Structural adaptation to low temperatures ) analysis of the subunit interface of oligomeric psychrophilic enzymes Daniele Tronelli1, Elisa Maugini1, Francesco Bossa1 and Stefano Pascarella1,2 ` Dipartimento di Scienze Biochimiche ‘A Rossi Fanelli’, Universita degli Studi di Roma ‘La Sapienza’, Rome, Italy ` Centro Interdipartimentale di Ricerca per la Analisi dei Modelli e dell’Informazione nei Sistemi Biomedici (CISB), Universita degli Studi di Roma ‘La Sapienza’, Rome, Italy Keywords cold-adapted enzymes; electrostatic and hydrophobic interactions; interface; protein quaternary structure; psychrophiles Correspondence S Pascarella, Dipartimento di Scienze ` Biochimiche, Universita ‘La Sapienza’, P le A Moro 5, 00185 Rome, Italy Fax: +39 06 49917566 Tel: +39 06 49917574 E-mail: Stefano.Pascarella@uniroma1.it Website: http://schubert.bio.uniroma1.it/ (Received June 2007, revised 12 July 2007, accepted 13 July 2007) doi:10.1111/j.1742-4658.2007.05988.x Enzymes from psychrophiles show higher catalytic efficiency in the 0–20 °C temperature range and often lower thermostability in comparison with meso ⁄ thermophilic homologs Physical and chemical characterization of these enzymes is currently underway in order to understand the molecular basis of cold adaptation Psychrophilic enzymes are often characterized by higher flexibility, which allows for better interaction with substrates, and by a lower activation energy requirement in comparison with meso ⁄ thermophilic counterparts In their tertiary structure, psychrophilic enzymes present fewer stabilizing interactions, longer and more hydrophilic loops, higher glycine content, and lower proline and arginine content In this study, a comparative analysis of the structural characteristics of the interfaces between oligomeric psychrophilic enzyme subunits was carried out Crystallographic structures of oligomeric psychrophilic enzymes, and their meso ⁄ thermophilic homologs belonging to five different protein families, were retrieved from the Protein Data Bank The following structural parameters were calculated: overall and core interface area, characterization of polar ⁄ apolar contributions to the interface, hydrophobic contact area, quantity of ion pairs and hydrogen bonds between monomers, internal area and total volume of non-solvent-exposed cavities at the interface, and average packing of interface residues These properties were compared to those of meso ⁄ thermophilic enzymes The results were analyzed using Student’s t-test The most significant differences between psychrophilic and mesophilic proteins were found in the number of ion pairs and hydrogen bonds, and in the apolarity of their subunit interface Interestingly, the number of ion pairs at the interface shows an opposite adaptation to those occurring at the monomer core and surface Many terrestrial environments present physical and chemical conditions that can be defined as extreme from a human point of view Among these, permanent cold environments are the most common In fact, about 70% of the earth’s surface is covered by the oceans, whose temperature is constantly at 4–5 °C below a depth of 1000 m, regardless of the latitude Moreover, polar regions constitute a further 15% of the earth, and there are also alpine regions and glaciers Ectothermic organisms that have colonized such environments are called psychrophiles, and, considering their spread, represent a considerable component of the biosphere, in terms of species diversity and biomass Psychrophilic organisms include eubacteria, archaea, protozoa, fungi and multicellular eukaryotes such as algae, invertebrates and fish [1,2] Abbreviations CS, citrate synthase; MDH, malate dehydrogenase; TIM, triose phosphate isomerase FEBS Journal 274 (2007) 4595–4608 ª 2007 The Authors Journal compilation ª 2007 FEBS 4595 Analysis of the oligomeric psychrophilic interface D Tronelli et al To survive at temperatures close to the freezing point of water, psychrophiles have evolved some important cellular adaptations, including mechanisms to maintain membrane fluidity [3,4], synthesis of coldacclimation proteins [5], freeze tolerance strategies [6], and cold-active enzymes Psychrophilic enzymes are of great interest in the scientific community, and are currently under study to characterize their physical and chemical properties in an attempt to understand the molecular basis of cold adaptation Low temperatures have a negative effect on enzyme kinetics: any decrease in temperature results in an exponential decrease in reaction rate For example, lowering the temperature by 10 C° causes a two-fold to four-fold decrease in enzyme activity [1,7] Therefore, enzymes from psychrophiles show high catalytic efficiency in the 0–20 °C temperature range, temperatures at which counterparts from mesophilic or thermophilic organisms not allow adequate metabolic rates to support life or cellular growth Such high activity balances the cold-induced inhibition of reaction rates However, the structure of cold-adapted enzymes is also heat-labile Indeed, low stability at moderate temperatures (usually > 40 °C) is the other peculiar characteristic of psychrophilic enzymes [8,9] This trend was revealed by calorimetric analysis of residual enzyme activities after incubation at increasing temperatures (it should be pointed out, however, that the loss of activity at moderate temperatures might not be always directly related to the loss of enzyme structure) It is generally believed that cold adaptation results from a combination of lack of selective pressure for thermostability and strong selection for high activity at low temperatures [1] Psychrophilic enzymes are often characterized by high flexibility [10], which allows better interaction with substrates, and by lower activation energy requirements in comparison with their mesophilic and thermophilic counterparts Hence, the presence of high flexibility could explain both thermolability and high catalytic efficiency at low temperatures [11] The higher structural flexibility of psychrophilic enzymes, as compared to their mesophilic and thermophilic counterparts, could be the result of a combination of several features: weakening of intramolecular bonds (fewer hydrogen bonds and salt bridges as compared to mesophilic and thermophilic homologs have been shown); a decrease in compactness of the hydrophobic core; an increase in the number of hydrophobic side chains that are exposed to the solvent; longer and more hydrophilic loops; a reduced number of proline and arginine residues; and a higher number of glycine residues [12–15] However, each protein family adopts its own strategy