Improvement of a Potential Anthrax Therapeutic by Computational Protein Design

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.

A poly‐γ‐d‐glutamic acid depolymerase that degrades the protective capsule of Bacillus anthracis

“…Degradation of PDGA is likely to occur due to a classical hydrolytic mechanism catalysed by one of the conserved domains identified by sequence analysis. In comparison, CapD degrades PDGA by a transpeptidation reaction that relies on the transfer of a γ‐glutamyl moiety from PDGA to a nucleophile acceptor such as an amino acid, a peptide or a free amino function by a non‐sequential ‘ping‐pong’ mechanism (Wu et al ., ). Thus, degradation of the polymer by CapD depends on the presence of suitable acceptor molecules for transpeptidation and these may not always be present within the in vivo milieu pertaining during infection.…”

“…Thus, degradation of the polymer by CapD depends on the presence of suitable acceptor molecules for transpeptidation and these may not always be present within the in vivo milieu pertaining during infection. Moreover, attempts to improve the hydrolytic properties of CapD relative to its transpeptidation activity led to a marked reduction in its capsule degrading capacity (Wu et al ., ).…”

“…CapD is a γ‐glutamyltranspeptidase that also functions as a PDGA depolymerase (Candela and Fouet, ). We and others (Candela and Fouet, ; Scorpio et al ., ) have noted that, while CapD removes the PDGA capsule from B. anthracis , it is markedly unstable and variable in activity, in part due to its mode of enzymatic action and stringent requirements for polymer hydrolysis (Wu et al ., ). CapD did afford some protection to mice when co‐injected intraperitoneally with B. anthracis (Scorpio et al ., ) but attempts to engineer pharmaceutical stability led to a marked reduction in CapD capsule degrading capacity (Wu et al ., ).…”

Repurposing citizen science games as software tools for professional scientists

“…While still in development, Foldit Standalone had already begun to play a critical role in both research and education [5, 41]. Students have used this tool to re-engineer the reaction specificity of proteases for the development of therapeutics for Anthrax [49] and Celiac Disease [19]. Foldit Standalone has been particularly successful in engaging undergraduate students within the context of the International Genetically Engineering Competition (iGEM).…”

Draft Genome Sequences of Pseudomonas fluorescens BS2 and Pusillimonas noertemannii BS8, Soil Bacteria That Cooperate To Degrade the Poly-γ- d -Glutamic Acid Anthrax Capsule

“…If successful, the capsule-stripping approach could confound attempts to render anthrax untreatable by the engineering of multidrug-resistant strains for unlawful dissemination. The poly-γ-glutamate-specific capsule depolymerase (CapD) produced by B. anthracis removes the capsule from the surface of the anthrax bacillus (4) and can protect against anthrax infection (5) but is markedly unstable (6) and therefore unlikely to be useful for therapeutic development.…”