Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India
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Abstract |
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Keywords: aromatic clusters/aromatic packing/protein engineering/protein folding/thermal stability
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Introduction |
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So far two major approaches have been made to investigate this aspect. The first approach was to compare the structures and sequences of homologous proteins from thermophiles and mesophiles, which has resulted in the understanding of various aspects related to the thermal stability, such as increased number of salt bridges, better hydrogen bonding, high internal packing, strengthening inter-subunit association, all of which has been compiled in two recent reviews (Jaenicke and Bohm, 1998; Ladenstein and Antranikian, 1998
). More recently, Szilagyi and Zavodszky (2000) analysed 13 structural parameters in order to understand the structural features underlying thermal stability.
The second major approach involved protein engineering methods (Fersht and Serrano, 1993), which provided new insights into the factors that contribute to the thermal stability of proteins, such as (1) stabilization of the dipoles of the
-helices (Nicholson et al., 1988
), (2) reducing the difference in entropy between the folded and unfolded states (Matthews et al., 1987
) and (3) increasing the number of hydrophobic interactions in the hydrophobic core (Yutani et al., 1987
) and reducing the area of water accessible hydrophobic surface (Wigley et al., 1987
). There have also been attempts to combine both of these approaches to study thermal stability (Serrano et al., 1993
). The final conclusion from all these studies is that there is no unique factor which determines protein thermal stability but instead it is a result of a number of subtle interactions characteristic for each protein species (Ladenstein and Antranikian, 1998
; Szilagyi and Zavodszky, 2000
).
We recently elucidated a graph spectral method to identify side-chain clusters in protein structures (Kannan and Vishveshwara, 1999). In the present study, we applied this method to identify aromatic clusters in a set of homologous thermophiles and mesophiles from different protein families. Our analysis shows the presence of additional aromatic clusters and aromatic networks in the thermophilic protein as compared with their mesophilic homologue. Although many other structural features have been reported to contribute to thermal stability (Querol et al., 1996
; Vogt and Argos, 1997
; Szilagyi and Zavodszky, 2000
), to our knowledge no study has highlighted the presence of additional aromatic clusters in thermophilic proteins.
Aromatic interactions are known to be important in the structural stabilization of proteins (Burley and Petsko, 1985; Anderson et al., 1993
). A pair of aromatic interaction contributes between 0.6 and 1.3 kcal/mol to the protein stability (Serrano et al., 1991
). Protein engineering methods have shown that introducing aromatic pairs and aromatic clusters in a protein increases the thermal stability (Burley and Petsko, 1985
; Serrano et al., 1991
) and more recently Georis et al. (2000) demonstrated that introducing an additional aromatic interaction improves the thermophilicity and thermostability of family 11 xylanase. Recently, a structure of thermophilic protein, Bacillus Ak.1, from the subtilisin family has been reported which shows the presence of additional aromatic clusters on the surface of the protein as compared to its mesophilic counterpart (Smith et al., 1999
).
The present study on a dataset of 24 thermophilic and corresponding mesophilic homologue from different protein families shows the presence of additional aromatic clusters in 17 out of 24 thermophilic families compared with the mesophilic families. The additional aromatic clusters found in the thermophiles are mostly located on the surface of the protein and are usually small in size. The topologically equivalent residues in the mesophiles are generally leucine, isoleucine or serine residues. The aromatic residues of the additional clusters in the thermophilic proteins generally emanate from different secondary structural regions of the protein and have low B values. In most of the thermophilic proteins, at least one additional aromatic cluster was found to be located close to the binding/active site of the protein. An analysis of the packing geometry of pairwise aromatic interaction in the additional aromatic clusters showed that a T-shaped perpendicular packing geometry is preferred. The present study highlights the presence of additional aromatic clusters as one of the major contributing factors to the thermal stability of thermophilic proteins and provides new insights for protein engineers to design thermostable proteins.
