Báo cáo Y học: Stepwise adaptations of citrate synthase to survival at life’s extremes From psychrophile to hyperthermophile pot

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 of this dimeric enzyme and loop regions are reinforced by complex electrostatic interactions, and there is a reduced exposure of hydrophobic surface. These observations reveal a progressive pattern of stabilization through multiple additional interactions at solvent exposed, loop and inter- facial regions. Keywords: citrate synthase; Sulfolobus; citrate synthase; thermostability; crystal structure; ion networks. Comparative structural analysis of the same protein isolated from mesophiles and thermophiles have highlighted many structural adaptations that confer protein thermostability [1–6]. The importance of electrostatic interactions at specific locations within the structure, and particularly the presence of ion-pair networks, is a feature that is common to almost all the hyperthermophilic proteins [7–10], although many other additional differences such as improved hydrophobic packing, compactness and additional hydrogen bonds have been observed in other proteins. For our analysis we have chosen the enzyme citrate synthase (CS) (EC 4.1.3.7), which catalyses the condensa- tion of oxaloacetate and acetyl-CoA to form citrate and CoA. The enzyme from psychrophilic, mesophilic and thermophilic sources has been intensively studied both kinetically [11–13] and structurally [3,14]. Crystal structures exist for CS from a psychrophilic Antarctic bacterium Arthrobacter strain DS2-3R (growth optimum ¼ 31 °C) [15], pig (37 °C) [16], and the Archaea Thermoplasma acidophilum (55 °C) [17] and Pyrococcus furiosus (100 °C) [18]. To extend our previous studies we have chosen the organism Sulfolobus solfataricus, a thermophilic Archaeon 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 45 °Cand58 °C, and the archaeal enzymes at the upper end with temperatures of 87 °C, 95 °Cand100°C. MATERIALS AND METHODS Crystallization and structure solution Recombinant SsCS(85) was purified as described previously [19]. Crystallization trials were carried out using the hanging-drop vapour diffusion method using the Hampton Research Screens. A single rod-like crystal of approximate dimensions 2 · 0.1 · 0.1 mm grew in a 6-lL drop contain- ing 2 lL SsCS(85) (10 mgÆmL )1 )with10m M citrate and Correspondence to G. Taylor, Centre for Biomolecular Sciences, University of St. Andrews, St. Andrews, KY16 9ST, UK. E-mail: glt2@st-and.ac.uk (Received 3 July 2002, revised 8 October 2002, accepted 4 November 2002) Eur. J. Biochem. 269, 6250–6260 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03344.x CoA, 2 lLof100m M Tris/HCl, pH 7.2, containing 17% (v/v) PEG 8K, and 2 lLof0.1 M CaCl 2 .Thecrystalgrewin a partially dried out drop after six months. X-ray data were collected at room temperature on a 30-cm Mar image plate 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 resolution range for the translation search, the top solution (33rd highest from the rotation search) had a correlation coefficient (CC) of 32.0 and R-factor of 53.4% (compared with the next highest peak with a background CC of 23 and R-factor of 56%). This solution was fixed, and a solution for the second dimer in the asymmetric unit was identified (CC of 37.7% and R-factor of 51.9%, compared to the next highest peak of 31 and 53%, respectively). After a rigid- body refinement in AMORE of the two dimers, the final solutions had a CC of 56.6 and R-factor of 41.3%. The failure to find a solution using the closed form of the homologous enzyme, but a clear solution with the open forms, strongly suggested that the SsCS(85) had unexpect- edly crystallised in the open, unliganded form. Refinement and validation The restrained refinement of SsCS(85) was performed using REFMAC [24]. The initial R-factor in REFMAC (after rigid