title: 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 (Trp(29)-Sec(52)-Gln(72)). 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 Ser(52)/Sec substitution is critical for the GPx activity of Se-scFv2F3. In addition, Phe(29)/Trp-Val(72)/Gln mutations enhance the reaction rate via functional cooperation with Sec(52). Molecular dynamics simulations showed that the designed catalytic triad is very stable and the conformational flexibility caused by Tyr(101) 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 Tyr(54), Asn(55), and Gly(56) 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 Trp(29)-Sec(52)-Gln(72) 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.
authors.affiliation: State Key Laboratory of Supramolecular Structure and Materials, Institute of Theoretical Chemistry, Jilin University, Changchun 130012, People's Republic of China. luoquan@jlu.edu.cn
authors.forename: Quan
authors.initials: Q
authors.lastname: Luo
authors.forename: Chunqiu
authors.initials: C
authors.lastname: Zhang
authors.forename: Lu
authors.initials: L
authors.lastname: Miao
authors.forename: Dongmei
authors.initials: D
authors.lastname: Zhang
authors.forename: Yushi
authors.initials: Y
authors.lastname: Bai
authors.forename: Chunxi
authors.initials: C
authors.lastname: Hou
authors.forename: Junqiu
authors.initials: J
authors.lastname: Liu
authors.forename: Fei
authors.initials: F
authors.lastname: Yan
authors.forename: Ying
authors.initials: Y
authors.lastname: Mu
authors.forename: Guimin
authors.initials: G
authors.lastname: Luo
mesh_terms: D000595:Amino Acid Sequence; D017151:Antibodies, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D005979:Glutathione Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D017151:Antibodies, Catalytic; D057127:Single-Chain Antibodies; D005979:Glutathione Peroxidase