Discovery of a thermophilic protein complex stabilized by topologically interlinked chains

A growing number of organisms have been discovered inhabiting extreme environments, including temperatures in excess of 100 degrees C. How cellular proteins from such organisms retain their native folds under extreme conditions is still not fully understood. Recent computational and structural studies have identified disulfide bonding as an important mechanism for stabilizing intracellular proteins in certain thermophilic microbes. Here, we present the first proteomic analysis of intracellular disulfide bonding in the hyperthermophilic archaeon Pyrobaculum aerophilum. Our study reveals that the utilization of disulfide bonds extends beyond individual proteins to include many protein-protein complexes. We report the 1.6 A crystal structure of one such complex, a citrate synthase homodimer. The structure contains two intramolecular disulfide bonds, one per subunit, which result in the cyclization of each protein chain in such a way that the two chains are topologically interlinked, rendering them inseparable. This unusual feature emphasizes the variety and sophistication of the molecular mechanisms that can be achieved by evolution.     

Disulfide bonds and protein folding

The applications of disulfide-bond chemistry to studies of protein folding, structure, and stability are reviewed and illustrated with bovine pancreatic ribonuclease A (RNase A). After surveying the general properties and advantages of disulfide-bond studies, we illustrate the mechanism of reductive unfolding with RNase A, and discuss its application to probing structural fluctuations in folded proteins. The oxidative folding of RNase A is then described, focusing on the role of structure formation in the regeneration of the native disulfide bonds. The development of structure and conformational order in the disulfide intermediates during oxidative folding is characterized. Partially folded disulfide species are not observed, indicating that disulfide-coupled folding is highly cooperative. Contrary to the predictions of "rugged funnel" models of protein folding, misfolded disulfide species are also not observed despite the potentially stabilizing effect of many nonnative disulfide bonds. The mechanism of regenerating the native disulfide bonds suggests an analogous scenario for conformational folding. Finally, engineered covalent cross-links may be used to assay for the association of protein segments in the folding transition state, as illustrated with RNase A.     

Elucidation of factors responsible for enhanced thermal stability of proteins: a structural genomics based study

Understanding the molecular basis for the enhanced stability of proteins from thermophiles has been hindered by a lack of structural data for homologous pairs of proteins from thermophiles and mesophiles. To overcome this difficulty, complete genome sequences from 9 thermophilic and 21 mesophilic bacterial genomes were aligned with protein sequences with known structures from the protein data bank. Sequences with high homology to proteins with known structures were chosen for further analysis. High quality models of these chosen sequences were obtained using homology modeling. The current study is based on a data set of models of 900 mesophilic and 300 thermophilic protein single chains and also includes 178 templates of known structure. Structural comparisons of models of homologous proteins allowed several factors responsible for enhanced thermostability to be identified. Several statistically significant, specific amino acid substitutions that occur going from mesophiles to thermophiles are identified. Most of these are at solvent-exposed sites. Salt bridges occur significantly more often in thermophiles. The additional salt bridges in thermophiles are almost exclusively in solvent-exposed regions, and 35% are in the same element of secondary structure. Helices in thermophiles are stabilized by intrahelical salt bridges and by an increase in negative charge at the N-terminus. There is an approximate decrease of 1% in the overall loop content and a corresponding increase in helical content in thermophiles. Previously overlooked cation-pi interactions, estimated to be twice as strong as ion-pairs, are significantly enriched in thermophiles. At buried sites, statistically significant hydrophobic amino acid substitutions are typically consistent with decreased side chain conformational entropy.     

Structural differences between mesophilic, moderately thermophilic and extremely thermophilic protein subunits: results of a comprehensive survey

BACKGROUND: Proteins from thermophilic organisms usually show high intrinsic thermal stability but have structures that are very similar to their mesophilic homologues. From prevous studies it is difficult to draw general conclusions about the structural features underlying the increased thermal stability of thermophilic proteins. RESULTS: In order to reveal the general evolutionary strategy for changing the heat stability of proteins, a non-redundant data set was compiled comprising all high-quality structures of thermophilic proteins and their mesophilic homologues from the Protein Data Bank. The selection (quality) criteria were met by 64 mesophilic and 29 thermophilic protein subunits, representing 25 protein families. From the atomic coordinates, 13 structural parameters were calculated, compared and evaluated using statistical methods. This study is distinguished from earlier ones by the strict quality control of the structures used and the size of the data set. CONCLUSIONS: Different protein families adapt to higher temperatures by different sets of structural devices. Regarding the structural parameters, the only generally observed rule is an increase in the number of ion pairs with increasing growth temperature. Other parameters show just a trend, whereas the number of hydrogen bonds and the polarity of buried surfaces exhibit no clear-cut tendency to change with growth temperature. Proteins from extreme thermophiles are stabilized in different ways to moderately thermophilic ones. The preferences of these two groups are different with regards to the number of ion pairs, the number of cavities, the polarity of exposed surface and the secondary structural composition.     