body refinement) was 48.3% (R free ¼ 48.6%) and final R-factor of 20.8% (R free ¼ 28.5%) for all data from 20.0 A ˚ to 2.7 A ˚ 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 observed in TaCS(55) [17]; this together with the absence of density for substrates in the active site, supports the previous speculation that the structure is the ÔopenÕ form of the enzyme. After 24 rounds of refinement in REFMAC , the final R-factor was 20.8% (R free ¼ 28.5%) (Table 2). The quality ofthefinalelectrondensityisshowninFig.1. Table 2. Table displaying native data collected and refinement statistics for the SsCS. Data in parentheses correspond to the high resolution data shell (2.82–2.71 A ˚ ). Space group P2 1 Unit cell dimensions a ¼ 77.3 A ˚ ,b¼ 97.9 A ˚ , c ¼ 119.3 A ˚ , b ¼ 107.6° Resolution limit 2.7 A ˚ Data completeness 88.6% (91.2%) R merge 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 Citrate and CoA 2.7 2.0 Thermoplasma acidophilum 55 87 – 2.5 Sulfolobus solfataricus 85 95 – 2.7 Pyrococcus furiosus 100 100 Citrate & CoA 1.9 a It should be noted that, although Arthrobacter DS23R was isolated from a habitat temperature of approximately 0 °C, this organism displays a relatively high optimum growth temperature; therefore, although it is described here as psychrophilic, it should perhaps more correctly be referred to as psychrotolerant. Ó FEBS 2002 Citrate synthase from psychrophile to hyperthermophile (Eur. J. Biochem. 269) 6251 The Ramachandran plot shows that for the four mono- mers in the asymmetric unit, 91.3% of residues lie in the most favoured regions, with only 8.7% in the allowed regions and no residues appearing in the generously allowed or disallowed regions (excluding glycine and proline residues). Atomic coordinates have been deposited in the Protein Databank with accession code 107x 3 . RESULTS Overall structural comparisons All the eukaryotic 4 , archaeal and Gram-positive bacteria CSs are homo-dimeric structures with each monomer consisting of a large and small domain. In addition, they are almost entirely a-helical; the pigCS contains 20 a-helices (A-T) with theenzymefromArCS(31), TaCS(55), SsCS(85) and PfCS(100) containing 16 a-helices, all of which have an 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) and K306 (loop Q-R), R259 and K262 (both loop O-P), with the third phosphate being co-ordinated by R355¢ from the second monomer. The catalytic residues H218, H258 and D313 (loop Q-R) are also present in SsCS(85) and are in a similar position to the PfCS(100) residues. It is likely therefore that SsCS(85) binds substrates in a similar man- ner to PfCS(100) and that the mechanism of catalysis is the same. The dimer interface of all the CSs is made up of two parts and comprises residues solely in the large domain; the main part is the eight a-helical sandwich of four antiparallel pairs of helices (F, G, M and L), with the second being the additional interaction of N- and C-terminal regions (Fig. 3). The pigCS is different from the other four CSs in terms of the topology of the C-terminal region. In the other four, the C-terminal arm of one monomer wraps around the other monomer, clasping the two together [18], and results in more extensive interactions, including those with the N-terminus. It is important to note that as the C-terminal arms of the TaCS(55) and SsCS(85) are not complete in the structures (see below), there may be additional interactions present that have not been observed. This also suggests that the C-terminal arm seems to be ordered only in the presence of substrates. Sequence and structural statistics Pairwise sequence alignments were carried out using the program BESTFIT from the Wisconsin GCG 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 spanning a wide-range of growth temperatures, the deter- mination of the SsCS(85) structure fills an important gap between the enzyme from