Abstract
Use of point mutagenesis technique to improve protein thermostability is a routine strategy in the protein engineering community and directed evolution approach has been widely utilized to fulfill this. However, directed evolution often does not assure a minimalist design for obtaining a desired property in proteins, and other traditional methods such as error-prone PCR and iterative saturation mutagenesis are also too time-consuming and expensive to carry out a systemic search for protein mutation space. In the current study, we performed mutatomics analysis of the systematic thermostability profile of Bacillus subtilis lipase A (LipA) using a virtual scanning strategy. In the procedure, a new characterization method was proposed to describe structural variations upon protein residue mutation, and the generated descriptors were then statistically correlated with protein thermostability change associated with the mutation based on a large panel of structure-solved, melting temperature-known protein mutation data. As a result, linear and nonlinear quantitative structure-thermostability relationship (QSTR) models were built and their statistical quality was verified rigorously through internal cross-validation and external blind test. It is suggested that the nonlinear support vector machine (SVM) performed much better than linear partial least squares (PLS) regression in correlating protein structure and thermostability information. Thus, the SVM model was employed to systematically scan the complete mutation profile of LipA protein, resulting in a 181×19 matrix that characterizes the change in theoretical thermostability of LipA due to the mutation of wild-type residue at each of the 181 sequence sites to other 19 amino acid types. From the profile most mutations were predicted to (i) destabilize LipA structure and (ii) address modest effect on LipA thermostability. Satisfactorily, several known thermostable mutations such as G80V, G111D, M134D, and N161Y were identified properly and, expectedly, a number of mutations including L55Y, A75V, and S162P that have never been reported previously were inferred as hotspot mutations that have high potential to enhance LipA thermostability. The structural basis and energetic property of the five promising mutations were further examined in detail using atomistic molecular dynamics (MD) simulations and molecular mechanics Poisson-Boltzmann/surface area (MM-PB/SA) analysis, revealing intensive nonbonded interaction networks created by these mutations.
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References
Eggert T, van Pouderoyen G, Dijkstra BW, Jaeger KE (2001) Lipolytic enzymes LipA and LipB from Bacillus subtilis differ in regulation of gene expression, biochemical properties, and three-dimensional structure. FEBS Lett 502:89–92
van Pouderoyen G, Eggert T, Jaeger KE, Dijkstra BW (2001) The crystal structure of Bacillus subtilis lipase: a minimal alpha/beta hydrolase fold enzyme. J Mol Biol 309:215–226
Hasan F, Shah AA, Hameed A (2006) Industrial applications of microbial lipases. Enzym Microb Technol 39:235–251
Ahmad S, Kamal MZ, Sankaranarayanan R, Rao NM (2008) Thermostable Bacillus subtilis lipases: in vitro evolution and structural insight. J Mol Biol 381:324–340
Yun HS, Park HJ, Joo JC, Yoo YJ (2013) Thermostabilization of Bacillus subtilis lipase A by minimizing the structural deformation caused by packing enhancement. J Ind Microbiol Biotechnol 40:1223–1229
Acharya P, Rajakumara E, Sankaranarayanan R, Rao NM (2004) Structural basis of selection and thermostability of laboratory evolved Bacillus subtilis lipase. J Mol Biol 341:1271–1281
Ni Z, Jin X, Zhou P, Wu Q, Lin XF (2011) A combination of computational and experimental approaches to investigate the binding behavior of B. sub lipase A mutants with substrate pNPP. Mol Inform 30:359–367
Ni Z, Jin R, Chen H, Lin X (2013) Just an additional hydrogen bond can dramatically reduce the catalytic activity of Bacillus subtilis lipase A I12T mutant: an integration of computational modeling and experimental analysis. Comput Biol Med 43:1882–1888