Characterization of a multifunctional protein disulfide oxidoreductase from Sulfolobus solfataricus

A potential role in disulfide bond formation in the intracellular proteins of thermophilic organisms has recently been ascribed to a new family of protein disulfide oxidoreductases (PDOs). We report on the characterization of SsPDO, isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. SsPDO was cloned and expressed in Escherichia coli. We revealed that SsPDO is the substrate of a thioredoxin reductase in S. solfataricus (K(M) 0.3 microm) and not thioredoxins (TrxA1 and TrxA2). SsPDO/S. solfataricus thioredoxin reductase constitute a new thioredoxin system in aerobic thermophilic archaea. While redox (reductase, oxidative and isomerase) activities of SsPDO point to its central role in the biochemistry of cytoplasmic disulfide bonds, chaperone activities also on an endogenous substrate suggest a potential role in the stabilization of intracellular proteins. Northern and western analysis have been performed in order to analyze the response to the oxidative stress.     

Reconsideration of an early dogma,saying “there is no evidence for disulfide bonds in proteins from archaea”

Stability and function of a large number of proteins are crucially dependent on the presence of disulfide bonds. Recent genome analysis has pointed out an important role of disulfide bonds for the structural stabilization of intracellular proteins from hyperthermophilic archaea and bacteria. These findings contradict the conventional view that disulfide bonds are rare in those proteins. A specific protein, known as protein disulfide oxidoreductase (PDO) is recognized as a potential key enzyme in intracellular disulfide-shuffling in hyperthermophiles. The structure of this protein consists of two combined thioredoxin-related units which together, in tandem-like manner, form a closed protein domain. Each of these units contains a distinct CXXC active site motif. Both sites seem to have different redox properties. A relation to eukaryotic protein disulfide isomerase is suggested by the observed structural and functional characteristics of the protein. Enzymological studies have revealed that both, the archaeal and bacterial forms of this protein show oxidative and reductive activity and are able to isomerize protein disulfides. The variety of active site disulfides found in PDO’s from hyperthermophiles is puzzling. It is assumed, that PDO enzymes in hyperthermophilic archaea and bacteria may be part of a complex system involved in the maintenance of protein disulfide bonds.     

Patterns of temperature adaptation in proteins from Methanococcus and Bacillus

It has long been known that amino acid substitutions in proteins of organisms living at moderate and high temperatures (mesophiles and thermophiles, respectively) are not all symmetrical; for example, more aligned sites have lysine in mesophiles and arginine in thermophiles than have the opposite pattern. This is generally taken to indicate that certain amino acids are favored over others by selection at different temperatures. Previous comparisons of protein sequences from mesophiles and thermophiles have used relatively small numbers of sequences from a diverse array of species, meaning that only the most common amino acid substitutions could be examined and any taxon-specific patterns would be obscured. Here, we compare a large number of proteins between mesophiles and thermophiles in the archaeal genus Methanococcus and the bacterial genus Bacillus. Each genus exhibits dramatically asymmetrical substitution patterns for many pairs of amino acids. There are several pairs of amino acids for which one amino acid is favored in thermophilic Bacillus and the other is favored in thermophilic Methanococcus; this appears to result from the higher G + C content of the DNA of thermophilic Bacillus, a complication not seen in Methanococcus.     

