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Improvement of a Potential Anthrax Therapeutic by Computational Protein Design*

Open AccessPublished:July 18, 2011DOI:https://doi.org/10.1074/jbc.M111.251041
      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.

      Introduction

      Bacillus anthracis, the causative agent of anthrax, poses a significant bioterrorist threat. This was demonstrated in 2001 by a series of attacks in which B. anthracis spores were sent via the United States Postal Service to various media outlets and government officials. Although a safe and effective vaccine is available (
      Institute of Medicine (United States). Committee to Assess the Safety and Efficacy of the Anthrax Vaccine
      ), there remain concerns about the potential for use of naturally occurring or genetically engineered antibiotic- and vaccine-resistant strains of B. anthracis (
      • Pomerantsev A.P.
      • Staritsin N.A.
      • Mockov YuV.
      • Marinin L.I.
      ,
      • Stepanov A.V.
      • Marinin L.I.
      • Pomerantsev A.P.
      • Staritsin N.A.
      ) as bioterrorist weapons. Anthrax inhalation of aerosolized spores seems the most likely scenario for a bioterrorist attack. The last case of inhalational anthrax reported in the United States prior to the 2001 attacks occurred in 1976 (
      • Inglesby T.V.
      • O'Toole T.
      • Henderson D.A.
      • Bartlett J.G.
      • Ascher M.S.
      • Eitzen E.
      • Friedlander A.M.
      • Gerberding J.
      • Hauer J.
      • Hughes J.
      • McDade J.
      • Osterholm M.T.
      • Parker G.
      • Perl T.M.
      • Russell P.K.
      • Tonat K.
      ). The rarity of this particular type of infection makes it difficult to study the efficacy of various response strategies. These concerns and the historically effective use of B. anthracis to cause disease intentionally have led the biomedical community to focus on developing novel therapeutics as prophylactic countermeasures to the potential threat of a bioterrorist attack.
      The potent virulence of B. anthracis is thought to be a result primarily of exotoxins (lethal and edema toxins) as well as an antiphagocytic γ-linked poly-d-glutamic acid (PDGA)
      The abbreviation used is: PDGA
      γ-linked poly-d-glutamic acid.
      capsule (
      • Green B.D.
      • Battisti L.
      • Koehler T.M.
      • Thorne C.B.
      • Ivins B.E.
      ,
      • Mikesell P.
      • Ivins B.E.
      • Ristroph J.D.
      • Dreier T.M.
      ). Although the precise mechanism by which the capsule inhibits phagocytosis is unknown, it has been well established that strains lacking the PDGA capsule have significantly diminished virulence (
      • Scorpio A.
      • Chabot D.J.
      • Day W.A.
      • O'Brien D.K.
      • Vietri N.J.
      • Itoh Y.
      • Mohamadzadeh M.
      • Friedlander A.M.
      ).
      The capsule is created by a γ-glutamyl transpeptidase, CapD, attached to the outer cell wall, which covalently anchors PDGA to the B. anthracis peptidoglycan layer (
      • Candela T.
      • Fouet A.
      ). Although the function of native CapD is to build the antiphagocytic capsule, it was shown recently that administration of high doses of recombinant CapD significantly decreased the mortality rate of mice infected with B. anthracis (
      • Scorpio A.
      • Tobery S.A.
      • Ribot W.J.
      • Friedlander A.M.
      ). The ability of recombinant CapD to counteract virulence (the opposite of its native function) is likely due to the removal of the outer antiphagocytic capsule of B. anthracis in vivo, as it is no longer attached to the outer cell wall and localized by the peptidoglycan layer. The ability to degrade the capsule has been demonstrated in vitro previously (
      • Scorpio A.
      • Chabot D.J.
      • Day W.A.
      • O'Brien D.K.
      • Vietri N.J.
      • Itoh Y.
      • Mohamadzadeh M.
      • Friedlander A.M.
      ,
      • Scorpio A.
      • Tobery S.A.
      • Ribot W.J.
      • Friedlander A.M.
      ,
      • Scorpio A.
      • Chabot D.J.
      • Day W.A.
      • Hoover T.A.
      • Friedlander A.M.
      ). Because of the ability to confer antibiotic resistance to anthrax in a laboratory setting, significant focus has been placed on the further development of CapD into a robust and effective next generation protein therapeutic.
      The structure of CapD was solved recently and has opened the possibility of applying computational protein design techniques to further enhance the potential therapeutic properties of this protein (
      • Wu R.
      • Richter S.
      • Zhang R.G.
      • Anderson V.J.
      • Missiakas D.
      • Joachimiak A.
      ). In this study, we used the Rosetta software suite (
      • Simons K.T.
      • Kooperberg C.
      • Huang E.
      • Baker D.
      ,
      • Leaver-Fay A.
      • Tyka M.
      • Lewis S.M.
      • Lange O.F.
      • Thompson J.
      • Jacak R.
      • Kaufman K.
      • Renfrew P.D.
      • Smith C.A.
      • Sheffler W.
      • Davis I.W.
      • Cooper S.
      • Treuille A.
      • Mandell D.J.
      • Richter F.
      • Ban Y.E.
      • Fleishman S.J.
      • Corn J.E.
      • Kim D.E.
      • Lyskov S.
      • Berrondo M.
      • Mentzer S.
      • Popović Z.
      • Havranek J.J.
      • Karanicolas J.
      • Das R.
      • Meiler J.
      • Kortemme T.
      • Gray J.J.
      • Kuhlman B.
      • Baker D.
      • Bradley P.
      ) to create a circularly permuted variant of CapD (termed CP) with significantly improved production properties and kinetic thermostability. In addition, we computationally designed a variant of CP that altered the reaction specificity of the enzyme such that the transpeptidation reaction rate was reduced, whereas the rate of its hydrolytic side reaction was essentially maintained. We then utilized the mutant to examine the mechanism of capsule degradation. The improved physical properties of CP, in addition to a more complete understanding of the native mechanism of capsule degradation, should lead to the development of improved protein-based therapeutics that can better withstand environmental stress and that have improved pharmacokinetic properties.