Thermoplasma (55 °C) and Pyrococcus (100 °C). With the structure reported in this paper, we can now look for trends in the structures that might correlate with the increasing thermostabilities of these enzymes. However, the complex nature of the stabilization of a protein structure lends itself to many types of comparative analysis, and the results presented below are those where significant differences exist between the struc- tures. Other types of analysis (e.g. of hydrogen bonds and helix capping) have been performed but are not included Fig. 3. Schematic drawings of CS. Fromtoptobottom:ArCS(31), PigCS(37), TaCS(55), SsCS(85) and PfCS(100). The right hand col- umn represent views obtained by rotating the images in the left hand column by 90° about a horizontal axis. N- and C-terminii are denoted by blue and red spheres, respectively. The small domains (helices N to R) are coloured in a lighted shade. Catalytic residues, and citrate and Co-A where appropriate, are shown in ball-and-stick representation. All figures were created using BOBSCRIPT [51] and GL _ RENDER (L. Esser and J. Deisenhofer, unpublished results). Ó FEBS 2002 Citrate synthase from psychrophile to hyperthermophile (Eur. J. Biochem. 269) 6253 due to there being no significant differences between the different structures. 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 A ˚ 3 ). All the CSs have a similar percentage of atoms buried, although the hyperthermophilic PfCS(100) exhibits the highest with 54.5%. Examination of the exposed hydrophobic area shows a more obvious trend. Despite all the archaeal and bacterial CSs having a similar overall ASA, there is quite a difference in hydrophobic exposure when comparing ArCS(31) with the other CSs; the closed conformation of the ArCS(31) has 7854 A ˚ 2 overall exposed hydrophobic area (representing 29% of the total ASA) compared with the closed PfCS(100) with only 4942 A ˚ 2 (18% of total ASA). Thus, on average, ArCS(31) exposes 23 A ˚ 2 per hydrophobic residue compared with 16 A ˚ 2 per hydrophobic residue in PfCS(100). The total amount (A ˚ 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) 1.60 (82) 1.55 (82) 1.49 (90) 0.87 (719) 2.03 (581) TaCS(55) 32% (54%) – 22% (48%) – 0.76 (256) 1.66 (247) 0.76 (104) 1.02 (95) SsCS(85) 34% (55%) – 27% (50%) 59% (76%) – 1.94 (597) 1.53 (245) 1.03 (96) PfCS(100) 40% (58%) – 31% (53%) 42% (62%) 46% (67%) – Table 4. Accessible surface area (ASA) and volume statistics of CSs. CS ArCS(31) (closed) PigCS(37) (open) PigCS(37) (closed) PigCS(37) (closed, N-terminally deleted) TaCS(55) (open) SsCS(85) (open) PfCS(100) (closed) ASA ( · 10 4 A ˚ 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 most interfacial ion pairs but both ArCS(31) and pigCS(37) have more intrasubunit interactions than the TaCS(55) and SsCS(85), and therefore the location of these ionic inter- actions may be particularly relevant. With respect to the ionic interactions at the dimer interface (Fig. 4), the five CSs show considerable variation. Interactions in the pig enzyme are all unique with respect to the other CSs and involve residues near the termini and outer helices (F and L) of the eight-helical sandwich; the central helices of the dimer interface have no ionic interactions associated with them. In marked contrast, the other four CSs all have ionic interactions associated with the two internal helices of the interface (G and M) as well as those involved with the N-terminus and the C- terminal arm. Firstly, with respect to the interfacial helices, one completely conserved ionic interaction in ArCS(31), TaCS(55), SsCS(85) and PfCS(100) is that between a conserved aspartate at the N-terminal end of helix M with a lysine at the C-terminal end of helix M in the other subunit (D205 and K218 in TaCS).Thelysineresidueis not conserved in the pig enzyme. This leads to a single ion pair at each end of helix