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