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Triple mutated antibody scFv2F3 with high GPx activity: insights from MD, docking, MDFE, and MM-PBSA simulation

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

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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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Correspondence to Quan Luo or Junqiu Liu.

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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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Keywords

  • Abzyme
  • GPx mimic
  • Docking
  • MD simulation
  • MDFE simulation
  • MM-PBSA