M in TaCS(55), whereas in SsCS(85) there is an additional salt bridge in close proximity with the first one, between E89 (loop F-G) of one monomer and K108 at the C-terminal end of helix G in the other; thus in SsCS(85) both central helices have ion pairs at either end. In PfCS(100), the G-M interhelical electrostatic interactions are even more pronounced than in either TaCS(55) or SsCS(85), with a five-residue ionic network comprising H93 and R99 (helix G) and D113 in 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 interactions with two four-residue networks (E189,D12, R356¢ and R358¢) and two single ion pairs (E11-R353¢). A cluster of six isoleucines, three from each monomer, was observed in this region for PfCS(100) [18], indicative of a strong hydrophobic interaction in this region. Two of the three isoleucines are conserved in TaCS(55) and SsCS(85), residues 15 and 357, suggesting a similar role in these enzymes. Ionic interactions at the termini Ionic interactions may also be important for prevention of fraying of the N- and C-termini; several of these interactions Table 5. Total number of ion pairs and ion pair networks in CS. A and B refer to the two subunits of the dimer. ArCS(31) PigCS(37) (closed) TaCS(55) SsCS(85) PfCS(100) Total Ion Pairs 52 36 43 45 43 Intra A 21 12 18 21 14 Intra B 21 12 21 18 12 Inter AB 10 12 4 6 17 Intra A networks 3 residue 2 1 0 3 0 4 residue 1 0 1 1 1 5 residue 0 1 0 1 0 Inter AB networks 2 residue 5 7 2 4 9 3 residue 1 2 0 1 0 4 residue 1 1 1 0 1 5 residue 0 0 0 0 1 Ó FEBS 2002 Citrate synthase from psychrophile to hyperthermophile (Eur. J. Biochem. 269) 6255 interlink the two terminal regions, and therefore they may 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 immediately obvious on comparison of the ionic interac- tions that there is considerable difference between the five enzymes. There is a distinct absence of ionic interactions in the loops of the pig enzyme, with the thermophilic and psychrophilic CSs showing more extensive interactions. Many of these ionic interactions are involved with the dimer interface as well as those that interconnect one loop region with another, thus possibly ÔpinningÕ loops together. The total number of ionic interactions present within the loop and dimer interface regions of the five CSs is: ArCS(31) 12, pigCS(37) 8, TpCS(55) 16, SsCS(85) 24 and PfCS(100) 18. As in evident, the thermophilic CSs have the highest number of these interactions, with SsCS(85) having the most. However, the interactions in PfCS(100) tend to be more complex, perhaps affording a greater degree of stabilization in particular areas of the protein structure; also, as detailed below, the PfCS(100) often has the shortest loops, reducing the need for a large number of ionic interactions in those specific regions. The results for individual loops are: Loop E-F. The loop in pigCS(37) is one residue shorter than in ArCS(31), SsCS(85), and PfCS(100), and two residues shorter than TaCS(55), but the loops superimpose Fig. 4. Residues involved in ion-pair interactions at the dimer interface. Spheres are drawn for at the Ca atoms of acidic (red) and basic (blue) side-chains involved in intersubunit ionic interactions. From top to bottom: ArCS(31), PigCS(37), TaCS(55), SsCS(85) and PfCS(100). 6256 G. S. Bell et al. (Eur. J. Biochem. 