Insights on a new PDI-like family: structural and functional analysis of a protein disulfide oxidoreductase from the bacterium Aquifex aeolicus

A potential role in disulfide bond formation in the intracellular proteins of thermophilic organisms has recently been attributed to a new family of protein disulfide isomerase (PDI)-like proteins. Members of this family are characterized by a molecular mass of about 26kDa and by two Trx folds, each comprising a CXXC active site motif. We report on the functional and structural characterization of a new member of this family, which was isolated from the thermophilic bacterium Aquifex aeolicus (AaPDO). Functional studies have revealed the high catalytic efficiency of this enzyme in reducing, oxidizing and isomerizing disulfide bridges. Site-directed mutagenesis experiments have suggested that its two active sites have similar functional properties, i.e. that each of them imparts partial activity to the enzyme. This similarity was confirmed by the analysis of the enzyme crystal structure, which points to similar geometrical parameters and solvent accessibilities for the two active sites. The results demonstrated that AaPDO is the most PDI-like of all prokaryotic proteins so far known. Thus, further experimental studies on this enzyme are likely to provide important information on the eukaryotic homologue.     

半胱氨酸氧化还原状态的协同性

以2002年4月份的Culled Protein Data Bank数据库中的639条蛋白质多肽链为研究对象,统计分析了其含有的584条二硫键的形成特征,发现半胱氨酸氧化还原状态表现出明显的协同性现象：含有二硫键的蛋白质中几乎所有的半胱氨酸都以氧化态形式存在。这一协同性可以通过蛋白质全局水平上的20种氨基酸组分的百分含量很好地加以说明,由此来预测半胱氨酸的氧化还原状态准确率最高可达84．5％。结果表明半胱氨酸是否形成二硫键主要取决于蛋白质全局的而非局部的结构信息。     

The stability of proteins in extreme environments

Three complete genome sequences of thermophilic bacteria provide a wealth of information challenging current ideas concerning phylogeny and evolution, as well as the determinants of protein stability. Considering known protein structures from extremophiles, it becomes clear that no general conclusions can be drawn regarding adaptive mechanisms to extremes of physical conditions. Proteins are individuals that accumulate increments of stabilization; in thermophiles these come from charge clusters, networks of hydrogen bonds, optimization of packing and hydrophobic interactions, each in its own way. Recent examples indicate ways for the rational design of ultrastable proteins.     

Stability and rigidity/flexibility—Two sides of the same coin?

Protein molecules require both flexibility and rigidity for functioning. The fast and accurate prediction of protein rigidity/flexibility is one of the important problems in protein science. We have determined flexible regions for four homologous pairs from thermophilic and mesophilic organisms by two methods: the fast FoldUnfold which uses amino acid sequence and the time consuming MDFirst which uses three-dimensional structures. We demonstrate that both methods allow determining flexible regions in protein structure. For three of the four thermophile–mesophile pairs of proteins, FoldUnfold predicts practically the same flexible regions which have been found by the MD/First method. As expected, molecular dynamics simulations show that thermophilic proteins are more rigid in comparison to their mesophilic homologues. Analysis of rigid clusters and their decomposition provides new insights into protein stability. It has been found that the local networks of salt bridges and hydrogen bonds in thermophiles render their structure more stable with respect to fluctuations of individual contacts. Such network includes salt bridge triads Agr-Glu-Lys and Arg-Glu-Arg, or salt bridges (such as Arg-Glu) connected with hydrogen bonds. This ionic network connects alpha helices and rigidifies the structure. Mesophiles can be characterized by stand alone salt bridges and hydrogen bonds or small ionic clusters. Such difference in the network of salt bridges results in different flexibility of homologous proteins. Combining both approaches allows characterizing structural features in atomic detail that determine the rigidity/flexibility of a protein structure. This article is a part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly.     

Importance of main-chain hydrophobic free energy to the stability of thermophilic proteins

Living organisms are found in the most unexpected places, including deep-sea vents at 100 degrees C and several hundred bars pressure, in hot springs. Needless to say, the proteins found in thermophilic species are much more stable than their mesophilic counterparts. There are no obvious reasons to say that one would be more stable than others. Even examination of the amino acids and comparison of structural features of thermophiles with mesophilies cannot bring satisfactory explanation for the thermal stability of such proteins. In order to bring out the hidden information behind the thermal stabilization of such proteins in terms of energy factors and their combinations, analysis were made on good resolution structures of thermophilic and their mesophilic homologous from 23 different families. From the structural coordinates, free energy contributions due to hydrophobic, electrostatic, hydrogen bonding, disulfide bonding and van der Waals interactions are computed. In this analysis, a vast majority of thermophilic proteins adopt slightly lower free energy contribution in each energy terms than its mesophilic counterparts. The major observation noted from this study is the lower hydrophobic free energy contribution due to carbon atoms and main-chain nitrogen atoms in all the thermophilic proteins. The possible combination of different free energy terms shows majority of the thermophilic proteins have lower free energy strategy than their mesophilic homologous. The derived results show that the hydrophobic free energy due to carbon and nitrogen atoms and such combinations of free energy components play a vital role in the thermostablisation of such proteins.     

