Is it possible to stabilize a thermophilic protein further using sequences and structures of mesophilic proteins: a theoretical case study concerning DgAS
© Liu et al.; licensee BioMed Central Ltd. 2013
Received: 28 February 2013
Accepted: 29 March 2013
Published: 10 April 2013
Incorporating structural elements of thermostable homologs can greatly improve the thermostability of a mesophilic protein. Despite the effectiveness of this method, applying it is often hampered. First, it requires alignment of the target mesophilic protein sequence with those of thermophilic homologs, but not every mesophilic protein has a thermophilic homolog. Second, not all favorable features of a thermophilic protein can be incorporated into the structure of a mesophilic protein. Furthermore, even the most stable native protein is not sufficiently stable for industrial applications. Therefore, creating an industrially applicable protein on the basis of the thermophilic protein could prove advantageous. Amylosucrase (AS) can catalyze the synthesis of an amylose-like polysaccharide composed of only α-1,4-linkages using sucrose as the lone energy source. However, industrial development of AS has been hampered owing to its low thermostability. To facilitate potential industrial applications, the aim of the current study was to improve the thermostability of Deinococcus geothermalis amylosucrase (DgAS) further; this is the most stable AS discovered to date. By integrating ideas from mesophilic AS with well-established protein design protocols, three useful design protocols are proposed, and several promising substitutions were identified using these protocols. The successful application of this hybrid design method indicates that it is possible to stabilize a thermostable protein further by incorporating structural elements of less-stable homologs.
Life flourishes almost everywhere on earth, from hydrothermal vents in the deep-sea to the tops of the Himalayas, from rain forests to the hot sands of the Sahara desert, and even from the boiling waters of hot springs to the cold ice field of Antarctica. Organisms that inhabit such harsh environments must evolve to adapt those living conditions. In relation to temperature adaptations, environmental stress generally cannot be avoided through compensatory mechanisms, as is the case for other types of adaptations . Therefore, cellular and cytoplasmic components, specifically proteins, must achieve thermostability . For this reason, much effort has been directed towards understanding how proteins from thermophilic organisms retain their structure and function at elevated temperatures [3–7]. Such understanding is essential for a theoretical description of the physicochemical principles underlying protein folding and stability, but it is also critical for designing proteins that can work at high temperatures or are more resistant to unfolding at certain working temperatures. High thermostability is required for several industrial applications including detergent manufacturing, food and starch processing, production of high fructose corn syrup and PCR [8–10]. Furthermore, thermophilic proteins are more resistant to proteolysis and chemical denaturation than their mesophilic homologs . In general, thermophilic proteins possess multiple features that are important for high thermostability including more hydrogen bonds and salt bridges, and higher contact order, than their mesophilic counterparts. In view of this, much research has focused on elevating the thermostability of mesophilic proteins through investigating the features of their thermophilic homologs. By substituting key residues or even motifs on the basis of the sequences of homologs, the thermostability of a mesophilic protein can be improved relatively easily [12, 13]. For example, Németh and colleagues improved the Tm of a cellulose C by 3°C , and in our opinion this approach is significantly more effective than well-established experimental approaches such as library screening and random site-directed mutagenesis.
Despite the effectiveness of this method, applications of it are often hampered. First, it requires alignment of the target mesophilic protein sequence with those of thermophilic homologs, but not every mesophilic protein has a thermophilic homolog. Even though one can find the sequences from corresponding thermophilic proteins, the design accuracy remains limited owing to a lack of structural information. Although structure prediction has become a routine step during protein engineering, it requires a very skillful computational biologist to perform this well. Second, not all favorable features of a thermophilic protein can be incorporated into the structure of a mesophilic protein, as the function of the target protein must remain intact. In industry, even so-called ‘thermophilic’ proteins are not stable enough for industrial applications, and in this situation it is difficult to create a more thermostable protein from a mesophilic one. Therefore, creating an industrially applicable protein on the basis of the thermophilic protein could prove advantageous. To attain this objective, experimental biologists usually utilize well-established methods including library screening and random site-directed mutagenesis. Although effective, these methods can be time-consuming and costly. Furthermore, traditional methods such as library screening can result in researchers obtaining the same sequence time and time again, owing to limited evolutionary selection pressure. With the development of molecular modeling theory and computer science, several rational design approaches have been developed, and rational designs have been progressively applied as a routine step during protein thermostability engineering. However, the success rate for purely rational design is largely discounted because of the lack of structural information and design experience. Therefore, the question of whether it is possible to learn something from those mesophilic proteins to improve the thermostability of thermophilic proteins further must be addressed.