to increase 4596 its overall or local structural flexibility by using one or a combination of these structural modifications Earlier studies on the structural adaptation of extremophilic enzymes [16–19] were based on comparative analysis, also using homology modeling in cases where no experimental three-dimensional structures were available [20,21] These approaches could give valuable information on rules to be followed by protein engineers to produce modified enzymes with suitable features for biotechnological applications [22] In fact, because of their high catalytic efficiency at low temperatures, psychrophilic enzymes are investigated for their high potential economic benefit: in particular, they could be utilized in industrial processes as energy savers, and in the detergent industry as additives [23,24] Also, the possibility of selecting and rapidly inactivating these enzymes, due to their high thermolability, makes psychrophilic enzymes extremely useful in biomolecular applications [25] Previous comparative studies investigated factors governing cold adaptation occurring in the protein structure core, in the enzyme active site, and in the overall protein structure However, although the molecular adaptation of enzymes to extreme conditions has been intensively studied, not very much is known about the adaptations that have occurred at the interface of oligomeric enzymes Even less is known regarding the adaptation of the psychrophilic interface of oligomeric enzymes Intersubunit interactions have special importance in the stability of oligomeric psychrophilic enzymes and their function Indeed, interface regions between protein monomers are mainly responsible for the maintenance of the quaternary structure in oligomeric enzymes The hydrophobic interaction is at the base of the process of folding and the stabilization of protein association [26,27] The hydrophobic interaction occurs when apolar residues aggregate in an aqueous environment, achieving a loss in free energy that stabilizes the protein structure During association of monomers, hydrophobic residues are buried in the interface region, minimizing the number of thermodynamically unfavorable solute–solvent interactions Other important features, such as interface extension, residue packing, hydrogen bonds, salt bridges and internal cavities, can play a significant role in the stability of the quaternary structure in coldadapted enzymes Our work is aimed at detecting the structural variation related to the cold adaptation of the subunit interface of oligomeric psychrophilic enzymes To our knowledge, this is the first study entirely focused on the analysis of the molecular adaptations that have occurred at the level of subunit interfaces of psychro- FEBS Journal 274 (2007) 4595–4608 ª 2007 The Authors Journal compilation ª 2007 FEBS D Tronelli et al Analysis of the oligomeric psychrophilic interface thermophilic and six hyperthermophilic structures, accounting for a total of 35 oligomeric enzymes corresponding to a total of 30 pairwise comparisons The proteins belong to five families: citrate synthase (CS) [17], triose phosphate isomerase (TIM) [28], malate dehydrogenase (MDH) [18], alkaline phosphatase [29] and glyceraldehyde-3-phosphate dehydrogenase [30] In total, 21 incomplete side chains distributed in six proteins (1cer, 1a59, 1ixe, 1ew2, 4gpd, 1bmd) were rebuilt as described in Experimental procedures The careful reconstruction of the 21 incomplete side chains was necessary to include in the working dataset as much information as possible Ideally, only complete coordinate sets should be used, to avoid any artefact However, the philic enzymes Some of the key questions are as follows Are these interfaces significantly different from the interfaces of mesophilic enzymes? Which structural features are mostly variable? Are the interface adaptations different from those occurring at the level of monomer hydrophobic core and surface? The answers to these questions can give indications about aspects of protein–protein interaction at low temperatures and suggest rules for interface engineering Results The main dataset utilized for the analysis (Table 1) contained five psychrophilic, 20 mesophilic, four Table List of enzymes used in the work (main dataset) AP, alkaline phosphatase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PDB, Protein Data Bank Family Source organism Growth temperature (°C) CS Antarctic bacterium DS2–3R Escherichia coli Sulfolobus solfataricus Thermus thermophilus Pyrococcus furiosus Homarus americanus Leishmania mexicana Oryctolagus cuniculus Achromobacter xylosoxidans Trypanosoma cruzi Escherichia coli Bacillus stearothermophilus Thermus aquaticus Thermotoga maritima Thermus thermophilus Aquaspirillium arcticum Sus scrofa Thermus flavus Pandalus borealis Homo sapiens Escherichia coli Vibrio marinus Saccharomices cerevisiae Homo sapiens Gallus gallus Oryctolagus cuniculus Trypanosoma cruzi Caenorhabditis elegans Escherichia coli Entamoeba histolytica Plasmodium falciparum Leishmania mexicana Trypanosoma brucei Bacillus stearothermophilus Thermotoga maritima 37 85 85 100 20 37 37 30 37 37 55 72 85 85 37 72 37 37 15 27 37 37 37 37 22 37 37 37 37 41 55 85 GAPDH MDH AP TIM a Structure resolution ˚ (A) Identitya (%) PDB ID Sequence length (monomer) No of subunits 2.09 2.20 2.70 2.30 1.90 2.80 2.80 2.40 1.70 2.75 1.80 1.80 2.50 2.50 2.60 1.90 2.40 1.90 1.92 1.82 1.75 2.65 1.90 2.80 1.80 1.50 1.83 1.70 2.60 1.50 2.20 1.83 1.80 2.40 2.85 Ref 31 32 41 40 Ref 58 73 45 54 65 53 49 50 48 Ref 51 62 Ref 42 33 Ref 43 41 41 41 41 46 65 41 38 39 39 42 39 1a59 1k3p 1o7x 1ixe 1aj8 4gpd 1a7k 1j0x 1obf 1qxs 1gad 1gd1 1cer 1hdg 1vc2 1b8p 5mdh 1bmd 1k7h 1ew2 1ed9 1aw2 1ypi 1hti 1tph 1r2r 1tcd 1mo0 1tre 1m6j 1ydv 1amk 1tpf 2btm 1b9b 377 426 379 376 371 333 358 332 335 359 330 334 331 332 331 327 333 327 476 479 449 255 248 248 245 248 248 249 255 261 246 250 250 250 252 2 2 4 4 4 4 4 2 2 2 2 2 2 2 2 2 2 Percentage residue identity to the psychrophilic reference (Ref.) homolog sequence FEBS Journal 274 (2007) 4595–4608 ª 2007 The Authors Journal compilation ª 2007 FEBS 4597 Analysis of the oligomeric psychrophilic interface D Tronelli et al 21 side chains represent in this case only 0.3% of all the interface residues contained in the working dataset Consequently, even if, in the worst case, the reconstruction was not correct, the potential effect on the final statistics would be negligible The average sequence identity was calculated between enzyme pairs of the same family, and gave a value of 49.0% over all protein families The differences in structural features observed between the