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Materials and methods |
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A dataset of 48 proteins belonging to 24 protein famlies was considered for cluster analysis (Table I). The dataset was constructed based on the earlier report of Facchiano et al. (1998) and the recent exhaustive search of Szilagyi and Zavodszky (2000). The pairs of thermophilic and mesophilic proteins which had very high sequence identity and structural similarity were selected, in order to ensure that the observed amino acid substitution and structural difference between the thermophiles and their corresponding mesophilic homologue is due to the difference in stability and not an artifact of evolutive effects (Fachiano et al., 1998). For protein families in which more than one homologous mesophilic structure was known, the one which had high structural similarity and sequence identity to the mesophilic protein was considered.
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Aromatic clusters for all the proteins in the dataset were obtained by a Graph Spectral method as described earlier (Kannan and Vishveshwara, 1999). The method detects aromatic clusters by considering all the Cß atoms of the aromatic residues in the protein as nodes of a graph and two interacting aromatic side chains were connected in the graph assigning an edge in weight corresponding to the distance between the respective Cß atoms. This connectivity information is represented in the form of a Laplacian matrix. The Laplacian matrix is diagonalized and clustering information is obtained from the eigenvectors corresponding to the second lowest eigenvalue. The details of the algorithm were given in our earlier paper (Kannan and Vishveshwara, 1999
).
In our earlier report of identifying side chain clusters we constructed a graph for the protein structure with the constraint that the degree of at least one of the two interacting side chain should be >1. This constraint was imposed in order to avoid two-residue clusters. In the present analysis we removed this constraint so as to detect all the two-residue aromatic clusters in addition to clusters of larger size.
Geometry of aromatic packing
The inter-planar orientation between two interacting aromatic side chains was determined by evaluating the angle between the normals of the two aromatic planes. The inter-planar angle was evaluated for two pairs of aromatic rings if the distance between the center of the two rings was <6.5 Å.
Structural alignment
The thermophile and the corresponding mesophilic homologue were superimposed using the STAMP program (Russell and Barton, 1992). A structure-based sequence alignment was obtained using this program in order to compare the topologically equivalent residues.
Identification of cluster location
The location of the aromatic residues in the protein structure was identified by three procedures: (a) graphically using the VMD package (Humphrey et al., 1996), (b) calculation of the solvent accessibility using Connolly's program (Connolly, 1993
) and (c) the graph spectral method of assigning residues to the hydrophobic core based on the contact criteria of the hydrophobic residues (Kannan and Vishveshwara, 1999
). Using this method we were able to identify residue clusters on the surface and in the buried core of the protein using a `hydrophobic contact criterion'. Hydrophobic residues in a cluster having high internal contact with themselves were identified to occur in the core of the protein. The accessible surface area calculation was performed only on the monomeric subunits if the thermophile or mesophile existed as an oligomer.
Identification of aromatic clusters close to the active site
The aromatic cluster in which at least one of the residue atoms was at a distance of <10 Å from any of the ligand atoms was considered to be close to the active/binding site. In those cases where the ligand coordinates were not specified, the distance was evaluated from the active site residues of the protein.
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Results |
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The aromatic clusters in the 24 pairs of proteins were determined as mentioned in the Materials and methods section. The numbers of aromatic clusters found in the thermophiles and in the corresponding mesophilic homologue are listed in Table I. Based on the number of aromatic clusters identified, the protein pairs can be classified into three different groups: (a) in 10 out of 24 families (1, 2, 3, 6, 7, 8, 9, 11, 18 and 24) the number of aromatic clusters is clearly greater in the thermophiles than the mesophiles (Table I
); (b) in 10 protein families (4, 5, 10, 12, 13, 14, 16, 17, 22 and 23) the number of aromatic clusters is the same in both the thermophiles and mesophiles; and (c) in protein families 15, 19, 20 and 21 mesophilic proteins have more aromatic clusters (Table I
).