      DISCUSSION

      The B. anthracis γ-linked d-glutamyl transpeptidase CapD is expressed as an inactive linear peptide chain, which must undergo autocatalytic processing to form the active enzyme. This post-translational processing results in a heterodimer with a large subunit (residues 1–324) and a small subunit (residues 325–521), as depicted in Fig. 1A. The N-terminal threonine of the small subunit serves as the catalytic residue for the degradation of the PDGA substrate. Recombinant expression of CapD results in a heterogeneous mixture of processed and unprocessed soluble proteins (Fig. 2). This production property is not ideal for a protein therapeutic because consistent structural integrity is required for Food and Drug Administration (FDA) approval (

      U. S. Department of Health and Human Services Food and Drug Administration (1997) Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use (Docket No. 94D-0259), www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/OtherRecommendationsforManufacturers/UCM153182.pdf.

      ). Specifically, the FDA requires that a combination of SDS-PAGE, isoelectric focusing, high-performance liquid chromatography, and mass spectrometry be used to show that proteins are not fragmented, aggregated, or otherwise modified (e.g. partial autocatalytic processing). To address the issue of partial and variable autocatalytic processing of CapD, the protein was circularly permuted such that it would be produced as a homogeneous monomeric protein.
      As depicted in Fig. 1A, the N terminus of the large subunit and the C terminus of the small subunit of CapD are in proximity to one another in the folded processed heterodimer. We hypothesized that the termini could be connected to create a single-chain monomeric protein, circumventing the need for autocatalytic processing. Using the FoldIt interface of the Rosetta software suite, we designed a short peptide linker that was predicted to connect the C terminus of the small subunit to the N terminus of the large subunit (Fig. 1B). In the circularly permuted variant, the catalytic N-terminal threonine is natively translated as the protein's N-terminal residue, abrogating the need for CapD to undergo autocatalytic cleavage to form an active enzyme.
      Experimental characterization confirmed that autocatalytic processing was no longer required for the CP variant (Fig. 2). The activities of CP for both hydrolysis and transpeptidation were analyzed and were observed to remain the same as for CapD, if not slightly improved (Table 1).
      It has been previously demonstrated that circular permutation of proteins can result in changes in thermostability (
      • Yu Y.
      • Lutz S.
      ). To characterize kinetic thermostability, CapD and CP were incubated at a range of temperatures, after which the remaining activity was measured at a series of time points. As depicted in Fig. 4 (A and B), the circular permutation resulted in a significant increase in overall kinetic thermostability. At 45 °C, CapD was rendered inactive in a manner of minutes, whereas CP remained 90% active after 0.5 h. However, the structural thermostability was determined by CD for CapD and CP. Both enzymes exhibited similar melting temperatures of 48.4 and 51.1 °C, respectively (supplemental Fig. S5B). In this case, there was a much smaller change in stability. We hypothesize that this difference in stability is due to a loss of quaternary structure (i.e. dissociation of the two subunits), resulting in a decrease in catalytic activity for CapD. Such a disruption of quaternary structure would result in an inactive enzyme without significantly affecting the signal observed by CD. At significantly higher temperatures, the secondary structures of both proteins were disrupted, resulting in the observed changes in the CD signal (supplemental Fig. S5B). The designed peptide linker that connects the two subunits in CP altered the quaternary structure of the enzyme such that it was converted from a heterodimeric protein into a single-chain monomeric protein. This change would be predicted to have a more significant effect on the dissociation of the two subunits than the protein's secondary structure. This is consistent with the circular permutation of CapD having an increased kinetic thermostability. The increase in stability is likely to improve the ease of handling and may enhance its in vivo pharmacokinetic properties.
      Another feature of CapD that has remained a mystery is the mechanism through which it catalyzes the capsular degradation of B. anthracis in mouse serum. The primary function of CapD is transpeptidation, in which the polyglutamyl chain is cleaved, resulting in an acyl-enzyme intermediate. An acceptor with a free amine (such as an amino acid or a peptide from the peptidoglycan layer) must then bind within the active site and subsequently accept the polyglutamyl chain. If no acceptor is present, hydrolysis of the acyl-enzyme intermediate occurs instead. In this case, a water molecule acts as the primary acceptor, resulting in the formation of a free acid and the regenerated enzyme (supplemental Fig. S7). The balance of transpeptidation versus hydrolysis is well established for this class of enzymes (
      • Tate S.S.
      • Meister A.
      ,
      • Silverman R.B.
      ).
