyme (Fig. 4). Firstly, Arg324 (pig) has been changed to Lys266 (Thermoplasma), which is also able to act as a ligand for the o-phosphate of coenzyme A (CoA). Secondly, Arg164' (pig), which acts as the ligand for the 3'-phosphate of CoA, is present in a surface loop that has been dramatically shortened in the T acidophilum citrate synthase. In the absence of a structure for the 'closed' form of the archaeal enzyme, the precise location of this ligand cannot be defined; nevertheless, an alternative ligand, Arg366' (Thermoplasma), is in a suitable position in the proposed CoA-binding site to be able to act as a CoA-binding residue. Finally, Arg46 (pig) acts as a ligand for the o-phosphate of CoA, but in T acidophilum citrate synthase Glul1 is present in an equivalent position. This negatively charged residue cannot therefore act as a ligand, but Arg364' (Thermoplasma) is in a suitable position in the active site to fulfil this role. If this is the case, Arg421' in pig citrate synthase, which acts as a ligand for citrate and which is predicted from sequence alignments to be equivalent to Arg364' in the T acidophilum enzyme, must be replaced by Arg361' in the archaeal citrate synthase. Interestingly, from sequence alignments, Arg46 does not seem to be conserved in bacterial citrate synthases, but they all contain arginine
Russell et al.
or lysine at the same positions (361, 364 and 366) as T acidophilum citrate synthase. Eukaryotic citrate synthases all contain an arginine at position 46 (pig) but not at position 361 (Thermoplasma). Therefore, although the conformation of the active site and the proposed active-site residues are very similar in the T acidophilum and pig citrate synthases, and although they share similar kinetic properties, the exact locations of the substrate-binding residues are different. Arg164' in pig citrate synthase is located in a surface loop, which, based on molecular dynamic simulations, has been proposed to be highly flexible [13], with adjacent residues (e.g. Tyrl67) undergoing large fluctuations during the conformational change after substrate binding. Therefore, the absence of this loop in T acidophilum citrate synthase, and replacement of Arg164' by a differently positioned ligand, may be attributable to the required balance between both flexibility and stability for optimal activity at elevated temperatures. Whether or not this variation on an active-site theme is observed in other archaeal enzymes can only be addressed upon the elucidation of their crystal structures. Features of T.acidophilum citrate synthase that may confer thermostability From the comparison of the structures of the eukaryal and archaeal citrate synthases, it is possible to identify those parts of the enzyme where the two show significant differences, and then to consider those differences in relation to the differing thermostabilities of the two proteins.
(a) ARG 329
ARG 329
ARG I
N
ARG 164-'
(b) LYS
J
26
LY
AR21 ,2 ASP 317
6ASP317 HIS 187
HIS 262
00'HIS 262 HIS 187
ARG344
ARG 344 HIS 222
Fig. 5. Stereoviews of (a) the active-site residues of pig heart citrate synthase and (b) the proposed active-site residues of T. acidophilum citrate synthase. Primed residues reside in the second monomer of the dimer.
ARG 361'
ARG 364' ARG 366'
LYs31
ARG 361'
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Structure 1994, Vol 2 No 12 aeruginosa [16]; however, they are not the dominant wild-type enzymes but are products of a second citrate synthase gene in each organism. From the limited aminoterminal sequence data available, they appear to be closer in sequence to the T acidophilum citrate synthase than their own counterparts. Therefore, the amino-terminal differences may reflect phylogenetic differences as opposed to stability differences, although the functional role of these initial 35 residues remains unassigned. Loop regions The functional role of loops in protein structure and stability has been a topic of wide debate [17]. Molecular dynamics simulations on bovine pancreatic trypsin inhibitor reveal that loop and turn regions undergo the largest deviations from the crystal structure in simulations carried out at room temperature, and higher temperature simulations reveal that these are likely to be the regions of the structure that unfold first during thermal denaturation [18]. The structure of T acidophilum citrate synthase reveals a marked reduction in the size of many of the loops compared with pig citrate synthase (Fig. 6). Four loops are absent from each monomer (Table 1), with part of one of the missing loops incorporating helix H (amino acids 153-160) from pig heart citrate synthase. The 'open' form of pig heart citrate synthase has three regions where specific residues have relatively high temperature factors: the amino terminus and two loops (residues 80-85 and 290-295), all of which correspond to regions that are absent from the T acidophilum citrate synthase. Therefore, the loops present in pig citrate synthase may act as weak points during thermal unfolding owing to their relatively high flexibility. Because the loops in T acidophilum citrate synthase are shorter they may afford this enzyme some form of resistance to thermal unfolding.
Fig. 6. Superposed Ca tracings of T. acidophilum (yellow) and pig heart (magenta) citrate synthases, highlighting a number of the shorter loops present in the former. Top: loop between helices G-I; bottom: loop between helices N-0 (see Table 1).
A molecular dynamics simulation of the conformational change of pig heart citrate synthase revealed that many helices, especially those within the small domain (e.g. O and Q, show a high degree of flexibility [13]. The loops adjacent to the equivalent helices in T acidophilm citrate synthase are shorter. Wrba et al. [19] have proposed that
The amino terminus One of the most striking differences between the pig heart and acidophilum citrate synthases is that the archaeal enzyme is shorter at the amino terminus by 35 amino acids. In the pig enzyme, this segment forms an extended helix that folds over the surface of the molecule. This shorter polypeptide is in fact common to citrate synthases from both halophilic and thermophilic Archaea and has also been found in the enzyme from a thermotolerant Bacillus species [14]. This feature cannot necessarily be linked to the stability of these enzymes, however, as two mesophilic citrate synthases have recently been found to lack'the 35 amino-terminal residues, although all other eukaryal and bacterial citrate synthases are of the longer type. The two shorter mesophilic citrate svnthases are from Escherichia coli I 15]I and Pseudomonas I
Table 1. Residues occurring in loop regions (assigned by DSSP) that have been shortened in T. acidophilum citrate synthase (CS) compared with pig heart citrate synthase (CS). Helices
Residues in loops of T. acidophilum CS
Residues in loops of pig heart CS
C -* D C G 1 N O Q R
44-46 110-119 237 241 308-316
78-88 147-166 292-299 364-374
The deletion between helices G and I also incorporates the deletion of helix H of pig heart citrate synthase.
Crystal structure of Thermoplasma acidophilum citrate synthase Russell et a/.
thermostable enzymes show lower flexibility than their mesophilic counterparts at mesophilic temperatures, but that at temperatures optimal for activity both enzymes exhibit similar levels of flexibility [19]. Therefore the shortening of loops from the thermophilic archaeal citrate synthase that are present in the mesophilic pig citrate synthase may be related to the balance between flexibility and stability. Cavities
The compactness of the thermostable citrate synthase compared with the mesophilic counterpart, caused by the shorter loops, is also reflected in a decrease in both number and size of internal cavities present. Detection of cavities was performed using V O I D 0 0 [20] with a 1.4 A probe and a 0.75 A grid. The cavity calculations were carried out on the dimer for both T. acidophilum and pig heart citrate synthases, using the multi-rotational approach suggested by Kleywegt and Jones [20] in order to reduce errors caused by the grid specifications. Two different forms of detection and calculation were used; probe-occupied (PO) (akin to the Connolly MS program [21]) and probe-accessible (PA), and the results are presented as means for 10 different orientations. P O calculations indicate the volume occupied by the probe whereas PA calculations indicate the volume accessible to the centre of the probe sphere. P O calculations revealed seven cavities in the pig citr:te synthase dimer, equivalent to a volume of 612 A ~ , whereas T. acidophilum citrate synthase has only four cavities, equivalent to a volume of 21 l A ~ The . percentages of the protein occupied by cavities for the pig and Thermoplasma enzymes are 0.62% and 0.25% respectively. PA calculations revealed 11 cavities in pig heart citrate synthase (96 A') and only seven in T. acidophilum citrate synthase (29 A3). Therefore, with either approach, the volume occupied by cavities in the thermostable citrate synthase is only 30-40% of that in the mesophilic enzyme at room temperature. Whether or not the volume of the cavities increases significantly at 55OC (the optimum growth temperature of T. acidophilum) remains to be determined.