Amylosucrase (AS) is a type of glucosyltransferases (E.C. 184.108.40.206) that belongs to the Glycoside Hydrolase (GH) Family 13. In the presence of an activator polymer, in vitro, AS can catalyze the synthesis of an amylose-like polysaccharide composed of only α-1,4-linkages, using sucrose as the lone energy source, making it a potential candidate for industrial applications. Other enzymes responsible for the synthesis of such amylose-like polymers require the addition of expensive nucleotide-activated sugars. Despite its potential, the industrial development of AS is limited owing to its weak thermostability. During this study, we wished to compare the structure of Deinococcus geothermalis amylosucrase (DgAS), the most thermostable amylosucrase identified to date, with those of mesophilic amylosucrases, namely Neisseria polysaccharea amylosucrase (NpAS), Deinococcus radiodurans (DrAS) and the newly determined Arthrobacter chlorophenolicus (AcAS) , in the hope of further improving its thermostability. Using these mesophilic homologs, we have identified some promising substitutions, and believe these designs are helpful for improving the thermostability of DgAS.
Materials and methods
DgAS and NpAS are the only two ASs whose structures have been experimentally determined, and their crystal structures can be accessed via the protein data bank (PDB, http://www.rcsb.org/). During this work, PDB files of 3UCQ  and 1G5A  were chosen for DgAS and NpAS, respectively, as in our previous work  and for the same reason. These two files were processed before further analysis, following the procedures outlined in . In relation to DrAS and the newly identified AcAS, the powerful modeling program I-TASSER [18, 19] was used. All parameters of I-TASSER were kept at default settings. The sequence identities between DrAS and NpAS/DgAS were 41% and 74%, respectively. For AcAS, the values were 57% and 44%.
Structure and sequence comparison
With the aim of discovering how to improve the thermostability of DgAS, the structure of DgAS was superimposed on to those of NpAS, DrAS and AcAS using the “Align and Superimpose Proteins” protocol provided by Discovery Studio (DS). Details concerning the comparisons between the structure of DgAS and those of the mesophilic homologs are described in the Results section.
Sequence alignment was not arbitrarily performed using the BLAST program ; we utilized structural similarity and human expertise during the sequence aligning process to correct the alignment generated by automatic methods.
H-bonds and salt-bridges
H-bonds and salt-bridges are important contributors to protein thermostability. Properly incorporating an H-bond or a salt-bridge (usually on the protein surface) can improve thermostability. These two values are calculated by VMD . The distance (between the donor and the acceptor atoms) and angle (donor-hydrogen-acceptor) cutoff for a H-bond were set to 3.0 Å and 150°, respectively. The distance cutoff for a salt bridge was set to 3.5 Å.
Contact orders of four AS were calculated with default parameters by the contact order calculation server (http://depts.washington.edu/bakerpg/contact_order/) provided in Washington University.
In a previous study, we demonstrated that the value of “number-of-contacts-per-residue (NCPR)” is related to protein thermostability and unfolding order . Herein, we rename NCPR ‘contact density’ for convenience. The contact density value corresponds directly to the compactness of a protein, which has been demonstrated to be critical for protein thermostability . The method for calculating contact density has been described previously .
Free energy calculation
Thermostability is strongly correlated to folding free energy. To evaluate the thermodynamic stability of wild type (WT) and mutant DgAS, the folding free energy changes were estimated utilizing the FoldX program [26–28], which uses a full atomic description of the structure of a protein. The predictive power of the FoldX force field has been tested on a very large set (more than a thousand) of point mutants spanning most of the structural environments found in proteins. Detailed descriptions of the energy function used by FoldX are addressed elsewhere [26–28].