269) Ó FEBS 2002 well and there are no ionic interactions in pigCS(37) compared with a single ion pair in ArCS(31). TaCS(55) 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). Loop Q-R. This loop is shortest in ArCS(31) and it has already been suggested that the reason for this is that it seems to allow greater accessibility to the active site [15]. However, recent site-directed mutagenesis studies to increase the length of this loop to mimic the situation in the PfCS(100), reveal that the cold activity of the ArCS(31) is not significantly compromised by the mutations [31]. DISCUSSION The determination of the crystal structure of SsCS(85), and its comparison with four other CSs from organisms that essentially span the temperature range over which life exists, have allowed a detailed structural analysis to be performed to investigate the structural mechanisms underlying protein thermal stability in this enzyme. This has been possible because, in general, the 3D structures of the CSs are highly similar and we have therefore not only been able to identify specific differences, but have also succeeded in finding trends in structural changes that correlate with increasing thermostability of the individual proteins. These identified structural differences are mainly concerned with the protein’s compactness, both in general and in the loop regions, and with the nature of the interactions at the dimer interface, possibly indicating that the respective protein thermostabilities are largely determined by these parts of the protein. General compactness The compactness of heat-stable proteins has often been found to be synonymous with their thermostability, and can be described in a number of ways. There is a tendency towards a smaller accessible surface area and volume when 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 (closed) TaCS SsCS PfCS Interface ASA ( A ˚ 2 ) 3403 4934 3154 3363 3698 % of total ASA 21.0 24.0 19.0 19.8 22.6 % polar atoms 34.8 37.8 32.6 32.3 39.2 % nonpolar atoms 65.2 62.2 67.4 67.7 60.8 Hydrogen bonds 44 42 24 28 54 Gap volume ( A ˚ 3 ) 10591 17164 6474 8474 9605 Gap volume index 1.52 1.74 1.03 1.26 1.29 Ó FEBS 2002 Citrate synthase from psychrophile to hyperthermophile (Eur. J. Biochem. 269) 6257 comparison of the ionic interactions in these regions: very few are present in pigCS(37), but a large number occur in the archaeal and bacterial proteins, with the thermophilic CSs, particularly SsCS(85), having the most extensive networks that cross-link loop regions. A significant increase in the number of long-range (in sequence terms) electrostatic interactions is also observed in SsCs(85) and PfCS(100), where they serve to tether different parts of the structure together. This compares with the observations of b-glyco- sidase from S. solfataricus [35], which was shown to have ionic interactions (specifically networks) over the surface of the protein such that they cross-linked areas of surface structure. Similarly, a mutational analysis of the hyperthermostable indoleglycerol-phosphate synthase from T. maritima, in which an ion-pair linking two a-helices was 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 actually across the interface). PfCS(100) also has the additional two four-residue networks near the active site region (with the other archaeal and bacterial enzymes displaying interactions to a lesser degree) as well as the isoleucine cluster [18] which is partly conserved in TaCS(55) and SsCS(85), again suggesting a better hydrophobic packing than with either the mesophilic or psychrophilic enzyme. Finally, the parts of the dimer interface associated with both termini seem to be stabilized by ionic interactions particularly in the PfCS(100), but also to some degree in ArCS(31) and SsCS(85). These conclusions with respect to the ionic interactions at the subunit interface and termini are supported by muta- genesis studies [38]. Analysis of chimeric mutants between the TaCS(55) and PfCS(100), where the large and small domains were swapped, demonstrated that the determinants of thermostability lie mainly with the large (subunit contact) domain, possibly correlating with the trend of increasing ionic interactions that are seen at the subunit interface as the thermostability of the enzyme increases. Additionally, mutagenesis of the PfCS(100) where we have disrupted the ionic network at the subunit interface, and have removed the C-terminal ionic