Fig. 7. Helical wheel of helix G of (a) T. acidophilum (residues 95-109) and (b) pig heart (residues 137-151) citrate synthases, produced with GCG [43]. Boxed residues indicate hydrophobic
residues.
Fig. 8. Typical section of a 2F,-F, electron-density map drawn at lm, displaying the number of aromatic interactions within the small domain of T. acidophilum citrate synthase. Carbon atoms are shown in yellow, oxygens in red. Electron density is blue. Produced using 0 [35].
A large number of mutational studies have highlighted the importance of the role of cavities in protein stability [22]. Cavity-creating mutations have consistently created proteins with decreased stability with respect to the wildtype [23]. Alternatively, in some instances, cavity-filling
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Structure 1994, Vol 2 No 12 mutations have been shown to cause an increase in the stability of the mutant [24]. Therefore, the tight packing apparent in the T acidophilum citrate synthase may be another factor responsible for its high thermal stability. Subunit interface The subunit interface of both citrate synthases is composed of an eight or-helical sandwich, comprising four antiparallel pairs of helices (F, G, L and M). McEvily and Harrison [25] have proposed that pig heart citrate synthase undergoes a dimer-to-monomer transition upon heating, and therefore the subunit interface in T acidophilum citrate synthase may feature specific differences with respect to the pig enzyme to account for their differing stabilities. Helices F, L and M in pig citrate synthase are hydrophobic on the sides facing the other monomer, whereas helix G is hydrophilic. The equivalent helices in T acidophilum citrate synthase are all hydrophobic. Helical wheels for helix G of T acidophilum and pig citrate synthases are shown in Fig. 7. The major difference between these helices is the increase in alanine residues (from three to eight) in the thermostable enzyme, in which, in the surface facing the other monomer, a serine and a threonine residue in pig citrate synthase have been changed to alanines in the T acidophilum enzyme. Protein mutagenesis and helical-peptide studies show that alanine has the most stabilizing effect on a helix as a result of the increased hydrophobicity and decreased flexibility that its presence induces [26-28]. MenendezArias and Argos [29] reported from sequence comparisons between mesophiles and thermophiles that serine/threonine--alanine changes occur with a high frequency and often occur in a-helices or at subunit interfaces, or both. Such differences were noted in a subunit-interface helix of lactate dehydrogenase from the mesophilic Bacillus megaterium and the thermophilic Bacillus stereothermophilus, and mutation of serine and threonine to alanine in the mesophilic enzyme caused its thermostability to increase dramatically (by 200 C) [30]. Therefore, the specific amino acid changes in the subunit interface of T acidophilum citrate synthase may contribute to maintaining its structural integrity at elevated temperatures. Other interactions A number of additional structural features have been identified that may contribute to the thermal stability of T acidophilum citrate synthase. For example, there is a marked increase in aromatic interactions, using the convention of Burley and Petsko [31], within the small domain (see Fig. 8), and a complete absence of cysteine residues. Such features have been noted in other thermostable proteins [32]. The structural features highlighted above may serve to stabilize the T acidophilum citrate synthase, but may also reflect the archaeal nature of the protein. The compactness
of the enzyme manifested by the decreased size of the loops may be a primitive characteristic of proteins but more crystal structures of archaeal enzymes must be elucidated before firmer conclusions can be drawn as to whether this structure reflects the minimum functional unit for a citrate synthase.
Biological implications The Archaea are a group of organisms, primitive in evolutionary terms, that inhabit a range of extreme environments. Structural studies of proteins from these organisms may therefore yield specific insights into the stabilizing features necessary for optimal activity under the extreme growth conditions. The crystal structure of a citrate synthase from the thermophilic Archaeon Thermoplasma acidophilum now allows the above issues to be addressed for this key metabolic enzyme. Despite low sequence identity, the archaeal enzyme exhibits a high degree of structural and functional similarity with the evolutionary-distant pig heart citrate synthase, therefore allowing useful direct structural comparisons. Although the active sites of the enzymes display an almost identical conformation and composition, putative differences in the locations of specific substrate-binding residues may reflect the need for achieving a balance between stability and flexibility in performing the same reaction but at different temperatures. Elucidation of the substrate-bound form of the thermophilic citrate synthase is currently in progress to allow the determination of the substrate-binding residues and any conformational changes that occur during catalysis. The consequences of structural differences observed in individual monomers and at the subunit interface of the active dimer between the thermophilic and mesophilic citrate synthases may now be explored via site-directed mutagenesis experiments to investigate the role of specific interactions on protein stability. Insights into the role of flexibility on stability may be made by using a combination of molecular dynamics simulations and X-ray data collection at higher temperatures. The determination of the T acidophilum citrate synthase structure at higher resolution is also under way which should allow a more detailed comparison of specific interactions. In addition to these structural considerations, the study of other archaeal citrate synthases may reveal insights into the phylogenetic relationships of the Archaea and further define the evolutionary status of this unique group of extremophilic organisms.
Crystal structure of Thermoplasma acidophilumcitrate synthase Russell et al.
Materials and methods Crystallization Monoclinic type 2 crystals were obtained (using the conditions previously reported [5]) belonging to space group P2 1 with unit cell dimensions of a=53.77 A, b=173.81 A, c=86.74 A and P=97.12 ° .
X-ray data collection Data were collected on a Siemens multiwire area detector mounted on a Siemens rotating anode at room temperature. Native crystals diffracted to a resolution limit of 2.4 A, but only data to 2.5 A were included in the refinement because of lack of completeness at 2.4 A. Data were analyzed with XDS [33] and the statistics are given in Table 2.
Molecular replacement The structure of T. acidophilum citrate synthase was solved by the method of molecular replacement. The volume of the asymmetric unit indicated that it contained two dimers. Many different molecular-replacement packages and altered search models were used, with the correct solution being identified using AMoRE [34]. The 'open' form of pig heart citrate synthase, which had been reduced to polyalanine to account for the very low sequence identity with the archaeal enzyme, was used as the search model. The successful search model was the dimeric form of the enzyme, but without its first 35 residues at the amino terminus (predicted from sequence alignments to be absent in T acidophilum citrate synthase) and the last 16 residues at the carboxyl terminus (which forms a helix in the pig enzyme and which is disconnected from the main body of the protein). Data in the resolution range of 20-4 A were used in both the rotation and translation functions. Results are discussed in terms of an AMoRE correlation coefficient,(CC) [34], with significance of peaks in terms of standard deviations above the mean. Using a Patterson cut-off radius of 25 A, a list of 20 rotation function peaks was obtained, with the top peak having an AMoRE CC of 15.3 (4.13ar). The first translation function to fix the position of the first dimer gave a top solution with a CC of 16.9 (3.86a), which used the eighth highest peak in the rotation function [CC of 11.3 (3.04ar)]. The next highest solution of the translation function had a CC of 15.0 (3.67(r). With the position of the first dimer fixed, the second translation function gave a top solution with a CC of 20.6 (6.91(r), which used the top peak of the initial rotation function. The next highest solution for the translation function had a CC of 18.0 (5.01cr). The two dimers were subjected to rigid-body refinement within AMoRE to give a CC of 50.1. The orientation and position of the two independent dimers were then identified in the asymmetric unit and no bad clashes with symmetryrelated molecules in the unit cell were present. The relatively large rmsd between the search model and the final refined structure (-2.3 A for Ca atoms) demonstrates the power of AMoRE in difficult molecular-replacement solutions.