Hydrophobic environment is a key factor for the stability of thermophilic proteins

The stability of thermophilic proteins has been viewed from different perspectives and there is yet no unified principle to understand this stability. It would be valuable to reveal the most important interactions for designing thermostable proteins for such applications as industrial protein engineering. In this work, we have systematically analyzed the importance of various interactions by computing different parameters such as surrounding hydrophobicity, inter‐residue interactions, ion‐pairs and hydrogen bonds. The importance of each interaction has been determined by its predicted relative contribution in thermophiles versus the same contribution in mesophilic homologues based on a dataset of 373 protein families. We predict that hydrophobic environment is the major factor for the stability of thermophilic proteins and found that 80% of thermophilic proteins analyzed showed higher hydrophobicity than their mesophilic counterparts. Ion pairs, hydrogen bonds, and interaction energy are also important and favored in 68%, 50%, and 62% of thermophilic proteins, respectively. Interestingly, thermophilic proteins with decreased hydrophobic environments display a greater number of hydrogen bonds and/or ion pairs. The systematic elimination of mesophilic proteins based on surrounding hydrophobicity, interaction energy, and ion pairs/hydrogen bonds, led to correctly identifying 95% of the thermophilic proteins in our analyses. Our analysis was also applied to another, more refined set of 102 thermophilic–mesophilic pairs, which again identified hydrophobicity as a dominant property in 71% of the thermophilic proteins. Further, the notion of surrounding hydrophobicity, which characterizes the hydrophobic behavior of residues in a protein environment, has been applied to the three‐dimensional structures of elongation factor‐Tu proteins and we found that the thermophilic proteins are enriched with a hydrophobic environment. The results obtained in this work highlight the importance of hydrophobicity as the dominating characteristic in the stability of thermophilic proteins, and we anticipate this will be useful in our attempts to engineering thermostable proteins. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Important inter-residue contacts for enhancing the thermal stability of thermophilic proteins

Proteins from thermophilic organisms exhibit high thermal stability, but have structures that are very similar to their mesophilic homologues. In order to gain insight into the basis of thermostability, we have analyzed the medium- and long-range contacts in mesophilic and thermophilic proteins of 16 different families. We found that the thermophiles prefer to have contacts between residues with hydrogen-bond-forming capability. Apart from hydrophobic contacts, more contacts are observed between polar and non-polar residues in thermophiles than mesophiles. Residue-wise analysis showed that Tyr has good contacts with several other residues, and Cys has considerably higher long-range contacts in thermophiles compared with mesophiles. Furthermore, the residues occurring in the range of 31-34 residues apart in the sequence contribute significant long-range contacts to the stability of thermophilic proteins.     

How Do Thermophilic Proteins and Proteomes Withstand High Temperature?

We attempt to understand the origin of enhanced stability in thermophilic proteins by analyzing thermodynamic data for 116 proteins, the largest data set achieved to date. We compute changes in entropy and enthalpy at the convergence temperature where different driving forces are maximally decoupled, in contrast to the majority of previous studies that were performed at the melting temperature. We find, on average, that the gain in enthalpy upon folding is lower in thermophiles than in mesophiles, whereas the loss in entropy upon folding is higher in mesophiles than in thermophiles. This implies that entropic stabilization may be responsible for the high melting temperature, and hints at residual structure or compactness of the denatured state in thermophiles. We find a similar trend by analyzing a homologous set of proteins classified based only on the optimum growth temperature of the organisms from which they were extracted. We find that the folding free energy at the temperature of maximal stability is significantly more favorable in thermophiles than in mesophiles, whereas the maximal stability temperature itself is similar between these two classes. Furthermore, we extend the thermodynamic analysis to model the entire proteome. The results explain the high optimal growth temperature in thermophilic organisms and are in excellent quantitative agreement with full thermal growth rate data obtained in a dozen thermophilic and mesophilic organisms.     

