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Improving the anti-toxin abilities of the CMG2-Fc fusion protein with the aid of computational design.

Xi Y, Wu X, Gao L, Shao Y, Peng H, Chen H, Chen H, Hu X, Yue J - PLoS ONE (2014)

Bottom Line: An experimental affinity assay revealed that the two variants showed increased binding affinity, and in vitro and in vivo toxin neutralization testing indicated that one of these mutants (CMG2-Fc(E117Q)) has superior activity against anthrax toxin and was suitable for further development as a therapeutic agent for anthrax infections.This study shows that the computational design of the PA binding interface of CMG2 to obtain CMG2-Fc variants with improving anti-toxin abilities is viable.Our results demonstrate that computational design can be further applied to generate other CMG2-Fc mutants with greatly improved therapeutic efficacy.

View Article: PubMed Central - PubMed

Affiliation: Beijing Institute of Biotechnology, Beijing, China.

ABSTRACT
CMG2-Fc is a fusion protein composed of the extracellular domain of capillary morphogenesis protein 2 (CMG2) and the Fc region of human immunoglobulin G; CMG2-Fc neutralizes anthrax toxin and offers protection against Bacillus anthracis challenge. To enhance the efficacy of CMG2-Fc against anthrax toxin, we attempted to engineer a CMG2-Fc with an improved affinity for PA. Using the automatic design algorithm FoldX and visual inspection, we devised two CMG2-Fc variants that introduce mutations in the CMG2 binding interface and improve the computationally assessed binding affinity for PA. An experimental affinity assay revealed that the two variants showed increased binding affinity, and in vitro and in vivo toxin neutralization testing indicated that one of these mutants (CMG2-Fc(E117Q)) has superior activity against anthrax toxin and was suitable for further development as a therapeutic agent for anthrax infections. This study shows that the computational design of the PA binding interface of CMG2 to obtain CMG2-Fc variants with improving anti-toxin abilities is viable. Our results demonstrate that computational design can be further applied to generate other CMG2-Fc mutants with greatly improved therapeutic efficacy.

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Predicted structures for designed mutations; green ribbon, PA backbone; magenta ribbon, CMG2 backbone.Carbon atom, cyan; Nitrogen atom, blue; Oxygen atom, red. Mutation of CMG2 117 Glu to Gln is shown in (A) and (B), residues 117, 52, 54 and PA 683 are highlighted as sticks and balls. (C) and (D) indicate CMG2 158Tyr to Gln, position 158 of CMG2 and residues 342–344 of PA are highlighted as sticks and balls. Binding energies are given beneath.
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pone-0104674-g002: Predicted structures for designed mutations; green ribbon, PA backbone; magenta ribbon, CMG2 backbone.Carbon atom, cyan; Nitrogen atom, blue; Oxygen atom, red. Mutation of CMG2 117 Glu to Gln is shown in (A) and (B), residues 117, 52, 54 and PA 683 are highlighted as sticks and balls. (C) and (D) indicate CMG2 158Tyr to Gln, position 158 of CMG2 and residues 342–344 of PA are highlighted as sticks and balls. Binding energies are given beneath.

Mentions: In the predicted CMG2(E117Q)-PA complex model, the repulsive electrostatic interaction between Asp683 of PA and position 117 of CMG2 did disappear (Figure 2). Together with diminishing the repulsive interaction, E117Q mutant broke the hydrogen bonding formed between the E117 of CMG2 and the D683 of PA, but the overall effect of the charge inversion at this position gave a positive contribution to the binding between CMG2 and PA. Binding free energy (ΔΔG binding) calculations indicated that this mutation produced a lower binding free energy relative to WT (Figure 2). Overall, substitution in position 117 of CMG2 increases the binding affinity between PA and CMG2.


Improving the anti-toxin abilities of the CMG2-Fc fusion protein with the aid of computational design.

Xi Y, Wu X, Gao L, Shao Y, Peng H, Chen H, Chen H, Hu X, Yue J - PLoS ONE (2014)

Predicted structures for designed mutations; green ribbon, PA backbone; magenta ribbon, CMG2 backbone.Carbon atom, cyan; Nitrogen atom, blue; Oxygen atom, red. Mutation of CMG2 117 Glu to Gln is shown in (A) and (B), residues 117, 52, 54 and PA 683 are highlighted as sticks and balls. (C) and (D) indicate CMG2 158Tyr to Gln, position 158 of CMG2 and residues 342–344 of PA are highlighted as sticks and balls. Binding energies are given beneath.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4125234&req=5

pone-0104674-g002: Predicted structures for designed mutations; green ribbon, PA backbone; magenta ribbon, CMG2 backbone.Carbon atom, cyan; Nitrogen atom, blue; Oxygen atom, red. Mutation of CMG2 117 Glu to Gln is shown in (A) and (B), residues 117, 52, 54 and PA 683 are highlighted as sticks and balls. (C) and (D) indicate CMG2 158Tyr to Gln, position 158 of CMG2 and residues 342–344 of PA are highlighted as sticks and balls. Binding energies are given beneath.
Mentions: In the predicted CMG2(E117Q)-PA complex model, the repulsive electrostatic interaction between Asp683 of PA and position 117 of CMG2 did disappear (Figure 2). Together with diminishing the repulsive interaction, E117Q mutant broke the hydrogen bonding formed between the E117 of CMG2 and the D683 of PA, but the overall effect of the charge inversion at this position gave a positive contribution to the binding between CMG2 and PA. Binding free energy (ΔΔG binding) calculations indicated that this mutation produced a lower binding free energy relative to WT (Figure 2). Overall, substitution in position 117 of CMG2 increases the binding affinity between PA and CMG2.

Bottom Line: An experimental affinity assay revealed that the two variants showed increased binding affinity, and in vitro and in vivo toxin neutralization testing indicated that one of these mutants (CMG2-Fc(E117Q)) has superior activity against anthrax toxin and was suitable for further development as a therapeutic agent for anthrax infections.This study shows that the computational design of the PA binding interface of CMG2 to obtain CMG2-Fc variants with improving anti-toxin abilities is viable.Our results demonstrate that computational design can be further applied to generate other CMG2-Fc mutants with greatly improved therapeutic efficacy.

View Article: PubMed Central - PubMed

Affiliation: Beijing Institute of Biotechnology, Beijing, China.

ABSTRACT
CMG2-Fc is a fusion protein composed of the extracellular domain of capillary morphogenesis protein 2 (CMG2) and the Fc region of human immunoglobulin G; CMG2-Fc neutralizes anthrax toxin and offers protection against Bacillus anthracis challenge. To enhance the efficacy of CMG2-Fc against anthrax toxin, we attempted to engineer a CMG2-Fc with an improved affinity for PA. Using the automatic design algorithm FoldX and visual inspection, we devised two CMG2-Fc variants that introduce mutations in the CMG2 binding interface and improve the computationally assessed binding affinity for PA. An experimental affinity assay revealed that the two variants showed increased binding affinity, and in vitro and in vivo toxin neutralization testing indicated that one of these mutants (CMG2-Fc(E117Q)) has superior activity against anthrax toxin and was suitable for further development as a therapeutic agent for anthrax infections. This study shows that the computational design of the PA binding interface of CMG2 to obtain CMG2-Fc variants with improving anti-toxin abilities is viable. Our results demonstrate that computational design can be further applied to generate other CMG2-Fc mutants with greatly improved therapeutic efficacy.

Show MeSH
Related in: MedlinePlus