Refinement Rigid-body refinement of the polyalanine dimers within X-PLOR was used to further refine the orientations and positions of the two dimers to a resolution limit of 4 A. The Rfactor at this stage was 53.4%. An initial 2Fo-F c map at 2.8 A was generated and side-chain density was apparent in areas of uninterrupted backbone density. Equivalent regions of pig heart and T. acidophilum citrate synthases were identified using a sequence alignment and, if the corresponding density was apparent, the side-chain atoms on the correctly orientated and positioned search model were mutated using the graphics package O [35]. Mutation erred on the side of caution. After all the residues whose position could be identified with confidence had been mutated, a round of simulated-annealing refinement within X-PLOR [36] was undertaken, using data between 8 A and 2.8 A. On average, 30-40 residues were mutated before each round of refinement, and in this boot-strapping fashion, the T. acidophilum citrate synthase sequence was fitted to the observed electron density. Non-crystallographic restraints were employed in the refinement until the majority of residues had been fitted. The R-factor for the complete model before noncrystallographic symmetry (ncs) restraints were omitted was 29.6% (F>2(r, for data between 8 A and 2.8 A). Removal of the ncs restraints produced no appreciable deviation of the main chain of the monomers in the two dimers of the asymmetric unit compared with that of the restrained monomer (rmsd between restrained and unrestrained monomers at the end of refinement is 0.5 A for all Ca atoms). The presence of ncs was exploited during the refinement procedure through the use of real-space averaging (using the RAVE software [37], with mask creation and manipulation using the MAMA software [37]. This technique of map improvement proved particularly useful in the later stages of refinement (after the majority of residues had been fitted), with the averaged map highlighting regions (140-160 and 275-290) where errors had occurred in initial rounds of manual model building. The final ncs R-factor was 18.7% with CCs for the ncs operators of 0.86, 0.85 and 0.85. The final model does not contain the initial five residues at the amino terminus or the final 14 residues at the carboxyl terminus because of poorly defined electron density. No solvent molecules have been included in the final model. Model assessment The final refined structure was initially assessed using PROCHECK [38]. A Ramachandran plot for a monomer of T. acidophilum citrate synthase has 89% of residues in the mostfavoured regions, and one residue (Lys237) in a disallowed region in all four individually refined monomers. This residue lies in a crystal contact. Three-dimensional/one-dimensional profile plots [39] for both the monomer and dimer show that all residues lie in acceptable environments. Fig. 9a shows a plot of
Table 2. Summary of data collection statistics. Resolution (A) No. of observations No. of unique observations No. of unique observations (>lo) Overall Rmerge (%) Completeness by shell (>lo)(%) Rmerge in shell (%)
oo-2.8
o-2.5
oo-2.4
107 689 36 744 35 926 6.3
125 777 49 276 47185 6.4
129 680 52 490 49941 6.4 -
-
8-2.8
2.8-2.5
2.5-2.4
-
-
-
-
-
-
91.8
71.7
6.8
10.9
39.0 14.6
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Structure 1994, Vol 2 No 12
Fig. 9. (a) Distance between C positions of the four monomers (A, B, C and D) of the two dimers in the asymmetric unit of T. acidophilum citrate synthase crystals, after least-squares superposition. (b) Superimposed plot of mainchain B-factors for the four monomers (A to D) of the two dimers in the asymmetric unit of T. acidophilum citrate synthase crystals. Secondary structural elements are shown at the top (circles for helices, triangles for strands). The coordinates have been deposited in the Brookhaven Protein Data Bank.
the rmsd for Ca atoms between the four individual monomers in the asymmetric unit. Residues displaying a high rmsd all reside in crystal contacts, and a plot of crystallographic B-factors (Fig. 9b) indicates that these residues also have relatively high mobility. The average main-chain B-factor for the large domain is 17.8 A2 but 23.3 A2 for the small domain. The final R-factor in X-PLOR is 19.7% for all reflections between 8 A and 2.5 A, after individual isotropic B-factor refinement (Table 3).
Acknowledgements: We are grateful to the BBSRC for financial support via a CASE studentship (with Zeneca BioProducts, Billingham, UK) to RJMR and a Biotechnology Directorate Research Grant to MJD, DWH and GLT. We also wish to thank Jamie John for useful discussions.
Table 3. Refinement statistics for all data.
References
R-factor (%)
19.7
Resolution (A) Unique reflections No. of protein atoms (non-hydrogen) Rmsd in bond lengths (A) Rmsd in bond angles ()
8-2.5 46 235 11444 0.019 3.6
1. Woese, C.R., Kandler, O. &Wheelis, M.L. (1990). Towards a natural system of organisms: proposals for the domains Archaea, Bacteria and Eucarya. Proc. Natl. Acad. Sci. USA 87, 4576-4579.
2. Muir, J.M., Hough, D.W. &Danson, M.J. (1994). Citrate synthases from the Archaea. Sys. Appl. Microbiol. 16, 528-533.
3. Sutherland, K.J., Henneke, C.M., Towner, P., Hough, D.W. & Danson, M.J. (1990). Citrate synthase from the thermophilic archaebacterium Thermoplasma acidophilum - cloning and sequencing of the gene. Eur.J. Biochem. 194, 839-844.