Local structure entropy (LSE)
Since structure conservation reflects the effects of intrinsically stable (context-independent) sequence patterns and long-range generic contributions (context dependent) from surrounding residues , structural entropy provides a convenient structural measure of thermostability. The LSE value of a protein is closely related to its intrinsic thermostability; in general, thermostable proteins have smaller LSE values than their mesophilic homologs. For detailed descriptions of the LSE method, please see Chan’s original work . In the current LSE was calculated for DgAS and its mutants using a JAVA program (in house script).
Modeling and validation of the structures of DrAS and AcAS
The structures of AcAS and DrAS were modeled using I-TASSER, based on the structural information relating to DgAS and NpAS. Models were sorted according to their C-scores, and the one with the best C-score were selected as the final model (Additional file 1: Figure S1, structural models). The C-scores of the final models for DrAS and AcAS are 1.39 and 1.02, respectively. The C-score is a confidence score for estimating the quality of predicted models by I-TASSER, and typically ranges from −5 to 2. A higher C-score signifies a model that can be regarded with confidence. Subsequently, the final structural models of AcAS and DrAS were evaluated using Ramachandran plots [31, 32] and Profiles-3D .
According to the final models produced by I-TASSER, only 1.1% and 1.0% of the residues in AcAS and DrAS, respectively, are located in the disallowed regions of the Ramachandran plot. In comparison, 0.2% and 0.1% residues of DgAS and NpAS, respectively, are located in the disallowed regions. On the basis of this comparison, the models of AcAS and DrAS are comparable in overall quality with the experimentally determined structures of DgAS and NpAS, at least in terms of backbone conformations.
VERIFY scores for the four AS structures
Structural comparison between DgAS and mesophilic homologs
The structures of DgAS and its mesophilic homologs were compared to gain insight into their differences and to discover rules important for thermostability design. The structure of DgAS was superimposed on to those of its mesophilic homologs and the root-mean-squared deviation (RMSD) between DgAS and the other ASs were calculated as 0.64, 2.38 and 1.47 Å (in the sequence DrAS, NpAS and AcAS). On the basis of this structure superimposition, sequence alignment (Additional file 2: Figure S2) for these ASs was adjusted accordingly to improve the design accuracy.
Comparing a protein with several homologs in great detail is extremely laborious, particularly when several deletions/insertions exist. We, therefore, will just address major differences here.
D2_Ac is also located in the B-domain, closely resembling the conformation of D1_Dr. D4_Ac is situated at the joint between the C-terminal of the B’-domain and the N-terminal of the α7-helix of the A-domain. According to structure and sequence comparisons, corresponding regions of DgAS and DrAS are eight residues longer than those of AcAS. According to the crystal structure of DgAS, E473 forms a salt-bridge with R545 (α8), improving connections between α7 and α8. However, this salt-bridge does not exist in AcAS owing to deletion and substitution in corresponding positions. The last major difference (D5_Ac) between DgAS and AcAS lies in a beta-turn of the C-domain, where AcAS is three residues longer than DgAS. The proline-enriched elongated beta-turn could help to stabilize the local structure of AcAS by increasing rigidity.
Factors critical for thermostability
Some generally accepted key factors for thermostability were calculated for the four ASs to analyze structural differences that cannot be detected visually. Under no circumstances could we produce successful designs without knowing why DgAS is more stable than other ASs. It should be noted that homology models were used to calculate structural properties. Although the models of AcAS and DrAS were predicted carefully, we cannot guarantee that all calculations based on these models are correct.
The distribution of proline residues in the four ASs among each domain
According to Table 2, proline numbers for these four ASs range from 30 to 43. Among these proline residues, only 17 are absolutely conserved. Surprisingly, AcAS has the most proline residues, whereas DgAS, the most stable AS identified so far, has only one more proline than NpAS. This demonstrates that the total proline number alone is not a good indicator of stability, a point stressed in our previous work , where we found that the proline number for each individual domain of the stable DgAS is not necessarily more than that of NpAS, although the total proline number of NpAS is six lower than that of DgAS. This provides the possibility of enhancing protein stability by substituting proper residues with proline residues based on less stable proteins. This point will be discussed further in the following section.
