Abstract
By combining computational design and site-directed mutagenesis, we have engineered a new catalytic ability into the antibody scFv2F3 by installing a catalytic triad (Trp29–Sec52–Gln72). The resulting abzyme, Se-scFv2F3, exhibits a high glutathione peroxidase (GPx) activity, approaching the native enzyme activity. Activity assays and a systematic computational study were performed to investigate the effect of successive replacement of residues at positions 29, 52, and 72. The results revealed that an active site Ser52/Sec substitution is critical for the GPx activity of Se-scFv2F3. In addition, Phe29/Trp–Val72/Gln mutations enhance the reaction rate via functional cooperation with Sec52. Molecular dynamics simulations showed that the designed catalytic triad is very stable and the conformational flexibility caused by Tyr101 occurs mainly in the loop of complementarity determining region 3. The docking studies illustrated the importance of this loop that favors the conformational shift of Tyr54, Asn55, and Gly56 to stabilize substrate binding. Molecular dynamics free energy and molecular mechanics-Poisson Boltzmann surface area calculations estimated the pK a shifts of the catalytic residue and the binding free energies of docked complexes, suggesting that dipole–dipole interactions among Trp29–Sec52–Gln72 lead to the change of free energy that promotes the residual catalytic activity and the substrate-binding capacity. The calculated results agree well with the experimental data, which should help to clarify why Se-scFv2F3 exhibits high catalytic efficiency.
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Abbreviations
- GPx:
-
Glutathione peroxidase
- MD:
-
Molecular dynamics
- MDFE:
-
Molecular dynamics free energy
- MM-PBSA:
-
Molecular mechanics-Poisson Boltzmann surface area
- ROS:
-
Reactive oxygen species
- GSH:
-
Glutathione
- Sec:
-
Selenocysteine
- EDTA:
-
Etheylenediaminetetraacetic acid
- NADPH:
-
Reduced nicotinamide adenine dinucleotide phosphate
- CDR3:
-
Complementarity determining region 3
- RMSD:
-
Root mean-square deviation
References
Bashford D, Case DA (2000) Generalized born models of macromolecular solvation effects. Annu Rev Phys Chem 51:129–152
Battin EE, Brumaghim JL (2009) Antioxidant activity of sulfur and selenium: a review of reactive oxygen species scavenging, glutathione peroxidase, and metal-binding antioxidant mechanisms. Cell Biochem Biophys 55:1–23
Bhabak KP, Mugesh G (2010) Functional mimics of glutathione peroxidase: bioinspired synthetic antioxidants. Acc Chem Res 43:1408–1419
Braman J, Papworth C, Greener A (1996) Site-directed mutagenesis using double-stranded plasmid DNA templates. Methods Mol Biol 57:31–44
Devasagayam TP, Tilak JC, Boloor KK, Sane KS, Ghaskadbi SS, Lele RD (2004) Free radicals and antioxidants in human health: current status and future prospects. J Assoc Physicians India 52:794–804
Esworthy RS, Swiderek KM, Ho YS, Chu FF (1998) Selenium-dependent glutathione peroxidase-GI is a major glutathione peroxidase activity in the mucosal epithelium of rodent intestine. Biochim Biophys Acta 1381:213–226
Frisch MJ, Trucks GW, Schlegel HB (2003) Gaussian 03 (Revision A.1) Gaussian Pittsburgh
Fujiwara S, Amisaki T (2008) Identification of high affinity fatty acid binding sites on human serum albumin by MM-PBSA method. Biophys J 94:95–103
Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18:2714–2723
Huang X, Liu XM, Luo Q, Liu JQ, Shen JC (2011) Artificial selenoenzymes: designed and redesigned. Chem Soc Rev 40:1171–1184
Huber RE, Criddle RS (1967) Comparison of the chemical properties of selenocysteine and selenocystine with their sulfur analogs. Arch Biochem Biophys 122:164–173
Hummer G, Szabo A (1996) Calculation of free-energy differences from computer simulations of initial and final states. J Chem Phys 105:2004–2010
Kollman P (1993) Free energy calculations: applications to chemical and biochemical phenomena. Chem Reviews 93:2395–2417
Kollman PA, Massova I, Reyes C, Kuhn B, Huo S, Chong L, Lee M, Lee T, Duan Y, Wang W, Donini O, Cieplak P, Srinivasan J, Case DA, Cheatham TE III (2000) Calculating structures and free energies of complex molecules: combining molecular mechanics and continuum models. Acc Chem Res 33:889–897
Laskowski RA, Macarthur MW, Moss DS, Thornton JM (1993) Procheck: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26:283–291