Crystal structure of Thermoplasma acidophilum citrate synthase Russell et a/. Sutherland, K.J., Danson, M.J., Hough, D.W. & Towner, P. (1991). Expression and purification of plasmid-encoded Thermoplasma acidophilum citrate synthase from Escherichia coli. FEBS Lett. 282, 132-1 34. Russell, R.J.M., Byrom, D., Danson, M.J., Hough, D.W. & Taylor, G.L. (1993). Crystallization and preliminary crystallographic study of citrate synthase from the thermophilic Archaeon Thermoplasma acidophilum. /. Mol. Biol. 232, 308-309. Russell, R.J.M. (1994). The crystal structure of Thermoplasma acidophilum citrate synthase [Ph.D. Thesis]. University of Bath, UK. Remington, S.J. (1992). Structure and mechanism of citrate synthase. Curr. Top. Cell Regul. 33, 209-229. Remington, S.J., Wiegand, G. & Huber, R. (1982). Crystallographic refinement and atomic models of two different forms of citrate synthase at 2.7 and 1.7 A resolution. 1. Mol. Biol. 158, 111-1 52. Liao, D.I., Karpusas, M. & Remington, S.J. (1991). Crystal structure of an open conformation of citrate synthase from chicken heart at 2.8 A resolution. Biochemistry 30, 6031-6036. Alter, C.M., Casazza, J.P., Zhi, W., Memeth, P., Srere, P.A. & Evans, C.T. (1990). Mutation of essential residues in pig heart citrate synthase. Biochemistry 29, 7557-7563. Kurz, L.C., Drysdale, C.R., Riley, M.C., Evans, C.T. & Srere, P.A. (1992). Catalytic strategy of citrate synthase: effects of amino acid changes in the acetyl-CoA binding site on transition-state analog inhibitor complexes. Biochemistry 31, 7908-791 4. Usher, K.C., Remington, S.J., Martin, D.P. & Drueckhammer, D.C. (1994). A very short hydrogen-bond provides only moderate stabilization of an enzyme-inhibitor complex of citrate synthase. Biochemistry 33, 7753-7759. El-Kettani, M.A.E., Zakrzewska, K., Durup, J. & Lavery, R. (1993). An analysis of the conformational paths of citrate synthase. Proteins 16, 393407. Schendel, F.J., August, P.R., Anderson, C.R., Hanson, R.S. & Flickinger, M.C. (1992). Cloning and nucleotide sequence of the gene encoding for citrate synthase from a thermotolerant Bacillus sp. Appl. Environ. Microbiol. 58, 335-345. Patton, A,, Hough, D.W., Towner, P. & Danson, M.J. (1993). Does Escherichia coli possess a second citrate synthase gene. Eur. 1. Biochem. 214, 75-81. Anderson, S.C.K., Powrie, R. & Mitchell, C.C. (1993). Pseudomonas citrate synthase: purification and characterisation. Biochem. Soc. Trans. 22, 415. Leszczynski, J.F. & Rose, G.D. (1986). Loops in globular proteins: a novel category of secondary structure. Science 234, 849-855. Daggett, V. & Levitt, M. (1993). Protein unfolding pathways explored through molecular dynamics simulations. /. Mol. Biol. 232, 600-61 9. Wrba, A,, Schweiger, A,, Schultes, V., Jaenicke, R. & Zavodsky, P. (1990). Extremely thermostable glyceraldehyde-3-phosphate dehydrogenase from the eubacterium Thermotaga maritima. Biochemistry 29, 7584-7592. Kleywegt, C.J. & Jones, A.T. (1994). Detection, delineation, measurement and display of cavities in macromolecular structures. Acta Crystallogr. D 50, 178-1 85. Connolly, M.L. (1983). Analytical molecular surface calculation. J. Appl. Crystallogr. 16, 548-558. Richards, F.M. & Lim, W.A. (1993). An analysis of packing in the protein folding problem. Q. Rev. Biophys. 26, 423498. Eriksson, A.E., et a/., & Matthews, B.W. (1992). Response of a protein structure to cavity-creating mutations and its relation to the hydrophobic effect. Science 255, 178-1 83. Ishikawa, K., Nakamura, H., Morikawa, K. & Kanaya, S. (1993). Stabilization of Escherichia coli ribonuclease H1 by cavity-filling mutations within the hydrophobic core. Biochemistry 32, 6171-61 78.
McEvily, A.J. & Harrison, J.H. (1986). Subunit equilibria of porcine citrate synthase - effects of enzyme concentration, pH and substrates. /. Biol. Chem. 261, 2593-2598. Serrano, L., Sancho, J., Hirshberg, H. & Fersht, A.R. (1992). Alphahelix stability in proteins. 1. Empirical correlations concerning substitution of side-chains at the N and C-caps and the replacement of alanine by glycine or serine at solvent exposed surfaces. /. Mol. Biol. 227, 544-549. Horovitz, A,, Matthews, J.M. & Fersht, A.R. (1992). Alpha-helix stability in proteins. 2. Factors that influence stability at an internal position. /. Mol. Biol. 227, 560-568. O'Neil, K.T. & DeCrado, W.F. (1990). A thermodynamic scale for the helix-forming tendencies of the commonly occurring aminoacids. Science 250,646-651. Menendez-Arias, L. & Argos, P. (1989). Engineering protein thermalstability - sequence statistics point to residue substitutions in alphahelices. ]. Mol. Biol. 206, 397-406. Kotik, M. & Zuber, H. (1993). Mutations that significantly change the stability, flexibility and quaternary structure of the L-lactate dehydrogenase from Bacillus megaterium. Eur. 1. Biochem. 211, 267-280. Burley, S.K. & Petsko, G.A. (1985). Aromatic-aromatic interaction - a mechanism of protein structure stabilization. Science 229, 23-28. Amaki, Y., Nakano, H. & Yamane, T. (1994). Role of cysteine residues in esterase from Bacillus stereothermophilus and increasing its thermostability by the replacement of cysteines. Appl. Microbiol. Biotech. 40, 664-668. Kabsch, W. (1988). Evaluation of single crystal X-ray diffraction data from a position-sensitivedetector. 1. Appl. Crystallogr. 21, 916-924. Navaza, J. (1992). AMoRE: a new package for molecular replacement. In Molecular Replacement. (Dodson, E.J., Cover, S. and Wolfe, W., eds), pp. 87-90, SERC Daresbury Laboratory, Warrington, UK. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. (1991). Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110-119. Brunger, A.T. (1988). Crystallographic refinement by simulated annealing: application to a 2.8 A resolution structure of aspartate aminotransferase.1. Mol. Biol. 203, 803-81 6. Kleywegt, G.A. & Jones, T.A. (1994). 'Halloween ... Masks and Bones.' In From First Map to Final Model. (Bailey, S., Hubbard, R., and Waller, D., eds), pp. 59-66, SERC Daresbury Laboratory, Warrington, UK. Morris, A.L., MacArthur, M.W. & Thornton, J.N. (1992). Stereochemical quality of protein structure coordinates. Proteins 12, 345-364. Luthy, R., Bowie, J.U. & Eisenberg, D. (1992). Assessment of protein models with 3-dimensional profiles. Nature 356, 83-85. Kraulis, P.J. (1991). MOLSCRIPT: a program to produce both detailed and schematic plots of proteins. /. Appl. Crystallogr. 24, 946-950. Kabsch, W. & Sander, C. (1983). Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22, 2577-2637. Barton, G.J. (1993). ALSCRIPT - a tool to format multiple sequence alignments. Protein Eng. 6, 37-40. Devereux, J., Haeberli, P. & Smithies, 0. (1984). A comprehensive set of sequence analysis programs for the Vax. Nucleic Acids. Res. 12, 387-395. Received: 8 Sep 1994; revision requested: 3 Oct 1994; revisions received: 10 Oct 1994. Accepted: 12 Oct 1994.
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Results: We have determined the crystal structure of Thermoplasma acidophilum citrate synthase to 2.5 A and have compared it with the citrate synthase from pig heart, with which it shares a high degree of structural homology, but little sequence identity (20%). Conclusions: The three-dimensional structural comparison of thermophilic and mesophilic citrate synthases has permitted catalytic and substrate-binding residues to be tentatively assigned in the archaeal, thermophilic enzyme, and has identified structural features that may be responsible for its thermostability.