A graphical examination of the superimposed structures and the cluster residues of the thermophile and its mesophilic homologue showed that in 10 protein families (group a) one or more aromatic clusters in the thermophilic protein was absent in the topologically equivalent position in the mesophilic homologue. A cluster in the thermophile for which a topologically equivalent cluster is not found in the mesophile is referred to as an additional aromatic cluster. Structure-based sequence alignment showed that the residues forming the additional aromatic cluster were mutated to non-aromatic residues in the mesophile. The superimposed structures and the additional clusters in the thermophile and the equivalent substitution in the mesophile are shown for the neutral protease (family 1) (Figure 1). A three-residue aromatic cluster of residues Tyr93, Tyr151 and Trp115 (dark) is found in the thermophile and the topologically equivalent residues in the mesophile are Ile94, Asn152 and Trp116. Owing to a non-aromatic mutation of Tyr93 and Tyr151 to Ile94 and Asn152, the aromatic cluster is absent in the mesophile. Also, a two-residue cluster comprising residues Tyr28 and Tyr24 is found in the thermophile but is absent in the mesophile owing to a non-aromatic substitution of Tyr24 to Leu24. This feature is also observed in nine other protein families of group a. In 10 protein families (group b) the number of aromatic clusters in both the thermophiles and mesophiles is the same. However, in seven of them (4, 5, 10, 12, 13, 16 and 22) the number of aromatic residues constituting the clusters was larger in the thermophiles than their mesophilic counterpart. For example, in the case of ribonuclease H two clusters were detected in the thermophile (1RIL) (Figure 2a
) and in the mesophile (2RN2) (Figure 2b
). Cluster 1 in both the proteins occurs in a topologically similar location but the cluster in the thermophile (Tyr73, Trp104, Phe78, Phe118, Phe120, Trp81, Trp85 and Trp90) is larger than that in the mesophile (Tyr73, Trp104, Trp81, Trp85, Trp90). The three additional aromatic residues Phe78, Phe118 and Phe120 in the first cluster of the thermophile are mutated to Ile78, Trp118 and Trp120 in the mesophile. Although Trp118 and Trp120 are equivalent aromatic substitutions in the mesophile, they do not form a part of the cluster as Phe78, which interacts with Phe118 and Phe120 in the thermophile is mutated to Ile78 in the mesophile. Since this interaction is lost, the cluster size reduces to five residues in the mesophile. The second cluster in the thermophile and in the mesophile do not occur in topologically similar positions. The second cluster constituting residues Phe8 and Tyr68 in the thermophile (Figure 2a
) (cluster 2) is smaller than that in the mesophile where a three-residue cluster of residues Tyr22, Phe35 and Tyr39 is observed (Figure 2b
) (cluster 2'). Therefore, even though the number of clusters in the two proteins is the same, the cluster sizes are different. There are additional aromatic interactions in the thermophile as the net number of aromatic residues in the clusters of thermophile is greater than that in the mesophile. This trend is also observed in protein families 5, 10, 12, 13, 16 and 22. However in families 14, 17 and 23 the number of clusters and also the number of residues constituting the cluster are the same in the thermophile and in the mesophile. Further, a reverse trend of more aromatic clusters in the mesophile than in the thermophile is observed in the protein families 15, 19, 20 and 21. This could possibly be because of the presence of other factors such as increased number of hydrogen bonds, increased number of salt bridge interactions across the inter-subunit interface and also a decrease in the enzyme to surface volume ratio which may contribute to thermal stability of this protein (Tanner et al., 1996
). Since the trend of additional aromatic interactions in the thermophiles is observed in 17 out of 24 cases, these additional aromatic clusters of the 17 protein families were further analysed in detail.
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The location of the aromatic clusters was identified by the three methods mentioned in the Materials and methods section. Their location in the thermophilic proteins was quantitatively analysed by calculating the accessible surface area of the cluster residues in addition to graphical visualization. In most of the protein families the additional cluster was found to be located on the protein surface with a partially accessible surface area (Figure 4). In three cases the clusters show an accessibility <5% and in most of the cases the accessible area is <15% and >40% (Figure 4
), indicating that the additional aromatic clusters are partially exposed. Our analysis by the graph spectral method showed that most of the additional clusters in the thermophiles are not part of the protein core and they occur as separate entities on the protein surface. Previously Heringa et al. (1995) had identified strong side chain clusters on the protein surface in the subtilisin family and predicted a few point mutations which increased the thermal stability of the protein. Generally, mutations on the surface of the protein do not have a large impact on the native structure and the folding intermediate. Hence, it appears that nature has chosen to engineer thermal stability by mutations on the surface of the protein.