The crystal structure of adenylosuccinate lyase from Pyrobaculum aerophilum reveals an intracellular protein with three disulfide bonds

Adenylosuccinate lyase catalyzes two separate reactions in the de novo purine biosynthetic pathway. Through its dual action in this pathway, adenylosuccinate lyase plays an integral part in cellular replication and metabolism. Mutations in the human enzyme can result in severe neurological disorders, including mental retardation with autistic features. The crystal structure of adenylosuccinate lyase from the hyperthermophilic archaebacterium Pyrobaculum aerophilum has been determined to 2.1 A resolution. Although both the fold of the monomer and the architecture of the tetrameric assembly are similar to adenylosuccinate lyase from the thermophilic eubacterium Thermotoga maritima, the archaebacterial lyase contains unique features. Surprisingly, the structure of adenylosuccinate lyase from P. aerophilum reveals that this intracellular protein contains three disulfide bonds that contribute significantly to its stability against thermal and chemical denaturation. The observation of multiple disulfide bonds in the recombinant form of the enzyme suggests the need for further investigations into whether the intracellular environment of P. aerophilum, and possibly other hyperthermophiles, may be compatible with protein disulfide bond formation. In addition, the protein is shorter in P. aerophilum than it is in other organisms. This abbreviation results from an internal excision of a cluster of helices that may be involved in protein-protein interactions in other organisms and may relate to the observed clinical effects of human mutations in that region.     

Structural features of thermozymes

Enzymes synthesized by thermophiles and hyperthermophiles are known as thermozymes. These enzymes are typically thermostable, or resistant to irreversible inactivation at high temperatures, and thermophilic, i.e. optimally active at elevated temperatures between 60 and 125 degrees C. Enzyme thermostability encompasses thermodynamic stability and kinetic stability. Thermodynamic stability is defined by the enzyme's free energy of stabilization (deltaG(stab)) and by its melting temperature (Tm). An enzyme's kinetic stability is often expressed as its halflife (t1/2) at defined temperature. DeltaG(stab) of thermophilic proteins is 5-20 kcal/mol higher than that of mesophilic proteins. The thermostability mechanisms for thermozymes are varied and depend on the enzyme; nevertheless, some common features can be identified as contributing to stability. These features include more interactions (i.e. hydrogen bonds, electrostatic interactions, hydrophobic interactions, disulfide bonds, metal binding) than in less stable enzymes and superior conformational structure (i.e. more rigid, higher packing efficiency, reduced entropy of unfolding, conformational strain release and stability of alpha-helix). Understanding of the stabilizing features will greatly facilitate reengineering of some of the mesozymes to more stable thermozymes.     

Patterns of temperature adaptation in proteins from the bacteria Deinococcus radiodurans and Thermus thermophilus

Asymmetrical patterns of amino acid substitution in proteins of organisms living at moderate and high temperatures (mesophiles and thermophiles, respectively) are generally taken to indicate selection favoring different amino acids at different temperatures due to their biochemical properties. If that were the case, comparisons of different pairs of mesophilic and thermophilic taxa would exhibit similar patterns of substitutional asymmetry. A previous comparison of mesophilic versus thermophilic Methanococcus with mesophilic versus thermophilic Bacillus revealed several pairs of amino acids for which one amino acid was favored in thermophilic Bacillus and the other was favored in thermophilic Methanococcus. Most of this could be explained by the higher G+C content of the DNA of thermophilic Bacillus, a phenomenon not seen in the Methanococcus comparison. Here, I compared the mesophilic bacterium Deinococcus radiodurans and its thermophilic relative Thermus thermophilus, which are similar in G+C content. Of the 190 pairs of amino acids, 83 exhibited significant substitutional asymmetry, consistent with the pervasive effects of selection. Most of these significantly asymmetrical pairs of amino acids were asymmetrical in the direction predicted from the Methanococcus data, consistent with thermal adaptation resulting from universal biochemical properties of the amino acids. However, 12 pairs of amino acids exhibited asymmetry significantly different from and in the opposite direction of that found in the Methanococcus comparison, and 21 pairs of amino acids exhibited asymmetry that was significantly different from that found in the Bacillus comparison and could not be explained by the greater G+C content in thermophilic Bacillus. This suggests that selection due to universal biochemical properties of the amino acids and differences in G+C content are not the only causes of substitutional asymmetry between mesophiles and thermophiles. Instead, selection on taxon-specific properties of amino acids, such as their metabolic cost, may play a role in causing asymmetrical patterns of substitution.