psychrophilic and mesophilic enzymes were compared with the differences between the same properties calculated from an oligomeric mesophilic reference dataset (Table 2) This nonredundant reference dataset contained 148 protein structures belonging to 43 oligomeric enzyme families, with an average sequence identity between enzyme pairs of the same family of 52.4% The dataset generated a total of 514 pairwise comparisons The whole dataset included 10 mainly-a domains, two mainly-b domains, and 53 a–b domains The taxonomic composition of the Table List of mesophilic enzymes used in the reference dataset Family Protein Data Bank IDs 01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 1deh, 1p1r, 1p0f, 1e3i, 1cdo 1d2c, 1r8x, 1r74 1get, 1bwc, 1onf, 1gxf 1xwk, 1b4p, 1c72, 1dug 7aat, 1ajs, 1yaa, 1art 1dsu, 3rp2 1m9n, 1pkx 1a4i, 1b0a 1ijl, 1pp2, 1vip, 1cL5, 1kvo 1gqn, 1sfl 1a7a, 1v8b 2tdm, 1tjs, 1sej, 2tsr, 1hw4, 1f28 1qm5, 1xoi, 1lwo, 1ygp 2crk, 1vrp, 1qh4, 1g0w 1cm7, 1cnz 1lld, 1ez4, 9ldb, 1i0z, 1v6a 1qo5, 1fba, 1epx, 1f2j, 1zah, 1a5c 1xim, 1s5n, 1qt1 1aq6, 1jud 1isa, 3sdp, 2awp, 2a03, 1uer, 1y67 1hL4, 1q0e, 1xso, 1to5, 1f1g 1bxk, 1g1a, 1ket, 1r66 1csh, 2cts 1bsr, 1z7x, 1rra 1ade, 1p9b 2ar7, 3adk, 4ake 1fro, 1fa8 1imb, 2bji 1e98, 1tmk 1ebh, 1te6, 1pdz, 1e9i, 1iyx 1qr2, 1qrd, 1dxq, 1d4a 1r2f, 1uzr 1ivy, 1wpx 1trk, 1r9j, 1qgd 1a06, 1h1w 1tc1, 1pzm, 1hgx 1grv, 1z7g, 1cjb 1pfk, 1zxx 1opy, 8cho 1dfo, 1ls3, 2a7v, 1eji 1ew2, 1ed9 1ypi, 1hti, 1tph, 1r2r, 1tcd, 1mo0, 1tre, 1m6j, 1ydv, 1amk, 1tpf 1a7k, 1j0x, 1obf, 1qxs,1gad Alcohol dehydrogenase Glycine-N-methyltransferase Glutathione-disulfide reductase Glutathione transferase Aspartate transaminase Serine protease AICAR transformylase Methylenetetrahydrofolate dehydrogenase Phospholipase A2 3-Dehydroquinate dehydratase Adenosylhomocysteinase Thymidylate synthase Phosphorylase Creatine kinase 3-Isopropylmalate dehydrogenase L-lactate dehydrogenase Fructose bisphosphate aldolase Xylose isomerase 2-Haloacid dehalogenase Superoxide dismutase (iron ⁄ manganese) Superoxide dismutase (copper ⁄ zinc) dTDP-glucose-4,6-dehydratase Citrate synthase Ribonuclease Adenylosuccinate synthase Adenylate kinase Lactoylglutathione lyase Inositol phosphate phosphatase dTMP kinase Phosphopyruvate hydratase NADPH dehydrogenase (quinone) Ribonucleoside diphosphate reductase Carboxypeptidase C Transketolase Protein kinase Hypoxanthine phosphoribosyltransferase (dimeric) Hypoxanthine phosphoribosyltransferase (tetrameric) 6-Phosphofructokinase Steroid D-isomerase Glycine hydroxymethyltransferase Alkaline phosphatase Triose phosphate isomerase Glyceraldehyde-3-phosphate dehydrogenase 4598 FEBS Journal 274 (2007) 4595–4608 ª 2007 The Authors Journal compilation ª 2007 FEBS D Tronelli et al Analysis of the oligomeric psychrophilic interface Table Statistical analysis of the differences in the number of interface ion pairs and hydrogen bonds The electrostatic interaction quantities showed were normalized by mean interface surface psy, psychrophilic; mes, mesophilic; therm, thermophilic Interface structural property Strong ion pairs psy versus mes psy versus mes Weak ion pairs psy versus mes psy versus mes Total ion pairs psy versus mes psy versus mes Hydrogen bonds psy versus mes psy versus mes t-value P-value (%) + therm 2.69 0.85 7.4 · 10)1 39.57 + therm 5.85 4.62 8.75 · 10)7 4.83 · 10)4 + therm 6.12 4.20 1.86 · 10)7 3.13 · 10)3 + therm ) 2.60 ) 3.02 9.6 · 10)1 2.6 · 10)1 0.7 Salt bridges per interface residue dataset included species belonging to the prokaryotes and eukaryotes (comprising protozoa and multicellular organisms such as invertebrates, fish, and mammals) The significance of the observed differences in structural properties, calculated as described in Experimental procedures, was measured by a t-value t-values ¼ +1.96 or t-values ¼ )1.96 with a number of degrees of freedom > 500 correspond to a P-value ¼ 5% that the null hypothesis is true This value represents the significance threshold adopted in our analyses The structural properties tested at the subunit interface were: interface and core interface extension; number of ion pairs and hydrogen bonds; fraction of apolar contact surface; atomic packing; volume and internal surface area of interface cavities; and fraction of apolar surface in the interface and in the core interface Whenever applicable, the properties were normalized by interface extension and number of interface residues However, as there was no difference between the two normalizations, only the former is considered here Table shows the t-values and the percentage probabilities relative to the structural differences in the number of strong, weak and total ion pairs and hydrogen bonds between psychrophilic and meso ⁄ thermophilic homologs The t-value relative to the strong ion pairs at the interface indicates a significant increase in these electrostatic interactions in the psychrophilic enzymes as compared to the mesophilic enzymes The same results were found for weak and total ion pairs The significance of this trend decreased in the comparison of psychrophilic with both mesophilic and thermo- 0.6 0.5 0.4 0.3 0.2 0.1 CS TIM AP GAPDH MDH Fig Normalized mean number of psychrophilic (white), mesophilic (gray) and thermophilic (black) interface total ionic interactions, calculated from protein structures for each family of the main dataset The number of total ion pairs at the interface was normalized by the number of residues composing the interface philic enzymes Figure shows the normalized mean number of total ionic interactions at the interface, calculated from psychrophilic, mesophilic and thermophilic protein structures for each family of the main dataset The number of total ion pairs at the interface was normalized by the number of residues composing the interface For each one of the five enzyme families considered, the number of ion pairs was higher in psychrophilic proteins than in mesophilic ones, whereas in three cases out of four (atomic coordinates of thermophilic AP are not available), the number of ion pairs was higher in thermophilic proteins than in mesophilic ones, with the exception of the TIM enzyme family No significant trend was found in the comparison of strong ion pairs between psychrophilic and meso ⁄ thermophilic homologs (Table 3) The t-value for hydrogen bonds (Table 3) showed a significant decrease in these interactions at the oligomeric interface of psychrophilic enzymes when compared to their mesophilic counterparts The trend held for the number of hydrogen bonds per unitary surface and per interface residue The inclusion of thermophilic enzymes in the comparison increased