Structure 15 December 1994, 2:1157-1167 Key words: Archaea, citrate synthase, crystal structure, thermophile, Thermoplasma acidophilum
Introduction It has been proposed that all living organisms belong to one of three phylogenetically distinct domains: the Eukarya, the Bacteria and the Archaea [1]. The Archaea inhabit a diverse range of extreme environments and phenotypically can be divided into three major groups: thermophiles, halophiles and methanogens, with some members possessing more than one of these phenotypes. Structural studies on archaeal enzymes may therefore reveal features necessary for stability under the extreme growth conditions of the organism, and in addition, possibly, features unique to archaeal proteins. In this respect, the study of homologous proteins from mesophilic and extremophilic hosts is one important approach. However, although sequence analysis can highlight specific amino acid changes between extremophilic and mesophilic proteins, the structural consequence of these changes with respect to additional specific interactions can only be confidently gained from comparisons of the threedimensional structures of the two homologous proteins followed by site-directed mutagenesis experiments to test the predictions. For our comparative studies we have chosen the enzyme citrate synthase [2], which catalyzes the entry of carbon into the citric acid cycle according to the scheme: oxaloacetate+acetyl-coenzyme A+H20--citrate+coenzyme A In the present paper we report the crystal structure of citrate synthase from the thermophilic Archaeon Thermoplasma acidophilum, which grows optimally at 55-60°C and pH 1-3.
T acidophilum citrate synthase exists as a dimer of Mr-90000, with each monomer comprising 384 amino acids. The gene for the enzyme has previously been cloned and sequenced and expressed in Escherichia coli [3,4]. A purification scheme and a preliminary crystallographic study for the recombinant enzyme have been reported [5]. Optimal activity is observed at 55°C although decreased activity is seen at room temperature, with kinetic parameters recorded at the elevated temperature being similar to those for pig heart citrate synthase at mesophilic temperatures. The enzyme is stable and active after incubation for 10 min at 78C, and no appreciable change in a-helical content is observed in circular dichroism (CD) spectra recorded at either 55 0 C or. 80 0C. In contrast, pig heart citrate synthase exhibits no activity after incubation for 10 min at 45 0 C [6]. The T acidophilum citrate synthase exhibits only 20% sequence identity with the dimeric pig heart citrate synthase, but a sequence alignment study reveals conservation of catalytically important residues and predictions from CD spectra suggest similar levels of secondary structural elements for these two phylogenetically diverse citrate synthases. The crystal structures of citrate synthase from pig and chicken heart muscle have been elucidated for 13 different crystalline complexes, including the unliganded enzyme and substrate/analogue complexes [7]. Each monomer, which is >60% ot-helical, can be divided into a small and a large domain. The active site of each monomer is situated between the two domains, comprising residues from both monomers. Upon binding of oxaloacetate to the 'open' (substrate entry/product release)
*Corresponding author. © Current Biology Ltd ISSN 0969-2126
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Structure 1994, Vol 2 No 12 conformation, the enzyme undergoes a large conformational change equivalent to a 190 rigid-body rotation of the small domain with respect to the large domain, thus forming the 'closed' (catalytically active) conformation of
the enzyme [8]. In the present paper, the crystal structure of the 'open' form of T acidophilum citrate synthase at 2.5 A resolution was determined by the method of molecular replacement using the pig enzyme as the search model. The asymmetric unit of the T acidophilum crystals contains two dimers. The structure of the thermophilic archaeal enzyme exhibits a high degree of structural homology with the
eukaryotic enzyme, and putative catalytic and substratebinding residues have been assigned. In addition, a number of structural differences have been observed that may contribute to the thermostability of T acidophilum citrate synthase.
Results and discussion The nomenclature proposed by Remington et al. [8] in naming the secondary structural elements has been adopted to describe the conformation of T acidophilum citrate synthase. Primed helices refer to the second monomer of the dimer.
Fig. 1. The T. acidophilum citrate synthase monomer. (a) Stereoview of the Ca backbone of the T. acidophilum citrate synthase monomer. (b) Schematic representation of the T. acidophilum citrate synthase monomer drawn with MOLSCRIPT [40] and Raster 3D (E Merritt, unpublished program). The secondary structure was assigned using DSSP [411. The large domain is coloured orange at the amino terminus and yellow at the carboxyl terminus, and the small domain is coloured blue. Strand 1, 21-24; strand 2, 29-32; strand 3, 35-36; helix C, 37-43; helix D, 47-56; helix E, 62-74; helix F, 80-87; helix G, 95-109; helix 1, 120-142; helix J, 156-165; helix K, 171-183; helix L, 191-200; helix M, 206-217; helix N, 225-236; helix 0, 242-249; helix P, 270-283; helix Q, 287-307; helix R, 317-327; helix S, 335-359.
Crystal structure of Thermoplasma acidophilum citrate synthase Russell et al. The overall folded conformation T acidophilum citrate synthase comprises 16 ot-helices per monomer, equivalent to 57% of residues, a value that correlates well with that predicted by CD spectra. Each monomer can be described as having two domains: a large domain containing 11 helices (residues 1-224 and 326-384; helices C-G, I-M and S), and a small domain containing five helices (residues 225-325; helices N-R). A small section of surface-accessible antiparallel -sheet, present as three small strands, is formed by residues 21-36 (Fig. 1). Upon dimerization, 20% of the accessible surface area of each monomer is calculated to be buried. The subunit interface consists of an eight ot-helix sandwich, with four pairs of antiparallel helices (FF', GG', LL' and MM') whose axes lie approximately perpendicular to the twofold axis relating the monomers (Fig. 2). Wrapped around the sandwich is a pair of antiparallel helices (I and S) both of which bend smoothly. Similar bending was observed in the equivalent helices in pig heart citrate synthase, although helix I of the pig enzyme contains a proline residue that is thought to induce the bending, whereas the archaeal citrate synthase contains a serine at the equivalent position. Comparison with pig citrate synthase The overall conformation Upon superposition onto the pig enzyme (Fig. 3), T acidophilum citrate synthase exhibits an overall root mean square deviation (rmsd) of 2.27 A for 356 Cot atoms with respect to the open form of pig heart citrate synthase, with the 16 helices in T acidophilum citrate synthase all possessing their structural counterparts in the pig enzyme, despite the lack of sequence identity between the two proteins (Fig. 4). The main areas of structural differences are found at loops connecting the ol-helices, and these are discussed in detail below in relation to the proposed features conferring thermal stability on T acidophilum citrate synthase. The subunit interface helices and helices I and S display the highest degree of structural conservation (rmsd for 96 Cot atoms 1.36 A), with most divergence occurring in surface-accessible regions.
Fig. 3. Stereoview of the superposed Ca backbone tracings of T.acidophilum (red) and pig heart (black) citrate synthases.
