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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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Structure of the CMG2-PA complex.(A) The crystal structure of the CMG2-PA complex (PDB ID: 1T6B). PA is shown in red, CMG2 is shown in green, and the interface residues on CMG2 are shown in blue. (B) Close views of the interactions between PA D683 and CMG2 E117. The two residues are shown as sticks and balls. PA D683 is depicted in yellow, and CMG2 E117 is depicted in blue. (C) Close views of the interactions between PA 344R and CMG2 Y158. The two residues are shown as sticks and balls. PA R344 is depicted in magenta, and CMG2 Y158 is depicted in blue.
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pone-0104674-g001: Structure of the CMG2-PA complex.(A) The crystal structure of the CMG2-PA complex (PDB ID: 1T6B). PA is shown in red, CMG2 is shown in green, and the interface residues on CMG2 are shown in blue. (B) Close views of the interactions between PA D683 and CMG2 E117. The two residues are shown as sticks and balls. PA D683 is depicted in yellow, and CMG2 E117 is depicted in blue. (C) Close views of the interactions between PA 344R and CMG2 Y158. The two residues are shown as sticks and balls. PA R344 is depicted in magenta, and CMG2 Y158 is depicted in blue.

Mentions: From the CMG2-PA complex model, we found that there were some unfavorable interactions between CMG2 and PA. One of these unfavorable interactions was caused by Glu117 of CMG2; Glu117 is located on the area of contact where CMG2 binds to PA and was opposite to the Asp683 residue of PA (Fig. 1b). When the two molecules are in close proximity, a repulsive force may form between these two negatively charged amino acids, which would be unfavorable for CMG2-PA interactions. The substitution of Glu117 with neutral polar or positively charged residues would decrease the repulsive force between the two sites. Because an increase in size of the amino acid would cause steric hindrance between the substituted residue with surrounding ones, in order to avoid causing unexpected steric hindrance, E117 of CMG2 should be mutated to an amino acid that has a similar side chain size to Glu. As an uncharged polar amino acid, Gln differs in charge property, but has similar size relative to Glu. The replacement of Glu117 in CMG2 with Gln may reduce the repulsive interaction with the negatively charged D683 of PA without causing a steric clash. We expected this replacement may improve the binding affinity between the two molecules.


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)

Structure of the CMG2-PA complex.(A) The crystal structure of the CMG2-PA complex (PDB ID: 1T6B). PA is shown in red, CMG2 is shown in green, and the interface residues on CMG2 are shown in blue. (B) Close views of the interactions between PA D683 and CMG2 E117. The two residues are shown as sticks and balls. PA D683 is depicted in yellow, and CMG2 E117 is depicted in blue. (C) Close views of the interactions between PA 344R and CMG2 Y158. The two residues are shown as sticks and balls. PA R344 is depicted in magenta, and CMG2 Y158 is depicted in blue.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104674-g001: Structure of the CMG2-PA complex.(A) The crystal structure of the CMG2-PA complex (PDB ID: 1T6B). PA is shown in red, CMG2 is shown in green, and the interface residues on CMG2 are shown in blue. (B) Close views of the interactions between PA D683 and CMG2 E117. The two residues are shown as sticks and balls. PA D683 is depicted in yellow, and CMG2 E117 is depicted in blue. (C) Close views of the interactions between PA 344R and CMG2 Y158. The two residues are shown as sticks and balls. PA R344 is depicted in magenta, and CMG2 Y158 is depicted in blue.
Mentions: From the CMG2-PA complex model, we found that there were some unfavorable interactions between CMG2 and PA. One of these unfavorable interactions was caused by Glu117 of CMG2; Glu117 is located on the area of contact where CMG2 binds to PA and was opposite to the Asp683 residue of PA (Fig. 1b). When the two molecules are in close proximity, a repulsive force may form between these two negatively charged amino acids, which would be unfavorable for CMG2-PA interactions. The substitution of Glu117 with neutral polar or positively charged residues would decrease the repulsive force between the two sites. Because an increase in size of the amino acid would cause steric hindrance between the substituted residue with surrounding ones, in order to avoid causing unexpected steric hindrance, E117 of CMG2 should be mutated to an amino acid that has a similar side chain size to Glu. As an uncharged polar amino acid, Gln differs in charge property, but has similar size relative to Glu. The replacement of Glu117 in CMG2 with Gln may reduce the repulsive interaction with the negatively charged D683 of PA without causing a steric clash. We expected this replacement may improve the binding affinity between the two molecules.

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