interaction, support the role of these electrostatic bonds in the stability of the enzyme. In nearly all cases, the catalytic parameters of the mutants were not significantly different from the wild-type enzyme, supporting the contention that we have not grossly altered the structure of the enzyme but have merely disrupted stabilizing ionic 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 method of stabilization of proteins at high temperatures, particularly for oligomeric proteins. Recent thermodynamic studies on a mesophilic and thermophilic pair of CheY proteins, have suggested that a reduced change in heat capacity upon unfolding is a possible indicator of thermostability [48], also supported by 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 thermostability. This may be true for small monomeric proteins such as CheY and RnaseH, but for oligomeric proteins that make up > 85% of intracellular proteins, the nature of the oligomer interface is key. Ionic networks at interfaces, however, are not the exclusive means of gaining thermostability, as the tetrameric triosephosphate isomerase structure from P. furiosus has shown [50]. Nevertheless, for the family of CSs presented here, increased ionic interac- tions either between loops or at the dimer interface do appear to correlate with increasing thermostability. The results presented here lay the foundation for a suite of site- directed mutagenesis experiments to investigate the precise role of each of the sets of individual interactions in the five dimeric CSs. Preliminary experiments that remove interac- tions and destabilize the enzyme without affecting catalytic activity have already been successfully carried out, but the real ÔproofÕ of their importance is now to introduce ionic bonds and networks into the less stable CSs to increase their thermostability. 6258 G. S. Bell et al. (Eur. J. Biochem. 269) Ó FEBS 2002 ACKNOWLEDGEMENTS This work was supported by the Biotechnology and Biological Sciences Research Council. REFERENCES 1. Russell, R.J. & Taylor, G.L. (1995) Engineering thermostability: lessons from thermophilic proteins. Curr. Opin. Biotechnol. 6, 370– 374. 2. Rees, D.C. & Adams, M.W.W. (1995) Hyperthermophiles: taking the heat and loving it. Structure Folding Design. 3, 251–254. 3. Danson, M.J. & Hough, D.W. (1998) Structure, function and stability of enzymes from the Archaea. Trends Microbiol. 6, 307– 314. 4. Maes, D., Zeelen, J.P., Thanki, N., Beaucamp, N., Alvarez, M., Thi, M.H., Backmann, J., Martial, J.A., Wyns, L., Jaenicke, R. & Wierenga, R.K. (1999) The crystal structure of triosephosphate isomerase (TIM) from Thermotoga maritima:acomparative thermostability structural analysis of ten different TIM structures. Proteins. 37, 441–453. 5. Sterner, R. & Liebl, W. (2001) Thermophilic adaptation of pro- teins. Crit Rev. Biochem. Mol. Biol. 36, 39–106. 6. Vieille, C. & Zeikus, G.J. (2001) Hyperthermophilic enzymes: Sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65, 1–43. 7. Xiao, L. & Honig, B. (1999) Electrostatic contributions to the stab- ility of hyperthermophilic proteins. J. Mol. Biol. 289, 1435–1444. 8. Kumar, S., Tsai, C.J. & Nussinov, R. (2000) Factors enhancing protein thermostability. Protein Eng. 13, 179–191. 9. Cambillau, C. & Claverie, J M. (2000) Structural and genomic correlates of hyperthermostability. J. Biol. Chem. 275, 32383– 32386. 10. Karshikoff,A.&Ladenstein,R.(2001)Ionpairsandthether- motolerance of proteins from hyperthermophiles: a Ôtraffic ruleÕ for hot roads. Trends Biochem. Sci. 26, 550–556. 11. Kurz, L.C., Drysdale, G.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, 7908–7914. 12. Kurz, L.C., Drysdale, G., Riley, M., Tomar, M.A., Chen, J., Russell, R.J. & Danson, M.J. (2000) Kinetics and mechanism of the citrate synthase from the thermophilic archaeon Thermo- plasma acidophilum. Biochemistry 39, 2283–2296. 