Fig. 2. Schematic representation of the T. acidophilum citrate synthase dimer drawn with MOLSCRIPT [40] and Raster 3D (E Merritt, unpublished program). The view is down the two-fold axis. Putative catalytic residues (His222, His262 and Asp317) are highlighted. Colour scheme as for Fig. 1b. The small domains of both citrate synthases comprise five equivalent helices, but the large domain of the T acidophilum enzyme contains four fewer helices (helices A, B, H and T) than that of pig heart; this difference may actually be two helices (A and H), as the amino and carboxyl termini of T acidophilum citrate synthase have very poorly defined electron density (i.e. in the regions corresponding to helices B and T of pig citrate synthase). This reduction in the number of helices and loops in the archaeal citrate synthase results in a markedly more compact enzyme. The orientation of the two domains with respect to each other is subtly different in the two citrate synthases. Within the small domain, helices R and N in the T
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Fig. 4. Structure-based sequence alignment of pig heart (PigCS) and T. acidophilum (TpCS) citrate synthases. Helices are indicated by horizontal lines, conserved residues by shading and active-site residues in inverse type. The small domain of each enzyme is shown in smaller type (residues 280-394). Residues from the second monomer involved in the active site are asterisked. The numbers in parentheses indicate residue numbers. (Figure produced using ALSCRIPT [42].) acidophilum citrate synthase show only small differences to the equivalent helices in the pig enzyme, but the remaining three helices (0, P and Q) are all shifted in the direction of the small domain closure observed in pig citrate synthase upon substrate binding, resulting in a more compact enzyme. The conformational change upon substrate binding in the archaeal citrate synthase cannot be determined until the structure of the 'closed' form is elucidated; however, a conformational change probably does occur in the T acidophilum citrate synthase, as addition of the substrate, oxaloacetate, to native crystals causes the crystals to crack and disintegrate. Doubts have been raised as to whether the 'open' form of citrate synthase represents a stable conformation of the enzyme or is just an artefact of the crystallization conditions. The structure reported here supports the findings of Liao et al. [9] that the 'open' form must represent a natural conformation of citrate synthase. A main-chain temperature factor plot
(see Materials and methods section) indicates increased flexibility of the small domain with respect to the large domain. This trend is also observed in the 'open' form of pig heart citrate synthase and may therefore reflect the conformational change that the enzyme undergoes upon binding of substrates. The active site The active sites of pig citrate synthase lie between the large and small domains of each monomer, and each is composed of residues from both monomers. The activesite residues of the pig enzyme have been elucidated by the combinatorial approach of mutagenesis studies and determination of the crystal structures of substrate-bound forms (see [7,10-12] and references therein). Three residues, His274, His320 and Asp375, have been shown to act in a concerted acid-base mechanism, and all three have their structural counterpart in T acidophilum citrate
Crystal structure of Thermoplasma acidophilum citrate synthase
synthase (His222, His262 and Asp317), suggesting conservation of the catalytic mechanism (Fig. 5). A number of positively charged residues have been shown to be essential in the binding of both substrates in pig citrate synthase, and all are maintained except for three putative modifications in the archaeal enzyme (Fig. 4). Firstly, Arg324 (pig) has been changed to Lys266 (Thermoplasma), which is also able to act as a ligand for the o-phosphate of coenzyme A (CoA). Secondly, Arg164' (pig), which acts as the ligand for the 3'-phosphate of CoA, is present in a surface loop that has been dramatically shortened in the T acidophilum citrate synthase. In the absence of a structure for the 'closed' form of the archaeal enzyme, the precise location of this ligand cannot be defined; nevertheless, an alternative ligand, Arg366' (Thermoplasma), is in a suitable position in the proposed CoA-binding site to be able to act as a CoA-binding residue. Finally, Arg46 (pig) acts as a ligand for the o-phosphate of CoA, but in T acidophilum citrate synthase Glul1 is present in an equivalent position. This negatively charged residue cannot therefore act as a ligand, but Arg364' (Thermoplasma) is in a suitable position in the active site to fulfil this role. If this is the case, Arg421' in pig citrate synthase, which acts as a ligand for citrate and which is predicted from sequence alignments to be equivalent to Arg364' in the T acidophilum enzyme, must be replaced by Arg361' in the archaeal citrate synthase. Interestingly, from sequence alignments, Arg46 does not seem to be conserved in bacterial citrate synthases, but they all contain arginine
Russell et al.
or lysine at the same positions (361, 364 and 366) as T acidophilum citrate synthase. Eukaryotic citrate synthases all contain an arginine at position 46 (pig) but not at position 361 (Thermoplasma). Therefore, although the conformation of the active site and the proposed active-site residues are very similar in the T acidophilum and pig citrate synthases, and although they share similar kinetic properties, the exact locations of the substrate-binding residues are different. Arg164' in pig citrate synthase is located in a surface loop, which, based on molecular dynamic simulations, has been proposed to be highly flexible [13], with adjacent residues (e.g. Tyrl67) undergoing large fluctuations during the conformational change after substrate binding. Therefore, the absence of this loop in T acidophilum citrate synthase, and replacement of Arg164' by a differently positioned ligand, may be attributable to the required balance between both flexibility and stability for optimal activity at elevated temperatures. Whether or not this variation on an active-site theme is observed in other archaeal enzymes can only be addressed upon the elucidation of their crystal structures. Features of T.acidophilum citrate synthase that may confer thermostability From the comparison of the structures of the eukaryal and archaeal citrate synthases, it is possible to identify those parts of the enzyme where the two show significant differences, and then to consider those differences in relation to the differing thermostabilities of the two proteins.
(a) ARG 329
ARG 329
ARG I
N
ARG 164-'
(b) LYS
J
26
LY
AR21 ,2 ASP 317
6ASP317 HIS 187
HIS 262
00'HIS 262 HIS 187
ARG344
ARG 344 HIS 222
Fig. 5. Stereoviews of (a) the active-site residues of pig heart citrate synthase and (b) the proposed active-site residues of T. acidophilum citrate synthase. Primed residues reside in the second monomer of the dimer.
ARG 361'
ARG 364' ARG 366'
LYs31
ARG 361'
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Structure 1994, Vol 2 No 12 aeruginosa [16]; however, they are not the dominant wild-type enzymes but are products of a second citrate synthase gene in each organism. From the limited aminoterminal sequence data available, they appear to be closer in sequence to the T acidophilum citrate synthase than their own counterparts. Therefore, the amino-terminal differences may reflect phylogenetic differences as opposed to stability differences, although the functional role of these initial 35 residues remains unassigned. Loop regions The functional role of loops in protein structure and stability has been a topic of wide debate [17]. Molecular dynamics simulations on bovine pancreatic trypsin inhibitor reveal that loop and turn regions undergo the largest deviations from the crystal structure in simulations carried out at room temperature, and higher temperature simulations reveal that these are likely to be the regions of the structure that unfold first during thermal denaturation [18]. The structure of T acidophilum citrate synthase reveals a marked reduction in the size of many of the loops compared with pig citrate synthase (Fig. 6). Four loops are absent from each monomer (Table 1), with part of one of the missing loops incorporating helix H (amino acids 153-160) from pig heart citrate synthase. The 'open' form of pig heart citrate synthase has three regions where specific residues have relatively high temperature factors: the amino terminus and two loops (residues 80-85 and 290-295), all of which correspond to regions that are absent from the T acidophilum citrate synthase. Therefore, the loops present in pig citrate synthase may act as weak points during thermal unfolding owing to their relatively high flexibility. Because the loops in T acidophilum citrate synthase are shorter they may afford this enzyme some form of resistance to thermal unfolding.
Fig. 6. Superposed Ca tracings of T. acidophilum (yellow) and pig heart (magenta) citrate synthases, highlighting a number of the shorter loops present in the former. Top: loop between helices G-I; bottom: loop between helices N-0 (see Table 1).