both tendencies The volume of the interface internal cavities, as well as the amino acid packing at the interface, did not show any measurable difference between psychrophilic and mesophilic proteins, and therefore the results are not shown Psychrophilic enzymes (Table 4) showed a significant decrease in the apolar fraction of interface in comparison with their mesophilic counterparts (t-value of ) 2.12, P ¼ 3.44) The trend was strengthened by the inclusion of thermophilic enzymes in the comparison Figure shows the percentage of apolar interface, FEBS Journal 274 (2007) 4595–4608 ª 2007 The Authors Journal compilation ª 2007 FEBS 4599 Analysis of the oligomeric psychrophilic interface D Tronelli et al Table Statistical analysis of the differences in the percentage of apolar interface, the hydrophobic contact area of interface, and the percentage of overall interface area psy, psychrophilic; mes, mesophilic; therm, thermophilic Table Statistical validation of the reference mesophilic dataset Interface structural property % not significanta Interface structural property Strong ion pairs Weak ion pairs Total ion pairs Hydrogen bonds Percentage of apolar interface Hydrophobic contact area in the interface Percentage of overall interface area Percentage of apolar core interface Percentage of overall core interface area 79.1 84.7 82.8 85.3 74.0 83.7 78.1 85.5 84.5 t-value ) 2.12 ) 3.29 3.44 1.1 · 10)1 ) 2.34 ) 3.55 Percentage of apolar interface psy versus mes psy versus mes + therm Hydrophobic contact area in the interface psy versus mes psy versus mes + therm Percentage of overall interface area psy versus mes psy versus mes + therm Percentage of apolar core interface psy versus mes psy versus mes + therm Percentage of overall core interface area psy versus mes psy versus mes + therm P-value (%) 1.96 4.0 · 10)2 ) 1.01 ) 2.19 31.29 2.90 1.66 1.00 9.75 31.77 ) 1.11 ) 1.83 26.75 6.78 Apolar interface area / Total interface area 80 70 60 50 40 30 20 10 CS TIM AP GAPDH MDH Fig Percentage of psychrophilic (white), mesophilic (gray) and thermophilic (black) apolar interface, calculated from protein structures for each family of the main dataset Each apolar interface area was normalized by the total interface area calculated from psychrophilic, mesophilic and thermophilic protein structures for each family of the main dataset Each apolar interface area was normalized by the total interface area With the exception of the CS enzyme family, the percentage of apolar interface was lower in cold-adapted enzymes than in mesophilic ones, whereas in three cases out of four, the percentage of apolar interface was higher in thermophilic proteins than in mesophilic ones, with the exception of the MDH enzyme family A similar significant trend was 4600 a Fraction of randomized trials that resulted in a nonsignificant t-value found for the hydrophobic contact area at the interface (Table 4) No significant trend was detected in the comparison of the percentage of overall interface area between psychrophilic and mesophilic proteins However, this trend became significant upon inclusion of thermophilic enzymes (t-value of ) 2.19) in the comparison (Table 4) Psychrophilic enzymes (Table 4) did not show significant variation of the core interface area and of the core interface apolar atomic composition when compared to mesophilic counterparts and to meso ⁄ thermophilic counterparts Table reports the results of the validation of the reference mesophilic dataset to exclude potential statistical bias on the t-tests applied to the psychrophilic proteins The number of randomized tests out of 1000 trials that resulted in a nonsignificant t-value were recorded for each structural properties On average, a structural property obtained a nonsignificant t-value in 820 out of 1000 randomized tests This suggests that the reference dataset appropriately represents the mesophilic proteins in the main dataset Discussion This research was aimed at elucidating the adaptations that have occurred at the interface of oligomeric enzymes synthesized by psychrophilic microorganisms We analyzed the structural differences between the oligomeric interfaces of psychrophilic and meso ⁄ thermophilic homologs Psychrophilic oligomeric enzymes must maintain high structural flexibility and, at the same time, the correct quaternary structure Hence, the comparison was focused on those physicochemical characteristics of the interface that are related to structural stability, namely: apolar contact surface; number of ionic interactions and hydrogen bonds; atomic packing; presence of cavities; percentage of apolar FEBS Journal 274 (2007) 4595–4608 ª 2007 The Authors Journal compilation ª 2007 FEBS D Tronelli et al surface; and interface and core interface extension It could be argued that cold interface adaptations may be similar to those occurring at the level of the monomer hydrophobic core and surface, which have already been thoroughly studied However, it was previously shown that hydrophobic interactions play a less relevant role in protein binding than in protein folding [31] Furthermore, in 1997, Xu and colleagues found that hydrophilic bridges, established between charged or polar atoms, can result in stronger stabilization in monomer–monomer binding than in the interior of monomers, due to the different environments to which such interactions are exposed [32] All of these findings prompted one of our initial questions: are the psychrophilic interface adaptations different from those occurring at the level of the protein hydrophobic core and surface? We believe that our comparative analysis can correctly answer this question However, one of the criticisms of such comparative studies is the way in which the statistical significance of the differences is assessed Therefore, in this analysis, for each structural feature, a robust reference distribution of the differences observed in the comparison of 148 mesophilic protein interfaces belonging to 43 oligomeric enzyme families was calculated Such a distribution answers the question of what difference should be expected for the structural property if the interfaces of two homologous mesophilic enzymes were compared The t-test should then establish whether the magnitude of the differences detected between the psychrophilic enzyme and the meso ⁄ thermophilic counterparts is significantly different from that expected from a mesophilic–mesophilic comparison The features that showed a significant difference from the reference sample were: (a) increase in the number of ionic interactions; (b) decrease in the number of hydrogen bonds; (c) decrease in the fraction