13. Evans, C.T., Kurz, L.C., Remington, S.J. & Srere, P.A. (1996) Active site mutants of pig citrate synthase: effects of mutations on the enzyme catalytic and structural properties. Biochemistry 35, 10661–10672. 14. Danson, M.J. & Hough, D.W. (2001) Citrate synthase from hyperthermophilic Archaea. Methods Enzymol. 331, 3–12. 15. Russell,R.J.,Gerike,U.,Danson,M.J.,Hough,D.W.&Taylor, G.L. (1998) Structural adaptations of the cold-active citrate syn- thase from an Antarctic bacterium. Structure 6, 351–361. 16. Remington, S., 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. J. Mol. Biol. 158, 111–152. 17. Russell, R.J., Hough, D.W., Danson, M.J. & Taylor, G.L. (1994) The crystal structure of citrate synthase from the thermophilic archaeon, Thermoplasma acidophilum. Structure. 2, 1157–1167. 18. Russell, R.J., Ferguson, J.M., Hough, D.W., Danson, M.J. & Taylor, G.L. (1997) The crystal structure of citrate synthase from the hyperthermophilic archaeon Pyrococcus furiosus at 1.9 A ˚ resolution. Biochemistry 36, 9983–9994. 19. Connaris, H., West, S.M., Hough, D.W. & Danson, M.J. (1998) Cloning and overexpression in Escherichia coli of the gene encoding citrate synthase from the hyperthermophilic Archaeon Sulfolobus solfataricus. Extremophiles 2, 61–66. 20. Muir, J.M., Russell, R.J., Hough, D.W. & Danson, M.J. (1995) Citrate synthase from the hyperthermophilic Archaeon, Pyrococcus furiosus. Protein Eng. 8, 583–592. 21. Gerike, U., Danson, M.J., Russell, N.J. & Hough, D.W. (1997) Sequencing and expression of the gene encoding a cold-active citrate synthase from an Antarctic bacterium, strain DS2-3R. Eur. J. Biochem. 248, 49–57. 22. Otwinowski, Z. & Minor, W. (1997) Processing of X-ray diffrac- tion data collected in oscillation mode. Method Enzymol. 276, 307– 326. 23. Navaza, J. (1994) AMoRe: an automated package for molecular replacement. Acta Crystallogr. A50, 157–163. 24. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. (1997) Refine- ment of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D53, 240–255. 25. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldaaard, M. (1991) Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A47, 110–119. 26. Nichols, A., Sharp, K.A. & Honig, B. (1991) Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296. 27. Kleywegt, G.J. & Jones, T.A. (1994) Detection, delineation, measurement and display of cavities in macromolecular structures. Acta Crystallogr. D50, 178–185. 28. Barlow, D.J. & Thornton, J.M. (1983) Ion pairs in proteins. J. Mol. Biol. 168, 867. 29. Daggett, V. & Levitt, M. (1993) Protein unfolding pathways explored through molecular dynamics simulations. J. Mol. Biol. 232, 600–619. 30. Hutchinson, E.G. & Thornton, J.M. (1996) PROMOTIF – a program to identify and analyze structural motifs in proteins. Protein Sci. 5, 212–220. 31. Gerike, U., Danson, M.J. & Hough, D.W. (2001) Cold-active citrate synthase: mutagenesis of active-site residues. Protein Eng. 14, 655–661. 32. Lonhienne, T., Gerday, C. & Feller, G. (2000) Psychrophilic enzymes: revisiting the thermodynamic parameters of activation may explain local flexibility. Biochim. Biophys. Acta. 1543, 1–10. 33. Sheridan, P.P., Panasik, N., Coombs, J.M. & Brenchley, J.E. (2000) Approaches for deciphering the structural basis of low temp- erature enzyme activity. Biochim. Biophys. Acta. 1543, 417–433. 34. Thompson, M.J. & Eisenberg, D. (1999) Transproteomic evidence of a loop-deletion mechanism for enhancing protein thermo- stability. J. Mol. Biol. 290, 595–604. 35. Aguilar, C.F., Sanderson, I., Moracci, M., Ciaramella, M., Nucci, R., Rossi, M. & Pearl, L.H. (1997) Crystal structure of the beta- glycosidase from the hyperthermophilic archeon Sulfolobus sol- fataricus: resilience as a key factor in thermostability. J. Mol. Biol. 271, 789–802. 