Li LW, Uversky NV, Dunker K, Meroueh SO (2007) A computational investigation of allostery in the catabolite activator protein. J Am Chem Soc 129:15668–15676
Liu JQ, Luo GM, Mu Y (2012) Selenoproteins and mimics. Springer, Berlin
Lu SY, Jiang YJ, Zou JW, Wu TX (2012) Effect of double mutations K214/A-E215/Q of FRATide on GSK3β: insights from molecular dynamics simulation and normal mode analysis. Amino Acids 43:267–277
Luo Q, Han WW, Zhou YH, Yao Y, Li ZS (2008) The 3D structure of the defense-related rice protein Pir7b predicted by homology modeling and ligand binding studies. J Mol Model 14:559–569
Luo Q, Zhou YH, Yao Y, Li ZS (2010) Theoretical design of catalytic domain of abzyme Se-scFv2F3 by introducing a catalytic triad. Chem Res Chin Univ 26:118–121
Lüthy R, Bowie JU, Eisenberg D (1992) Assessment of protein models with three-dimensional profiles. Nature 356:83–85
Maiorino M, Aumann KD, Brigelius-Flohé R, Doria D, van den Heuvel J, McCarthy J, Roveri A, Ursini F, Flohé L (1995) Probing the presumed catalytic triad of selenium-containing peroxidases by mutational analysis of phospholipid hydroperoxide glutathione peroxidase (PHGPx). Biol Chem Hoppe-Seyler 376:651–660
Maiorino M, Aumann KD, Brigelius-Flohé R, Doria D, van den Heuvel J, McCarthy J, Roveri A, Ursini F, Flohé L (1998) Probing the presumed catalytic triad of selenium-containing peroxidases by mutational analysis. Z Ernahrungswiss 37:118–121
Matés JM, Pérez-Gómez C, Núñez de Castro I (1999) Antioxidant enzymes and human diseases. Clin Biochem 32:595–603
Pastor RW, Brooks BR, Szabo A (1988) An analysis of the accuracy of Langevin and molecular dynamics algorithms. A Mol Phys 65:1409–1419
Ponder JW, Case DA (2003) Force fields for protein simulations. Adv Prot Chem 66:27–85
Ren X, Gao S, You D, Huang H, Liu Z, Mu Y, Liu J, Zhang Y, Yan G, Luo G, Yang T, Shen J (2001) Cloning and expression of a single-chain catalytic antibody that acts as a glutathione peroxidase mimic with high catalytic efficiency. Biochem J 359:369–374
Scheerer P, Borchert A, Krauss N, Wessner H, Gerth C, Höhne W, Kuhn H (2007) Structural basis for catalytic activity and enzyme polymerization of phospholipid hydroperoxide glutathione peroxidase-4 (GPx4). Biochemistry 46:9041–9049
Simmerling C, Strockbine B, Roitberg AE (2002) All-atom structure prediction and folding simulations of a stable protein. J Am Chem Soc 124:11258–11259
Simonson T, Carlsson J, Case DA (2004) Proton binding to proteins: pKa calculations with explicit and implicit solvent models. J Am Chem Soc 126:4167–4180
Stanfield RL, Wilson IA (1994) Antigen-induced conformational changes in antibodies: a problem for structural prediction and design. Trends Biotechnol 12:275–279
Thannickal VJ, Fanburg BL (2000) Reactive oxygen species in cell signaling. Am J Physiol Lung Cell Mol Physiol 279:L1005–L1028
Thomas JP, Maiorino M, Ursing F, Girutti AW (1990) Protective action of phospholipid hydroperoxide glutathione peroxidase against membrane-damaging lipid peroxidation. In situ reduction of phospholipid and cholesterol hydroperoxides. J Biol Chem 265:454–461
Toppo S, Flohé L, Ursini F, Vanin S, Maiorino M (2009) Catalytic mechanisms and specificities of glutathione peroxidases: variations of a basic scheme. Biochim Biophys Acta 1790:1486–1500
Tuccinardi T, Manetti F, Schenone S, Martinelli A, Botta M (2007) Construction and validation of a RET TK Catalytic domain by homology modeling. J Chem Inf Model 47:644–655
Wilson SR, Zucker PA, Huang RRC, Spector A (1989) Development of synthetic compounds with glutathione peroxidase activity. J Am Chem Soc 111:5936–5939
Wu ZP, Hilvert B (1990) Selenosubtilisin as a glutathione peroxidase mimic. J Am Chem Soc 112:5647–5648
Acknowledgments
This work was financially supported by the Natural Science Foundation of China (No: 21234004, 91027023, 20921003, 21004028) and 111 project (B06009). We gratefully acknowledge professor David A. Case et al. for giving us the AMBER 11 software as a freeware.
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Luo, Q., Zhang, C., Miao, L. et al. Triple mutated antibody scFv2F3 with high GPx activity: insights from MD, docking, MDFE, and MM-PBSA simulation. Amino Acids 44, 1009–1019 (2013). https://doi.org/10.1007/s00726-012-1435-3
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DOI: https://doi.org/10.1007/s00726-012-1435-3