Improvement of a Potential Anthrax Therapeutic by Computational Protein Design |
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Authors: | Sean J Wu Christopher B Eiben John H Carra Ivan Huang David Zong Peixian Liu Cindy T Wu Jeff Nivala Josef Dunbar Tomas Huber Jeffrey Senft Rowena Schokman Matthew D Smith Jeremy H Mills Arthur M Friedlander David Baker Justin B Siegel |
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Institution: | From the ‡Department of Biochemistry and ;¶Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195 and ;the §United States Army Medical Research Institute of Infectious Diseases, Frederick, Maryland 21702 |
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Abstract: | Past anthrax attacks in the United States have highlighted the need for improved measures against bioweapons. The virulence of anthrax stems from the shielding properties of the Bacillus anthracis poly-γ-d-glutamic acid capsule. In the presence of excess CapD, a B. anthracis γ-glutamyl transpeptidase, the protective capsule is degraded, and the immune system can successfully combat infection. Although CapD shows promise as a next generation protein therapeutic against anthrax, improvements in production, stability, and therapeutic formulation are needed. In this study, we addressed several of these problems through computational protein engineering techniques. We show that circular permutation of CapD improved production properties and dramatically increased kinetic thermostability. At 45 °C, CapD was completely inactive after 5 min, but circularly permuted CapD remained almost entirely active after 30 min. In addition, we identify an amino acid substitution that dramatically decreased transpeptidation activity but not hydrolysis. Subsequently, we show that this mutant had a diminished capsule degradation activity, suggesting that CapD catalyzes capsule degradation through a transpeptidation reaction with endogenous amino acids and peptides in serum rather than hydrolysis. |
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Keywords: | Antibiotics Enzyme Mutation Enzymes Hydrolases Protein Stability Anthrax CapD Circular Permutation Rosetta |
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