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Thermophilic enzymes are stable and fully active at elevated temperatures but are not functional at room temperature (Hecht et al., 1989). This ability of an enzyme to exhibit activity at high temperature is defined as thermophilicity (Georis et al., 2000
). The inactivation of an enzyme at room temperature has been attributed to the restriction of conformational fluctuations necessary for the catalytic function (Zavodszky et al., 1998
). We investigated this aspect by identifying the presence of additional aromatic clusters close to the active site of the thermophilic proteins. We found that in most of the thermophilic proteins at least one additional aromatic cluster was found close to the active site of the enzyme (Table II
). The presence of additional aromatic clusters near the active site should help in retaining the conformational features of the active sites residues required to bind the substrate at high temperatures and thus contributing to the high thermophilicity of the thermostable proteins.
In Figure 5a is shown a two-residue aromatic cluster of Phe8 and Tyr68 close to the active site (formed by residues Asp10, Asp70 and Glu248). This two-residue aromatic cluster is located in the two parallel ß-sheets on which the active site residues Asp10 and Asp70 are located (Figure 5a
) of the thermophilic protein (1RIL) belonging to the ribonuclease H family. This cluster, which interacts in a parallel offset geometry, is probably important for the conformational rigidity of the thermophile at room temperature. In the mesophile, Tyr68 is mutated to a Ser (Table II
), allowing for the conformational flexibility of the active site residues. Similarly, in the case of lactate dehydrogenase (Figure 5b
), two additional aromatic clusters are found close to the active site. The cluster residues emanate from three different helices marked 1, 2 and 3 (Figure 5b
). It can be seen that the N-terminal residues of helix 1 interact with the ligand and probably the flexibility of helix 1 is important for ligand binding. The two additional aromatic clusters involving the N-terminal (Phe30) and the C-terminal (Phe37) residues of helix 1 impart rigidity to this helix. However, in the case of the mesophile the cluster residues Phe30 and Tyr248 are mutated to Ala30 and Ser246, respectively (Table II
), and the other cluster residues Phe65 and Phe37 are mutated to Leu65 and Ile37, thus allowing for the conformational flexibility of helix 1 in the mesophilic protein.
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Vihinen (1987) showed an inverse correlation between thermal stability and protein flexibility by calculating flexibility indices from the thermal factors of the side chains in known 3D structures, and more recently Parthasarathy and Murthy (2000) showed that serine and threonine residues which show a high composition in the thermophiles have a low B factor compared with the mesophiles. In the present study, we analysed the thermal factors of the residues in the additional aromatic clusters. The flexibility of the aromatic clusters was studied by evaluating the temperature factor (B factor) of the aromatic residues forming clusters. Since most of the additional aromatic clusters are located on the protein surface, the average B factor of the aromatic residues forming clusters was compared with the average B factor of the partially buried residues in each of the thermophilic proteins. Interestingly, in most cases the average thermal factor of the cluster residues is less than or equal to the average B factor of the partially buried residues. Menendez and Argos (1989) showed that the regions which show reduced flexibility corrspond to the helical regions in the protein. The secondary structural location of the additional aromatic residues was analysed in our present dataset. We found that the aromatic residues forming the additional cluster emanate from different secondary structural regions of the protein, stabilizing the tertiary fold. Almost 38% of the aromatic residues in the additional aromatic clusters emanate from helices, 32% from strands, 21% from coil and 9% from the loop regions of the thermophilic proteins. However, the secondary structural composition of the thermophilic proteins in the dataset show that nearly 54% of the secondary structures are coils or loops and nearly 26% are strands and only 20% are helices. It is clear from the statistics of the general secondary structural composition and the secondary structural location of the aromatic residues that the additional aromatic clusters occur in regular secondary structures, implying their location to be in more rigid regions of the protein.