of apolar interface; and (d) decrease in the apolar contact surface However, the cold-adapted enzymes considered here also showed a significant decrease in the overall interface area, when compared with both mesophilic and thermophilic homologs, but this trend disappeared after comparison with the sole mesophilic oligomers (Table 4) This suggests that the thermophilic enzymes need a wider interface to maintain oligomer stability, but psychrophilic counterparts obtain no advantage from the shrinkage of the interface extension The increase in the number of strong, weak and total ion pairs at the interface of psychrophilic enzymes is important in maintaining the quaternary structure, whereas the strength of hydrophobic interactions is diminished at low temperature It should be considered Analysis of the oligomeric psychrophilic interface that, at moderate temperatures, hydrophobic interactions are the most relevant forces for the preservation of enzyme structure On the other hand, the trend for apolar surfaces to interact with other apolar surfaces, rather than with water, decreases at low temperatures, because solvation of nonpolar surface is thermodynamically favored at low temperatures This effect can lead to cold-induced denaturation, particularly of the most hydrophobic proteins, as well as oligomeric enzymes [33] Moreover, it was previously observed [34] that psychrophilic enzymes are affected by the weakening of hydrophobic interactions at low temperatures Hence, in these conditions, hydrophobic interactions are less relevant in maintaining the quaternary structure, and this phenomenon is reflected in the significant decrease in apolar components at the interface that we have found for oligomeric psychrophilic enzymes Moreover, our analysis underlines a significant decrease in the interface apolar contact area when comparing psychrophilic and mesophilic enzymes or psychrophilic and mesophilic plus thermophilic enzymes The same trend was previously found in the analysis of hydrophobicity in core residues of psychrophilic proteins [35] Indeed, buried residues in psychrophilic enzymes show weaker hydrophobicity than those in their mesophilic homologs, making the protein interior less compact and more flexible Therefore, a lower degree of hydrophobic interaction renders the role of salt bridges more relevant in stabilizing the protein quaternary structure of oligomeric cold-adapted enzymes, particularly if we consider that, as the formation of ion pairs is an exothermic electrostatic interaction, they are particularly strong at low temperatures A similar hypothesis was put forward by Russell et al [17] with regard to coldactive CS in comparison with the hyperthermophilic homolog The authors observed an increase in psychrophilic intramolecular ion pairs, but paradoxically also a reduced number of interface ion pairs They concluded that a large number of intramolecular ion pairs may serve to counteract the reduced thermodynamic stabilization due to hydrophobic interaction at low temperatures, preventing the cold denaturation of psychrophilic CS However, the psychrophilic enzyme showed a reduction in the extent of intersubunit ion pairs in comparison with the hyperthermophilic homolog Another comparative study of psychrophilic MDH and its thermophilic counterpart revealed the same trend: the cold-adapted enzyme had more intrasubunit and fewer intersubunit ion pairs [18] This is in apparent contrast with our results Indeed, in our analysis, we observed a significant increase in interface ion pairs for psychrophilic enzymes when compared exclusively to mesophilic homologs (Fig 1) The significance of FEBS Journal 274 (2007) 4595–4608 ª 2007 The Authors Journal compilation ª 2007 FEBS 4601 Analysis of the oligomeric psychrophilic interface D Tronelli et al this trend decreased when psychrophilic enzymes were compared to mesophilic plus thermophilic proteins This suggests that, in general, psychrophilic enzymes establish, on average, a number of ionic interactions at the subunit interface that is slightly lower or comparable with that observed in thermo ⁄ hyperthermophilic counterparts, but definitely higher than in mesophilic proteins It should be noted, therefore, that although psychrophilic and thermophilic enzymes are adapted to opposite temperature conditions, the structural adaptation strategies, relying on ionic interactions, appear to be similar A consistently higher number of interface salt bridges, in comparison to that present in mesophilic oligomers (Fig 1), could therefore be useful to improve the cohesion between monomers and to avoid both cold-induced and heat-induced unfolding in psychrophilic and thermophilic enzymes, respectively These results underline the fact that the comparative analyses for determining the structural differences related to thermal adaptation of psychrophiles should always include both mesophilic and thermophilic counterparts to ensure that the significant structural differences are appreciated The role of hydrogen bonds in psychrophilic protein adaptation is widely accepted Previous studies showed that cold-adapted enzymes have fewer total hydrogen bonds than their meso ⁄ thermophilic homologs Accordingly, we found a significant decrease in the number of hydrogen bonds at the interface of psychrophilic oligomers when compared with mesophilic enzymes This trend increased when thermo ⁄ hyperthermophilic enzymes were included in the working dataset Our findings underline the role of this kind of electrostatic interaction in determining greater stability of the quaternary structure in mesophilic and thermophilic proteins; in heat-labile cold-adapted enzymes, the number of interface hydrogen bonds is lower At the moment, no satisfactory mechanistic explanation for the decrease in the number of interface hydrogen bonds has been proposed Other structural features analyzed did not show any significant trend In particular, no significant trend was detected in the comparison of the percentage of core interface area and in the comparison of the core interface apolar atomic composition between psychrophilic and mesophilic proteins These results, showing that the percentage of core interface area does not show a significant difference, could be interpreted in the light of the work of Bahadur et al [36] These authors studied the subunit interfaces of 122 homodimers, and showed that the distribution of the area between the rim and core interface varies widely from one oligomer 4602 to another This could lead to a large value for the standard deviation of distributions of the rim and core interface extension, and, as a