H-bonds and salt-bridges
H-bonds and salt-bridges of the four ASs
H-bonds (3.0Å, 150°)
Salt-bridges (3.5 Å)
DgAS has more H-bonds than any other AS, particularly in relation to backbone-backbone type H-bonds. It also has the most salt-bridges. Surprisingly, we discovered that DrAS and AcAS have fewer backbone-backbone H-bonds and salt-bridges than DgAS and NpAS. To minimize errors introduced by the structure modeling process, a series of rigorous refinements and minimizations were carried out, but the results were comparable. In addition to the modeling error, we propose that the large difference in H-bonds and particularly in salt-bridges could have resulted for other reasons.
During the current study we identified that DgAS has more charged residues than any other AS. Although DrAS has the second most charged residues, almost 20% of these are not conserved between DgAS and DrAS. From visual inspection it was determined that DrAS has more unpaired charged residues than DgAS, owing to their different locations; other than NpAS, AcAS has the fewest positively-charged residues. Although AcAS has the second highest number of negatively-charged residues, several are unpaired with positively-charged residues owing to their locations.
The electrostatic interactions of these four ASs were calculated with the CHARMm [34, 35] module embedded in DS. On the basis these calculations, electrostatic interactions in DgAS are significantly stronger than in the other ASs (DgAS > DrAS, NpAS > AcAS). This implies that the strong electrostatic interactions in DgAS are partially responsible for its superior stability.
Contact order and contact density
Contact orders and contact densities of the four ASs
Potential energy and corresponding VDW and electrostatic contribution of the four ASs
Design trials and computational validation
With the rapid development of theory concerning protein structure and computer science, the domain of protein design, or more specifically redesign, has grown increasingly prosperous. Several design tools and skills have been proposed over the past few decades. In this section, we have divided the design processes into three parts (protocols) and given representative cases for each design protocol. All designs were evaluated by free energy calculation and LSE.
The easy way: substitution of specific residues with proline residues
Mutants designed by the second protocol (X-Pro substitutions)
φ / ψ (°)
Empirically, substitutions with negative ΔΔGf and negative ΔLSE are more likely to enhance the stability of the engineered protein than substitutions with either solely negative ΔΔG or negative ΔLSE (in-house data). Substitutions with ΔΔGf > −0.5 kcal⋅mol-1 are excluded from further consideration for the reason explained in the part of the methods section concerning free energy calculation. According to these criteria, only six substitutions remain for subsequent experimental validation (not included in this paper). For the sake of clarity, all substitutions that passed all filters in each design trial were nominated as ‘promising substitutions’.
The confused way: should we substitute glycine residues with bigger ones or vice versa?
Glycine is the most flexible of the 20 naturally occurring amino acid residues. It is usually located at turns or loops in proteins, and can access larger conformational spaces than other residues. Because glycine is small, it is prone to facilitate the motions of local structures around it and increase the conformational entropy of any state. The unfolded state is an ensemble of several non-native states, so the overall effect of introducing a glycine residue into a protein should be unfavorable for stability. Since glycine is the only residue whose backbone can adopt φ > 0 with little steric hindrance, it should, as far as possible, occur in the right half of the Ramachandran plot. A glycine residue with negative φ should be replaced by other residues to elevate thermostability if the space around it is large enough. For the sake of convenience, we nominate the glycine residues with positive φ as φ+ glycine residues, and the ones with negative φ as φ- glycine residues.
During the course of evolution, selection pressure has rendered the sequences and structures of native proteins optimal or thereabouts in terms of function and stability. However, if native proteins are nearly optimal, why are there still many seemingly unnecessary glycine residues in them? A likely explanation is that these “unnecessary” glycine residues are actually indispensable for the trade-off between function and stability. Previously, we identified only one dispensable glycine residue among the 49 in DgAS , indicating that several glycine residues are indispensable for the functioning and stability of this protein. However, substituting a normal (i.e. any except Gly and Pro) residue with glycine can improve the stability of the target protein. Previously, during stability engineering on a human-source antibody, we substituted an alanine residue located at a β-turn (whose φ is positive) with a glycine residue. The half-inactivation temperature of the engineered antibody was elevated by 1.2°C (unpublished data). In the current study we attempted to substitute residues with positive φ with glycine residues.