36. Merz, A., Knochel, T., Jansonius, J.N. & Kirschner, K. (1999) The hyperthermostable indoleglycerol phosphate synthase from Ther- motoga maritima is destabilised by mutational disruption of two solvent-exposed salt bridges. J. Mol. Biol. 288, 753–763. 37. Dill, K.A. (1990) Dominant forces in protein folding. Biochemistry 29, 7133–7155. 38. Arnott, M.A., Michael, R.A., Thompson, C.R., Hough, D.W. & Danson, M.J. (2000) Thermostability and thermoactivity of citrate synthases from the thermophilic and hyperthermophilic archaea, Thermoplasma acidophilum and Pyrococcus furiosus. J. Mol. Biol. 304, 657–668. 39. Yip, K.S., Stillman, T.J., Britton, K.L., Artymiuk, P.J., Baker, P.J., Sedelnikova, S.E., Engel, P.C., Pasquo, A., Chiaraluce, R. & Consalvi, V. (1995) The structure of Pyrococcus furiosus gluata- mate dehydrogenase reveals a key role for ion-pair networks in maintaining enzyme stability at extreme temperatures. Structure 3, 1147–1158. Ó FEBS 2002 Citrate synthase from psychrophile to hyperthermophile (Eur. J. Biochem. 269) 6259 [...]... Ladenstein, R (1997) Crystal structure of glutamate dehydrogenase from the ˚ hyperthermophilic eubacterium Thermotoga maritima at 3.0 A resolution J Mol Biol 267, 916–932 42 Pappenberger, G., Schurig, H & Jaenicke, R (1997) Disruption of an ionic network leads to accelerated thermal denaturation of D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima J Mol... Fleming, T.M., Dalby, A.R., Crowhurst, G.S., Bourne, P.C & Littlechild, J.A (1999) Crystal structure of the glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Sulfolobus solfataricus J Mol Biol 291, 651–660 44 Zhang, X., Meining, W., Fischer, M., Bacher, A & Ladenstein, R (2001) X-ray structure analysis and crystallographic refinement of lumazine synthase from the hyperthermophile. ..6260 G S Bell et al (Eur J Biochem 269) 40 Britton, K.L., Yip, K.S., Sedelnikova, S.E., Stillman, T.J., Adams, M.W., Ma, K., Maeder, D.L., Robb, F.T., Tolliday, N., Vetriani, C., Rice, D.W & Baker, P.J (1999) Structure determination of the glutamate dehydrogenase from the hyperthermophile Thermococcus litoralis and its comparison with that from Pyrococcus furiosus J Mol Biol 293, 1121–1132 41 Knapp,... sac7d from Sulfolobus acidocaldarius: contribution of salt bridges to thermostability J Mol Biol 285, 1811–1830 47 Das, R & Gerstein, M (2000) The stability of thermophilic proteins: a study based on comprehensive genome comparison Funct Integr Genomics 1, 76–88 48 Deutschman, W.A & Dahlquist, F.W (2001) Thermodynamic basis for the increased thermostability of CheY from the hyperthermophile Thermotoga... Aquifex aeolicus at ˚ 1.6 A resolution: determinants of thermostability revealed from structural comparisons J Mol Biol 306, 1099–1114 Ó FEBS 2002 45 Elcock, A.H (1998) The stability of salt bridges at high temperatures: implications for hyperthermophilic proteins J Mol Biol 284, 489–502 46 de Bakker, P.I., Hunenberger, P.H & McCammon, J.A (1999) Molecular dynamics simulations of the hyperthermophilic... Biochemistry 40, 13107–13113 49 Hollien, J & Marqusee, S (1999) A thermodynamic comparison of mesophilic and thermophilic ribonucleases H Biochemistry 38, 3831–3836 50 Walden, H., Bell, G.S., Russell, R.J., Siebers, B., Hensel, R & Taylor, G.L (2001) Tiny TIM: a small, tetrameric, hyperthermostable triosephosphate isomerase J Mol Biol 306, 745–757 51 Esnouf, R.M (1997) BOBSCRIPT: an extensively modified... & Taylor, G.L (2001) Tiny TIM: a small, tetrameric, hyperthermostable triosephosphate isomerase J Mol Biol 306, 745–757 51 Esnouf, R.M (1997) BOBSCRIPT: an extensively modified version of MOLSCRIPT that includes greatly enhanced coloring capabilities J Mol Graph Model 15, 132–143 . Stepwise adaptations of citrate synthase to survival at life’s extremes From psychrophile to hyperthermophile Graeme S. Bell 1 ,. regions. Keywords: citrate synthase; Sulfolobus; citrate synthase; thermostability; crystal structure; ion networks. Comparative structural analysis of the