consequence, could lead to the small t-value An analysis of amino acid packing in mesophilic and thermophilic enzymes was performed by Karshikoff & Ladenstein [37] to determine the role of packing density in thermostability They concluded that mesophilic and thermophilic proteins not differ in the degree of packing Likewise, our analysis did not find any measurable difference in the amino acid packing at the interface of psychrophilic enzymes and in the total volume of internal interface cavities, and for this reason the results are not shown In conclusion, the answers to our initial questions reveal that the interfaces of oligomeric psychrophilic enzymes are significantly different from those of their mesophilic and thermophilic homologs The most variable features are the increase in the number of ionic interactions, the decrease in the number of hydrogen bonds, the decrease in the fraction of apolar interface, and the decrease in the apolar contact surface Therefore, the structural adaptations observed are similar to those occurring at the monomer core and surface, with the notable exception of the increase in the number of ionic interactions Indeed, it has been reported that, in general, the flexibility of the monomeric structure is often achieved via a reduction of electrostatic interactions in psychrophiles Our results suggest that the interfaces of oligomeric psychrophilic enzymes need to be stabilized by the introduction of additional ion pairs It should be considered that relatively few structures of oligomeric psychrophilic enzymes are presently available Therefore, although the conclusions reported here are correct from the statistical point of view, particularly considering the robust testing procedure adopted, the results may change with the availability of significantly more data To confirm the results described here, the analysis should be repeated when more structures of psychrophilic oligomeric enzymes are available Several other analyses of the structural basis of enzyme cold adaptation have recently appeared in the literature For example, Jahandideh et al [38] reported a statistical analysis of the sequence and structural parameters enhancing adaptation of proteins to low temperatures Their work was aimed at the detection of variations in structural properties for the entire enzyme molecule, without any focus on the subunit interface Indeed, they considered both monomeric and oligomeric enzymes in their dataset, which included 13 pairs of homologous psychrophilic and mesophilic FEBS Journal 274 (2007) 4595–4608 ª 2007 The Authors Journal compilation ª 2007 FEBS D Tronelli et al proteins The structural properties tested were residue frequencies, helical and tight turn content, backbone hydrogen bonds, and disulfide bonds They assessed the significance of the differences between the average values of each structural property taken into account, calculated over 13 psychrophilic and mesophilic homologs Moreover, they utilized a t-test with a significance threshold lower than that which we used in our analysis, corresponding to a P-value equal to 0.1 with 24 degrees of freedom These differences make it difficult to relate the results presented here to those reported by Jahandideh et al [38], as well as to those of previous analyses Indeed, to our knowledge, the analysis described here is the first systematic study of cold adaptation at the level of the subunit interface Nonetheless, Jahandideh et al [38] came to the conclusion that the number of hydrogen bonds and, generally, the number of electrostatic interactions are decreased in psychrophilic proteins It should be noted that, although each enzyme family has its own strategy to increase flexibility by using one or a combination of the above alterations in structural features [1], even with a relatively limited number of psychrophilic enzyme structures available, some general trends involved in the maintenance of both structural flexibility and quaternary stucture in oligomeric psychrophilic enzymes can be appreciated by comparative analysis In conclusion, this kind of comparative analysis can contribute to the elucidation of structural determinants of adaptation of proteins to extreme conditions, and can give useful hints on how to modulate, through protein engineering, the stability and catalytic features of enzymes of biotechnological interest Experimental procedures Analysis of the oligomeric psychrophilic interface psychrophilic sequence were considered Only unique structures were retrieved, and when there were alternative structures for the same protein, only those displaying the best resolution and without point mutations were collected Proteins from plants were not taken into consideration, owing to the ambiguous definition of ‘optimum temperature’ for such organisms In order to assess the structural similarity within each collected family, we performed a structural alignment using the ce-mc program [42] Sequences of the selected proteins were aligned to each psychrophilic homolog The alignments were then manually corrected by inspection of the superimposed structures All the programs were written in PERL language and run under IRIX 6.5 or RED HAT ENTERPRISE LINUX 4.0 operating systems Crystallographic structure quality assessments ˚ All structures showing a resolution worse than 2.85 A were excluded from the main dataset All the incomplete interface side chains were rebuilt using the program biopolymer of the insightii package (version 2005; Accelrys, San Diego, CA, USA) The side chain rotamer displaying the lowest nonbond energy was kept and treated as experimental Ligands (cofactors, inhibitors, substrate analogs, etc.) and solvent molecules were always removed from the structures The quality assessment of the crystallographic structures was carried out using procheck software [43] Only two structures, 4gpd and 1ypi, did not pass the procheck stereochemical quality check, showing an overall average G-factor below ) 0.5, which is the lowest threshold for acceptable quality For these, an energetic minimization was performed using the program modeller [44] of the insightll package, using the highest optimization level After the energy minimization, the quality of the two structures was evaluated using the program prosaII [45] The refined structures showed overall average G-factors of ) 0.47 and ) 0.02, respectively Collection of main dataset The crystallographic structures of the available cold-active oligomeric enzymes were found in the Brookhaven Protein Data Bank [39] The search was carried out with the keywords ‘psychro’, ‘cold’, ‘arctic’, ‘antarctic’ and the like Only