Mutants designed by the second protocol (X-Gly substitutions)
φ / ψ (°)
As Table 7 demonstrates, all possible substitutions are located in coils or β-turns. Surprisingly, LSE results suggested all substitutions have an adverse effect on stability. This may be attributed, at least in part, to the statistical nature of the LSE method. The LSE method only takes the local sequence (4 residues) into consideration despite proteins being three-dimensional. Long-range contacts are always observed, not only in large, multi-domain proteins but also in many small proteins. To avoid this defect, we propose that the LSE method should be used together with structure-based methods when possible.
The moderate way: enhance interactions among target residues
Mutants designed by the third protocol (enhancing interactions)
Enhancing electrostatic interactions among residues is important during stability engineering, and this can be achieved by increasing the number of H-bonds and salt-bridges. A285R, A287K, A415E and V444Q are good examples of this; V444Q (Figure 6C) was selected on the basis of sequences of the less-stable AS (the corresponding position in DrAS is Q436).
A helix has an overall dipole moment caused by the cumulative effect of individual dipoles from the well-ordered amide groups pointing along the helix axis. This can lead to destabilization of the helix through entropic effects. To ease this adverse effect, the N or C caps of an α-helix can be modified to compensate for the dipole caused by the periodic nature of the α-helix. N413 is the N-cap of α-helix413-426 in the B’-domain (Figure 6D), and substituting it with an aspartate residue can effectively neutralize the positively-charged N-terminal of the helix, thereby removing the unfavorable dipole.
ΔLSE for these selected substitutions were calculated. In this type of design, it was determined that the LSE results did not correlate well with the free energy calculation. Chan’s work  suggests that this lack of correlation between the two methods can be attributed, at least in part, to the statistical nature of the LSE method.
During this study, the sequence and structure of DgAS was compared with those of DrAS, NpAS and the newly identified AcAS, and it was discovered that DgAS has favorable structural properties that can, in part, account for its superior stability. First, DgAS has the highest contact density, reflecting its strong VDW interactions. Second, the electrostatic energy of DgAS is much greater than that of the other ASs, which can be largely attributed to its excessive number of backbone-backbone H-bonds and salt-bridges.
Several groups have demonstrated that incorporating structural elements of thermostable proteins into their mesophilic homologs can improve stability. However, few protein designers have tried to find useful structural elements from less-stable proteins. This work not only focused on identifying allowable substitutions for DgAS stability engineering, but attempted to utilize useful structural elements from the other three ASs. By comparing the structure and sequence of DgAS with those of the others, promising substitutions were identified. In the first design trial (proline design), we attempted to introduce additional proline residues into the DgAS sequence, using the sequences of the other three ASs as references. Subsequently, in the second design trial (glycine design), the comparative design method was employed together with an empirical method to determine more designable positions. Finally, during the third design trial, we took advantage of automatic semi-saturation scanning and identified more allowable substitutions. On the basis of our analyses, some structural elements of less-stable proteins are better than their counterparts in the stable protein. This is not surprising, as a protein of 600 amino acid residues, theoretically, has an astronomical number (10780) of possible combinations. Therefore, even evolutionary selection pressure cannot guarantee that the most stable protein is constituted by the best structural elements. It is because of this that can we further improve the stability of the stable DgAS by utilizing structural elements from less-stable ASs. Given our experience of protein design (the author once designed hundreds of mutants for elevating stability, and the total accuracy was more than 40%), we believe several of these selected substitutions could enhance the stability of DgAS. All mutants shown here will be validated by subsequent experiments, the results of which will be presented in the near future.
In conclusion, we found that it is possible to stabilize a protein from thermophilic bacteria further by incorporating structural elements from less-stable proteins. On the basis of this work, it appears that the X-to-proline method can be easily integrated with information from other homologs. For proteins with few allowable positions for proline residues, the semi-saturation scanning method would be suitable. Although the glycine substitution method is not as effective as the other two, it could complement other methods. In addition to the design protocols mentioned above, semi-reasonable methods such as peptide fragment substitutions or domain swaps could also be used. However, the applications of these methods are limited, as protein engineers run the risk of impairing the functions and/or expression levels of the target protein. From our experience of daily design, a very complicated combination of single-site mutants is much more effective than the seemingly simple peptide fragment substitution method.
The authors of this paper appreciate helpful suggestions from Bin Liu, Ping Li, Jianping Hu, Liang Wang and Xing Huang.
This work was supported by National Natural Science Foundation (81202438 and 11204267).
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