Geometry of aromaticaromatic interaction
The packing geometries of the aromatic residues in the additional aromatic clusters was investigated by evaluating the inter-planar angles (see Materials and methods) for all pairwise aromatic interactions of the residues which were mutated to non-aromatic ones in the mesophile. The distributions of inter-planar angles were categorized in three regions, namely (030) denoting near-parallel face to face interaction, (3060) denoting tilted geometry and (6090) denoting orthogonal or T-shaped packing geometry. In 51 out of a total of 100 pairs, the aromatic residues interact pairwise in T-shaped orthogonal geometry and in 29 cases in a tilted geometry. In only 18 cases were the aromatic residues in the additional aromatic clusters found to interact in near-parallel geometry. A simple pairwise energy calculation of aromatic residues also had shown a preference for T-shaped packing (Burley and Petsko, 1985). Ab initio calculations of pairwise aromatic aromatic packing have shown that aromatic pairs can pack together in any of the two energetically favorable geometries, namely off-centred parallel displaced geometry or a T-shaped perpendicular geometry (Chipot et al., 1996
; Hobza et al., 1996
; Jaffe and Smith, 1996
).
The investigations of Singh and Thornton (1991) using more detailed energy calculations had shown that a T-shaped packing geometry of aromatic residues is preferred. However, more recently a study by McGanghey et al. (1998) showed that aromatic pairs favor an off-centered parallel orientation. The authors also pointed out that this deviation in the results is because the earlier workers had not separated out pairwise aromatic interactions and that looking at pairwise interactions in aromatic clusters (greater than two residues) dilutes the effect of TT stacking. We performed an analysis on all two-residue aromatic clusterd for the present dataset of 24 thermophiles and found that the pairwise aromatic interaction geometries show a preference for T-shaped or tilted geometry as opposed to a purely parallel geometry.
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Discussion |
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Moreover, the additional clusters in the thermophiles mostly involve pairwise interaction and occur on the protein surface. Additional stabilization on the surface of the protein by aromatic interactions could be essential to prevent the native structure from thermal denaturation. Since protein thermal denaturation is known to start with unfolding of the outer surface which leads to the exposure of the hydrophobic core (Calflish and Karplus, 1994), this denaturation can possibly be prevented by stabilizing the protein surface with aromatic interactions.
Further, in a few protein families (4, 5, 10, 12 and 15) there were additional aromatic clusters found on the mesophile but a topologically equivalent cluster was not found in the thermophile. The aromatic clusters found in the mesophile but not in the thermophile are listed in Table III. Interestingly, all the equivalent residues found in the thermophile are also in solvent-accessible positions. These residues in the thermophiles could again be possible targets of mutation for protein engineers in order to increase further the stability of the thermophilic proteins, i.e. to convert a thermophile to a hyperthermophile. For example, Leu35 and Glu39 in ribonuclease H (family 4) can be possibly mutated to Phe and Tyr, respectively, to enhance further the stability of the protein.
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This study on 24 pairs of structurally similar thermophilic and mesophilic proteins has shown that the thermophilic proteins have a large number of pairwise aromatic interactions compared with the mesophilic homologue. Certain families also show a presence of additional aromatic clusters which are larger in size. The additional clusters are located on the protein surface and are more rigid regions of the surface. The topologically equivalent mutations in the mesophiles are usually to non-aromatic Leu or Ile residues if the replacement is for Phe and either to Ser or Leu if a Tyr residue is mutated. The presence of at least one additional aromatic cluster close to the active site of the thermophile provides a plausible explanation for the high thermophilicity exhibited by most thermostable enzymes. During the course of evolution, the organisms had probably achieved viability by carefully mutating the surface residues and hence introducing an aromatic pair or an additional aromatic cluster in the protein.
Although the dataset considered in this study is limited, the consistent observation of additional aromatic clusters in nearly 70% of the protein families is significant. Also, the fact that this could be an elegant way of increasing thermal stability suggests that nature could probably have taken this simpler and elegant approach to adapt itself to higher temperatures. This study also provides new insights for protein engineers to design thermophilic and thermostable proteins.
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Notes |
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Acknowledgments |
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References |
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Received May 16, 2000; revised September 11, 2000; accepted September 28, 2000.