Ni Z, Lin X (2013) Insight into substituent effects in Cal-B catalyzed transesterification by combining experimental and theoretical approaches. J Mol Model 19:349–358
Kumar MD, Bava KA, Gromiha MM, Parabakaran P, Kitajima K, Uedaira H, Sarai A (2006) ProTherm and ProNIT: thermodynamic databases for proteins and protein-nucleic acid interactions. Nuleic Acids Res 34:D204–D206
Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE (2000) The protein data bank. Nucleic Acids Res 28:235–242
Potapov V, Cohen M, Schreiber G (2009) Assessing computational methods for predicting protein stability upon mutation: good on average but not in the details. Protein Eng Des Sel 22:553–560
Razvi A, Scholtz JM (2006) Lessons in stability from thermophilic proteins. Protein Sci 15:1569–1578
Ku T, Lu P, Chan C, Wang T, Lai S, Lyu P, Hsiao N (2009) Predicting melting temperature directly from protein sequences. Comput Biol Chem 33:445–450
Masso M, Vaisman II (2008) Accurate prediction of stability changes in protein mutants combining machine learning with structure based computational mutagenesis. Bioinformatics 24:2002–2009
Krivov GG, Shapovalov MV, Dunbrack RL (2009) Improved prediction of protein side-chain conformations with SCWRL4. Proteins 77:778–795
Duan Y, Wu C, Chowdhury S, Lee MC, Xiong G (2003) A point-charge force field for molecular mechanics simulations of proteins. J Comput Chem 24:1999–2012
Tian F, Lv Y, Yang L (2012) Structure-based prediction of protein-protein binding affinity with consideration of allosteric effect. Amino Acids 43:531–543
Case DA, Cheatham TE, Darden T, Gohlke H, Luo R, Merz KM (2005) The Amber biomolecular simulation programs. J Comput Chem 26:1668–1688
Hou T, McLaughlin W, Lu B, Chen K, Wang W (2011) Prediction of binding affinities between the human amphiphysin-1 SH3 domain and its peptide ligands using homology modeling, molecular dynamics and molecular field analysis. J Proteome Res 5:32–43
Hou T, Chen K, McLaughlin WA, Lu B, Wang W (2006) Computational analysis and prediction of the binding motif and protein interacting partners of the Abl SH3 domain. PLoS Comput Biol 2:e1
Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML (1983) Comparison of simple potential functions for simulating liquid water. J Chem Phys 79:926–935
Darden T, York D, Pedersen L (1993) Particale mesh Ewald and N.log(N) method for Ewald sums in large systems. J Chem Phys 98:10089–10092
Kollman PA, Massova I, Reyes C, Kuhn B, Huo SH (2000) Calculating structures and free energies of complex molecules: combining molecular mechanics and continuum models. Acc Chem Res 33:889–897
Golbraikh A, Tropsha A (2002) Beware of q2! J Mol Graph Model 20:269–276
Acharya P, Rajakumara E, Sankaranarayanan R, Rao NM (2004) Structural basis of selection and thermostability of laboratory evolved Bacillus subtilis lipase. J Mol Biol 341:1271–1281
Zhou P, Wang C, Ren Y, Yang C, Tian F (2013) Computational peptidology: a new and promising approach to therapeutic peptide design. Curr Med Chem 20:1985–1996
Acknowledgments
This work was supported by the One Hundred Person Project of Young Teacher in Southwest Jiaotong University (No. SWJTU12BR025) (for F.T.), the Open Research Fund Program of State Key Laboratory of Trauma, Burns and Combined Injury (for F.T.), the China Postdoctoral Science Foundation (for F.T.), the National Natural Science Foundation of China (No. 31200993) (for P.Z.), the Young Teacher Doctoral Discipline Fund of Ministry of Education of China (Nos. 20130184120034 and 20120185120025) (for F.T. and P.Z.), the Fundamental Research Funds for the Central Universities (No. ZYGX2012J111) (for P.Z.) and the Scholarship Award for Excellent Teachers of University of Electronic Science and Technology of China (for P.Z.).
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Tian, F., Yang, C., Wang, C. et al. Mutatomics analysis of the systematic thermostability profile of Bacillus subtilis lipase A. J Mol Model 20, 2257 (2014). https://doi.org/10.1007/s00894-014-2257-x
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DOI: https://doi.org/10.1007/s00894-014-2257-x