psychrophilic enzyme structures, for which exceptional high cold activity and low thermostability have previously been shown, were considered The protein structures corresponding to the biological units were collected from the Protein Quaternary Structure databank [40] Homologous structures from mesophilic and thermophilic organisms were subsequently retrieved from the Protein Data Bank and Protein Quaternary Structure databank by means of the program blast [41] To ensure structural homology, only sequences sharing ‡ 30% residue identity to the Identification of interface residues In each oligomeric enzyme, the interface region was defined as being composed of those residues that change their solvent accessibility area in the monomeric and in the oligomeric state (Fig 3) Solvent accessibility computation [47] was performed with naccess [48] The change in solvent accessibility area for each residue in the monomeric state and in the oligomeric state was calculated using a PERL script The interface residues were defined as those residues that show a change in solvent accessibility area upon monomer association Those residues for which the change was more than 90% were defined as composing the core interface [36] FEBS Journal 274 (2007) 4595–4608 ª 2007 The Authors Journal compilation ª 2007 FEBS 4603 Analysis of the oligomeric psychrophilic interface D Tronelli et al swiss-pdbviewer iterative magic fit tool [50] and the insightII package (version 2005; Accelrys) Surface characteristics Fig Interface and core interface of the dimer of thymidylate synthase from Lactobacillus casei (2tdm) The rim interface residues, in green, show a change in solvent accessibility area upon monomer association that is smaller than 90% Core interface residues, in red, show a change greater than 90% The remaining solventaccessible residues are shown in gray Drawn with PYMOL [46] naccess was utilized to calculate the percentages of the overall surface composing the interface and the core interface, the percentages of polar and nonpolar atomic contributions to the interface, and the percentages of polar and nonpolar atomic contributions to the core interface The overall hydrophobic contact area between residues of different monomers was calculated using the program pdb_np_cont [51] with the aid of a PERL script The pdb_np_cont program calculates pairwise atom contact areas between apolar atoms using a set of 512 points located on a sphere around each atom The sphere interaction radius of each atom is equal to the sum of the van der Waals radius of the atom type plus the radius of a water molecule Then, for each atom, the closest interacting atom is found for every point that is not buried by other atoms of the same residue Hydrogen bonds and ion pairs Fig Structural superimposition of 14 TIMs Interface regions are in red Drawn with INSIGHTII (version 2005, Accelrys, Cambridge, UK) The structural similarity of the subunit interfaces within each protein family was evaluated on the basis of the multiple structure alignment To ensure that the interface was structurally conserved within each family and the selected structural data were comparable, the interface Ca carbons of each mesophilic and thermophilic member were superimposed on the equivalent atoms from the psychrophilic ˚ homolog Only interfaces showing rmsd £ 1.3 A were considered to be similar (Fig 4) This threshold is within the expected structural variation corresponding to the range of sequence similarities of the multiple structure alignments [49] Indeed, the expected value of rmsd for a pair of homologous proteins whose sequence identity is 30% is ˚ equal to 1.42 A rmsds were calculated using the deepview– 4604 Hydrogen bonds were calculated using hbplus [52] with the default parameters, except for the maximum distance ˚ between donor and acceptor, which was set to 3.5 A ˚ , to be closer to that proposed in 1984 by instead of 3.9 A ˚ Baker & Hubbard (3.1–3.2 A) [53] Ion pairs at the interface were identified using a PERL script, on the basis of calculation of atomic distance Two residues with opposite charges are considered a strong ion ˚ pair if the distance between charged atoms is less than A This distance threshold is generally accepted after systematic analysis on a sample of protein structures [54] Weak ˚ ion pairs, which are established up to a distance of A [16], were also considered Positively charged atoms were arginine and lysine side chain nitrogens Negatively charged atoms were side chain carboxylate oxygens of glutamate and aspartate Complex salt bridges, defined as ion pair interactions joining more than two side chains, and simple salt links involving two side chains but more than two charged atoms were not considered in the calculations as a single interaction; rather, each individual atomic interaction (single ion pair) was counted [14] Ion pairs involving histidine were not considered because of the ambiguous assignment of its protonation state in the proteins Once all interactions were found, only those between residues of different monomers were considered Packing density, cavity volume and cavity internal surface The atomic packing of interface residues was computed using the os program [55] The os package calculates the FEBS Journal 274 (2007) 4595–4608 ª 2007 The Authors Journal compilation ª 2007 FEBS D Tronelli et al occluded surface, defined as the molecular surface that is ˚ less than 2.8 A from the surface of neighboring nonbonded atoms If a water molecule cannot fit between two atoms, they occlude each other To calculate the occluded surface, normals at the molecular surface were extended outwards until they intersected neighboring van der Waals surfaces The collection of extended normals and their respective lengths were used to define the packing of each atom in an enzyme structure A combination of occluded surface area and average length of the normals was used to calculate the occluded surface packing value for each residue These residue occluded surface packing values were used to calculate the average occluded surface packing value for interface and core interface residues The total surface area and the overall volume of internal cavities between different monomers in each oligomer were calculated using the program castp [56], which detects pro˚ tein pockets and cavities using a water probe of 1.4 A radius The detection of internal cavities was carried out using atomic coordinates of the residues previously identified as composing the subunit interface Analysis of the oligomeric psychrophilic interface shared ‡ 30% and < 90% sequence identity Moreover, in each protein