A molecular dynamics simulation of the conformational change of pig heart citrate synthase revealed that many helices, especially those within the small domain (e.g. O and Q, show a high degree of flexibility [13]. The loops adjacent to the equivalent helices in T acidophilm citrate synthase are shorter. Wrba et al. [19] have proposed that
The amino terminus One of the most striking differences between the pig heart and acidophilum citrate synthases is that the archaeal enzyme is shorter at the amino terminus by 35 amino acids. In the pig enzyme, this segment forms an extended helix that folds over the surface of the molecule. This shorter polypeptide is in fact common to citrate synthases from both halophilic and thermophilic Archaea and has also been found in the enzyme from a thermotolerant Bacillus species [14]. This feature cannot necessarily be linked to the stability of these enzymes, however, as two mesophilic citrate synthases have recently been found to lack'the 35 amino-terminal residues, although all other eukaryal and bacterial citrate synthases are of the longer type. The two shorter mesophilic citrate svnthases are from Escherichia coli I 15]I and Pseudomonas I
Table 1. Residues occurring in loop regions (assigned by DSSP) that have been shortened in T. acidophilum citrate synthase (CS) compared with pig heart citrate synthase (CS). Helices
Residues in loops of T. acidophilum CS
Residues in loops of pig heart CS
C -* D C G 1 N O Q R
44-46 110-119 237 241 308-316
78-88 147-166 292-299 364-374
The deletion between helices G and I also incorporates the deletion of helix H of pig heart citrate synthase.
Crystal structure of Thermoplasma acidophilum citrate synthase Russell et a/.
thermostable enzymes show lower flexibility than their mesophilic counterparts at mesophilic temperatures, but that at temperatures optimal for activity both enzymes exhibit similar levels of flexibility [19]. Therefore the shortening of loops from the thermophilic archaeal citrate synthase that are present in the mesophilic pig citrate synthase may be related to the balance between flexibility and stability. Cavities
The compactness of the thermostable citrate synthase compared with the mesophilic counterpart, caused by the shorter loops, is also reflected in a decrease in both number and size of internal cavities present. Detection of cavities was performed using V O I D 0 0 [20] with a 1.4 A probe and a 0.75 A grid. The cavity calculations were carried out on the dimer for both T. acidophilum and pig heart citrate synthases, using the multi-rotational approach suggested by Kleywegt and Jones [20] in order to reduce errors caused by the grid specifications. Two different forms of detection and calculation were used; probe-occupied (PO) (akin to the Connolly MS program [21]) and probe-accessible (PA), and the results are presented as means for 10 different orientations. P O calculations indicate the volume occupied by the probe whereas PA calculations indicate the volume accessible to the centre of the probe sphere. P O calculations revealed seven cavities in the pig citr:te synthase dimer, equivalent to a volume of 612 A ~ , whereas T. acidophilum citrate synthase has only four cavities, equivalent to a volume of 21 l A ~ The . percentages of the protein occupied by cavities for the pig and Thermoplasma enzymes are 0.62% and 0.25% respectively. PA calculations revealed 11 cavities in pig heart citrate synthase (96 A') and only seven in T. acidophilum citrate synthase (29 A3). Therefore, with either approach, the volume occupied by cavities in the thermostable citrate synthase is only 30-40% of that in the mesophilic enzyme at room temperature. Whether or not the volume of the cavities increases significantly at 55OC (the optimum growth temperature of T. acidophilum) remains to be determined.
Fig. 7. Helical wheel of helix G of (a) T. acidophilum (residues 95-109) and (b) pig heart (residues 137-151) citrate synthases, produced with GCG [43]. Boxed residues indicate hydrophobic
residues.
Fig. 8. Typical section of a 2F,-F, electron-density map drawn at lm, displaying the number of aromatic interactions within the small domain of T. acidophilum citrate synthase. Carbon atoms are shown in yellow, oxygens in red. Electron density is blue. Produced using 0 [35].
A large number of mutational studies have highlighted the importance of the role of cavities in protein stability [22]. Cavity-creating mutations have consistently created proteins with decreased stability with respect to the wildtype [23]. Alternatively, in some instances, cavity-filling
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Structure 1994, Vol 2 No 12 mutations have been shown to cause an increase in the stability of the mutant [24]. Therefore, the tight packing apparent in the T acidophilum citrate synthase may be another factor responsible for its high thermal stability. Subunit interface The subunit interface of both citrate synthases is composed of an eight or-helical sandwich, comprising four antiparallel pairs of helices (F, G, L and M). McEvily and Harrison [25] have proposed that pig heart citrate synthase undergoes a dimer-to-monomer transition upon heating, and therefore the subunit interface in T acidophilum citrate synthase may feature specific differences with respect to the pig enzyme to account for their differing stabilities. Helices F, L and M in pig citrate synthase are hydrophobic on the sides facing the other monomer, whereas helix G is hydrophilic. The equivalent helices in T acidophilum citrate synthase are all hydrophobic. Helical wheels for helix G of T acidophilum and pig citrate synthases are shown in Fig. 7. The major difference between these helices is the increase in alanine residues (from three to eight) in the thermostable enzyme, in which, in the surface facing the other monomer, a serine and a threonine residue in pig citrate synthase have been changed to alanines in the T acidophilum enzyme. Protein mutagenesis and helical-peptide studies show that alanine has the most stabilizing effect on a helix as a result of the increased hydrophobicity and decreased flexibility that its presence induces [26-28]. MenendezArias and Argos [29] reported from sequence comparisons between mesophiles and thermophiles that serine/threonine--alanine changes occur with a high frequency and often occur in a-helices or at subunit interfaces, or both. Such differences were noted in a subunit-interface helix of lactate dehydrogenase from the mesophilic Bacillus megaterium and the thermophilic Bacillus stereothermophilus, and mutation of serine and threonine to alanine in the mesophilic enzyme caused its thermostability to increase dramatically (by 200 C) [30]. Therefore, the specific amino acid changes in the subunit interface of T acidophilum citrate synthase may contribute to maintaining its structural integrity at elevated temperatures. Other interactions A number of additional structural features have been identified that may contribute to the thermal stability of T acidophilum citrate synthase. For example, there is a marked increase in aromatic interactions, using the convention of Burley and Petsko [31], within the small domain (see Fig. 8), and a complete absence of cysteine residues. Such features have been noted in other thermostable proteins [32]. The structural features highlighted above may serve to stabilize the T acidophilum citrate synthase, but may also reflect the archaeal nature of the protein. The compactness
of the enzyme manifested by the decreased size of the loops may be a primitive characteristic of proteins but more crystal structures of archaeal enzymes must be elucidated before firmer conclusions can be drawn as to whether this structure reflects the minimum functional unit for a citrate synthase.
Biological implications The Archaea are a group of organisms, primitive in evolutionary terms, that inhabit a range of extreme environments. Structural studies of proteins from these organisms may therefore yield specific insights into the stabilizing features necessary for optimal activity under the extreme growth conditions. The crystal structure of a citrate synthase from the thermophilic Archaeon Thermoplasma acidophilum now allows the above issues to be addressed for this key metabolic enzyme. Despite low sequence identity, the archaeal enzyme exhibits a high degree of structural and functional similarity with the evolutionary-distant pig heart citrate synthase, therefore allowing useful direct structural comparisons. Although the active sites of the enzymes display an almost identical conformation and composition, putative differences in the locations of specific substrate-binding residues may reflect the need for achieving a balance between stability and flexibility in performing the same reaction but at different temperatures. Elucidation of the substrate-bound form of the thermophilic citrate synthase is currently in progress to allow the determination of the substrate-binding residues and any conformational changes that occur during catalysis. The consequences of structural differences observed in individual monomers and at the subunit interface of the active dimer between the thermophilic and mesophilic citrate synthases may now be explored via site-directed mutagenesis experiments to investigate the role of specific interactions on protein stability. Insights into the role of flexibility on stability may be made by using a combination of molecular dynamics simulations and X-ray data collection at higher temperatures. The determination of the T acidophilum citrate synthase structure at higher resolution is also under way which should allow a more detailed comparison of specific interactions. In addition to these structural considerations, the study of other archaeal citrate synthases may reveal insights into the phylogenetic relationships of the Archaea and further define the evolutionary status of this unique group of extremophilic organisms.
