title: 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.
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Sean J
authors.initials: SJ
authors.lastname: Wu
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Eiben
authors.forename: John H
authors.initials: JH
authors.lastname: Carra
authors.forename: Ivan
authors.initials: I
authors.lastname: Huang
authors.forename: David
authors.initials: D
authors.lastname: Zong
authors.forename: Peixian
authors.initials: P
authors.lastname: Liu
authors.forename: Cindy T
authors.initials: CT
authors.lastname: Wu
authors.forename: Jeff
authors.initials: J
authors.lastname: Nivala
authors.forename: Josef
authors.initials: J
authors.lastname: Dunbar
authors.forename: Tomas
authors.initials: T
authors.lastname: Huber
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Senft
authors.forename: Rowena
authors.initials: R
authors.lastname: Schokman
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Smith
authors.forename: Jeremy H
authors.initials: JH
authors.lastname: Mills
authors.forename: Arthur M
authors.initials: AM
authors.lastname: Friedlander
authors.forename: David
authors.initials: D
authors.lastname: Baker
authors.forename: Justin B
authors.initials: JB
authors.lastname: Siegel
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000881:Anthrax / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics; D001408:Bacillus anthracis / Q000201:enzymology* / Q000235:genetics; D016667:Bacterial Capsules / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use; D003198:Computer Simulation; D006801:Humans; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use; D005723:gamma-Glutamyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D005723:gamma-Glutamyltransferase