family, the sequence residue identity was calculated for every enzyme pair, in order to determine the sequence identity distribution and its mean The differences in the values of the same structural features considered in the analysis were calculated between all the possible pairs of homologous enzymes in this dataset, and a reference distribution was built for each structural property Likewise, the reference mean and the standard deviation of the distributions were calculated Each observed difference was taken twice, once with a positive and once with a negative sign, as there is an equal probability of subtracting a property of enzyme A from a property of enzyme B or a property of enzyme B from a property of enzyme A if they are both mesophilic Hence, the mean of the distribution is, by definition, null The mean and the standard deviation (Eqns 1–3) of the distribution of the differences of the structural properties calculated between each possible pair of psychrophilic and homologous mesophilic and thermophilic enzymes were calculated according to the following equations: i i DXpj ¼ Xp À Xji Data analysis For each oligomer, the number of hydrogen bonds, and strong, weak and total ion pairs at the interface, were normalized either by the mean interface area or by the number of residues composing the interface The hydrophobic contact area at the interface was normalized by the overall monomeric surface as calculated by naccess The overall volume of internal cavities was normalized by the interface residue volume calculated using the VADAR server [57] http://redpoll.pharmacy.ualberta.ca/vadar/ The total surface area of cavities was normalized by the mean interface area The structural parameters calculated on the main dataset for each psychrophilic enzymes were compared to the same property observed in the homologous mesophilic and thermophilic enzymes The comparison was carried out with and without the thermophilic homologs in order to assess the impact of the latter enzymes on the final statistics To test whether the observed differences were statistically significant or were instead within the expected range of variation, a new reference dataset of mesophilic oligomeric protein families was collected To ensure a nonredundant reference dataset, the cathsolid hierarchical domain classification tool available at the CATH server [58] (http:// www.cathdb.info/latest/index.html) was used Only families with different homologous superfamily domains (H-level) were considered Multidomain protein families were rejected for cases when all the homologous superfamily domains within the same family have been already encountered in other families In each protein family, enzymes F Ni PP DXp ¼ i¼1 jẳ2 F P 1ị i DXpj 2ị Ni 1ị i¼1 vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi u F Ni uP P i u ðDXpj DXp ị2 uiẳ1 jẳ2 rDXp ị ẳ u u F P t Ni 1ị 3ị iẳ1 where X is a generic property (e.g number of hydrogen bonds, percentage of apolar interface, etc.), F is the number i of families collected, Xp is the property calculated at the interface of the psychrophilic enzymes of the ith family, Xji is the interface property of the jth homolog in the same i i family (X1 coincides with Xp ), Ni is the number of members in the ith family, DXp is the average difference, and r(DXp ) is the standard deviation of the differences Under these conditions, a Student’s unpaired two-tailed t-test could be applied to assess whether the distribution of the differences of the structural features observed in the main dataset between the psychrophilic and mesophilic enzymes was significantly different from the distribution of the reference differences of the same properties observed between mesophilic enzymes of the reference dataset In these t-tests, the null hypothesis was that there was no difference in a given property between psychrophilic and mesophilic structures (Eqn 4) DX and r(DX) were calculated both in the main dataset containing cold-active enzymes, DXp and r(DXp ), and in the reference mesophilic dataset DXm and r(DXm ) FEBS Journal 274 (2007) 4595–4608 ª 2007 The Authors Journal compilation ª 2007 FEBS 4605 Analysis of the oligomeric psychrophilic interface D Tronelli et al t-values were calculated according to: ðDXp DXm ị t ẳ r ẵrDXp ị2 Np 4ị ỵ ẵrDXm ị Nm where Np and Nm are the numbers of differences calculated in the main dataset containing cold-active enzymes and in the reference mesophilic dataset; therefore, the number of degrees of freedom corresponds to Np + Nm ) Assessment of the reference dataset To test whether the mesophilic reference dataset adequately represents the properties of the mesophilic proteins included in the main dataset, the following procedure was applied All the extremophilic proteins in the main dataset were removed Subsequently, one of the mesophilic proteins in each of the main dataset family containing at least two members was randomly labeled as ‘psychrophilic’ Then, all the calculations involving Eqns (1–4) were repeated for each structural property considered The entire procedure was repeated 1000 times The number of times that each trial resulted in a t-value outside the one-sided tail of the distribution where the t-value from the real experiment fell was then counted For example, if the t-value for property X was ) 5.0, then the trial t-value was considered to be not significant if greater than ) 1.96 Finally, the percentage of randomized tests that gave a nonsignificant t-value were recorded for each structural property 10 11 12 Acknowledgements This work was partially supported by the funds from ‘Progetto Nazionale di Ricerche in Antartide’ of the ` Italian ‘Ministero dell’Istruzione, dell’Universita e della Ricerca’ The authors are grateful to Dr Giulio Gianese and Dr Alessandro Paiardini for help 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MB & Thornton JM (1997) CATH A hierarchic classification of protein domain structures Structure 5, 1093–1108 FEBS Journal 274 (2007) 4595–4608 ª 2007 The Authors Journal compilation ª 2007 FEBS ... known about the adaptations that have occurred at the interface of oligomeric enzymes Even less is known regarding the adaptation of the psychrophilic interface of oligomeric enzymes Intersubunit... coldadapted enzymes Our work is aimed at detecting the structural variation related to the cold adaptation of the subunit interface of oligomeric psychrophilic enzymes To our knowledge, this is the first... percentage of apolar interface, etc .), F is the number i of families collected, Xp is the property calculated at the interface of the psychrophilic enzymes of the ith family, Xji is the interface