Crystal structure of Thermoplasma acidophilumcitrate synthase Russell et al.
Materials and methods Crystallization Monoclinic type 2 crystals were obtained (using the conditions previously reported [5]) belonging to space group P2 1 with unit cell dimensions of a=53.77 A, b=173.81 A, c=86.74 A and P=97.12 ° .
X-ray data collection Data were collected on a Siemens multiwire area detector mounted on a Siemens rotating anode at room temperature. Native crystals diffracted to a resolution limit of 2.4 A, but only data to 2.5 A were included in the refinement because of lack of completeness at 2.4 A. Data were analyzed with XDS [33] and the statistics are given in Table 2.
Molecular replacement The structure of T. acidophilum citrate synthase was solved by the method of molecular replacement. The volume of the asymmetric unit indicated that it contained two dimers. Many different molecular-replacement packages and altered search models were used, with the correct solution being identified using AMoRE [34]. The 'open' form of pig heart citrate synthase, which had been reduced to polyalanine to account for the very low sequence identity with the archaeal enzyme, was used as the search model. The successful search model was the dimeric form of the enzyme, but without its first 35 residues at the amino terminus (predicted from sequence alignments to be absent in T acidophilum citrate synthase) and the last 16 residues at the carboxyl terminus (which forms a helix in the pig enzyme and which is disconnected from the main body of the protein). Data in the resolution range of 20-4 A were used in both the rotation and translation functions. Results are discussed in terms of an AMoRE correlation coefficient,(CC) [34], with significance of peaks in terms of standard deviations above the mean. Using a Patterson cut-off radius of 25 A, a list of 20 rotation function peaks was obtained, with the top peak having an AMoRE CC of 15.3 (4.13ar). The first translation function to fix the position of the first dimer gave a top solution with a CC of 16.9 (3.86a), which used the eighth highest peak in the rotation function [CC of 11.3 (3.04ar)]. The next highest solution of the translation function had a CC of 15.0 (3.67(r). With the position of the first dimer fixed, the second translation function gave a top solution with a CC of 20.6 (6.91(r), which used the top peak of the initial rotation function. The next highest solution for the translation function had a CC of 18.0 (5.01cr). The two dimers were subjected to rigid-body refinement within AMoRE to give a CC of 50.1. The orientation and position of the two independent dimers were then identified in the asymmetric unit and no bad clashes with symmetryrelated molecules in the unit cell were present. The relatively large rmsd between the search model and the final refined structure (-2.3 A for Ca atoms) demonstrates the power of AMoRE in difficult molecular-replacement solutions.
Refinement Rigid-body refinement of the polyalanine dimers within X-PLOR was used to further refine the orientations and positions of the two dimers to a resolution limit of 4 A. The Rfactor at this stage was 53.4%. An initial 2Fo-F c map at 2.8 A was generated and side-chain density was apparent in areas of uninterrupted backbone density. Equivalent regions of pig heart and T. acidophilum citrate synthases were identified using a sequence alignment and, if the corresponding density was apparent, the side-chain atoms on the correctly orientated and positioned search model were mutated using the graphics package O [35]. Mutation erred on the side of caution. After all the residues whose position could be identified with confidence had been mutated, a round of simulated-annealing refinement within X-PLOR [36] was undertaken, using data between 8 A and 2.8 A. On average, 30-40 residues were mutated before each round of refinement, and in this boot-strapping fashion, the T. acidophilum citrate synthase sequence was fitted to the observed electron density. Non-crystallographic restraints were employed in the refinement until the majority of residues had been fitted. The R-factor for the complete model before noncrystallographic symmetry (ncs) restraints were omitted was 29.6% (F>2(r, for data between 8 A and 2.8 A). Removal of the ncs restraints produced no appreciable deviation of the main chain of the monomers in the two dimers of the asymmetric unit compared with that of the restrained monomer (rmsd between restrained and unrestrained monomers at the end of refinement is 0.5 A for all Ca atoms). The presence of ncs was exploited during the refinement procedure through the use of real-space averaging (using the RAVE software [37], with mask creation and manipulation using the MAMA software [37]. This technique of map improvement proved particularly useful in the later stages of refinement (after the majority of residues had been fitted), with the averaged map highlighting regions (140-160 and 275-290) where errors had occurred in initial rounds of manual model building. The final ncs R-factor was 18.7% with CCs for the ncs operators of 0.86, 0.85 and 0.85. The final model does not contain the initial five residues at the amino terminus or the final 14 residues at the carboxyl terminus because of poorly defined electron density. No solvent molecules have been included in the final model. Model assessment The final refined structure was initially assessed using PROCHECK [38]. A Ramachandran plot for a monomer of T. acidophilum citrate synthase has 89% of residues in the mostfavoured regions, and one residue (Lys237) in a disallowed region in all four individually refined monomers. This residue lies in a crystal contact. Three-dimensional/one-dimensional profile plots [39] for both the monomer and dimer show that all residues lie in acceptable environments. Fig. 9a shows a plot of
Table 2. Summary of data collection statistics. Resolution (A) No. of observations No. of unique observations No. of unique observations (>lo) Overall Rmerge (%) Completeness by shell (>lo)(%) Rmerge in shell (%)
oo-2.8
o-2.5
oo-2.4
107 689 36 744 35 926 6.3
125 777 49 276 47185 6.4
129 680 52 490 49941 6.4 -
-
8-2.8
2.8-2.5
2.5-2.4
-
-
-
-
-
-
91.8
71.7
6.8
10.9
39.0 14.6
1165
1166
Structure 1994, Vol 2 No 12
Fig. 9. (a) Distance between C positions of the four monomers (A, B, C and D) of the two dimers in the asymmetric unit of T. acidophilum citrate synthase crystals, after least-squares superposition. (b) Superimposed plot of mainchain B-factors for the four monomers (A to D) of the two dimers in the asymmetric unit of T. acidophilum citrate synthase crystals. Secondary structural elements are shown at the top (circles for helices, triangles for strands). The coordinates have been deposited in the Brookhaven Protein Data Bank.
the rmsd for Ca atoms between the four individual monomers in the asymmetric unit. Residues displaying a high rmsd all reside in crystal contacts, and a plot of crystallographic B-factors (Fig. 9b) indicates that these residues also have relatively high mobility. The average main-chain B-factor for the large domain is 17.8 A2 but 23.3 A2 for the small domain. The final R-factor in X-PLOR is 19.7% for all reflections between 8 A and 2.5 A, after individual isotropic B-factor refinement (Table 3).
Acknowledgements: We are grateful to the BBSRC for financial support via a CASE studentship (with Zeneca BioProducts, Billingham, UK) to RJMR and a Biotechnology Directorate Research Grant to MJD, DWH and GLT. We also wish to thank Jamie John for useful discussions.
Table 3. Refinement statistics for all data.
References
R-factor (%)
19.7
Resolution (A) Unique reflections No. of protein atoms (non-hydrogen) Rmsd in bond lengths (A) Rmsd in bond angles ()
8-2.5 46 235 11444 0.019 3.6
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