      Although the native function of CapD is to build the antiphagocytic outer coat of B. anthracis through a transpeptidation reaction, the mechanism of capsular degradation could proceed through either a hydrolysis or transpeptidation reaction. In the case of transpeptidation, endogenous amino acids and peptides in the host serum could act as acceptors instead of the peptidoglycan layer. If CapD levels are elevated beyond a particular threshold, the low-level but irreversible hydrolysis side reaction could eventually overwhelm the dominant but reversible transpeptidation reaction to the peptidoglycan layer. In an effort to develop protein-based anthrax therapeutics, it will be useful to know through which of the two mechanisms the capsular degradation is occurring. One potential method to determine this is to re-engineer the reaction specificity of CapD such that the transpeptidation reaction is diminished but the hydrolytic activity of the enzyme remains intact.
      Using the Rosetta software suite, we created a computationally designed active site library of 84 mutants predicted to be compatible with the hydrolysis transition state. Each of the 84 mutants was generated and screened for both hydrolytic and transpeptidation activities. From the initial screen, a general preference of disrupting transpeptidation with concomitant maintenance of hydrolytic activity was observed (supplemental Fig. S6). One mutant, F24H, showed a particularly interesting property in that it essentially maintained hydrolytic activity while significantly decreasing the transpeptidation reaction rate. Kinetic characterization of the F24H variant suggested that this mutation accomplished the desired alteration of reaction specificity, as shown in Table 1.
      To use this protein as a tool to elucidate the in vivo mechanism of capsular degradation, it was important to validate that the rate of the transpeptidation reaction was diminished not only with respect to the amino acid acceptor glutamate but also for all of the 20 amino acids that may be present in serum. As depicted in Fig. 3, this mutation significantly decreased the transpeptidation activity for every amino acid. In addition, the mutation did not compromise kinetic thermostability (Fig. 4B), suggesting that the change of the reaction was due to an alteration of the chemistry occurring in the active site and not simply due to a global structural destabilization. The F24H model does not clearly indicate why this particular mutation would result in such a drastic change in reaction specificity, as the closest nitrogen atom of His24 is 7.1 Å away from the oxygen atom of the catalytic Thr1 (Thr1 of CP corresponds to Thr352 of CapD) (Fig. 5B). The change in specificity could be the result of a modulation of the electrostatic environment of the active site such that the attack of the acyl-enzyme intermediate by a neutral amine within the active site is disfavored. Further experimentation is required to develop a more complete understanding of how this mutation alters the reaction specificity.
      Assays were conducted to explore the mechanism of capsular degradation in serum using CapD, CP, and F24H. Degradation efficiency in the presence of serum was measured as a function of protein concentration, as shown in Fig. 6. F24H exhibited a >10-fold decrease in its ability to degrade the capsule relative to CP and CapD. This change in reaction specificity is consistent with the mechanism of CapD-mediated capsule degradation being transpeptidation. If a hydrolytic mechanism were employed, this assay would not have shown such a significant difference between the ability of F24H to degrade the capsule relative to CP. The abilities of CP and F24H to catalyze the degradation of a synthetic PDGA substrate in the presence or absence of serum was then explored. Again, F24H showed an ∼2-fold increase in activity in the presence of 25% serum, whereas CP and CapD showed a >8-fold increase in activity in the presence of 25% serum (supplemental Fig. S8). Assuming that capsular degradation in mouse serum is representative of an in vivo environment, these experimental results comparing F24H with CP are consistent with the in vivo capsule degradation mechanism being transpeptidation (likely using endogenous amino acids and peptides as PDGA acceptors), as opposed to hydrolysis. Although F24H exhibits a significant change in reaction specificity and has provided an essential tool for the investigation of the in vivo mechanism of capsular degradation, additional mutants with larger changes in reaction specificity will be required to conclude that the in vivo reaction catalyzed by CapD is done primarily through transpeptidation and not hydrolysis.
      It is important to generate improved anthrax therapeutics in the near future. The native B. anthracis protein CapD is a promising candidate for such a protein-based therapeutic but currently suffers from limitations that may prevent its general use. In this study, we have applied computational protein modeling techniques both to improve the production properties of CapD and to significantly increase its kinetic thermostability. In addition, we generated a novel enzyme that was then used to help elucidate how this protein functions in vivo, and our results will inform future studies aimed at converting this protein into a potent protein-based therapeutic. The improved properties of CP, as well as the enhanced knowledge of this protein's function, should lead to the more efficient development of anthrax therapeutics.

      Acknowledgments

      We thank the International Genetically Engineered Machine (iGEM) Competition for promoting undergraduate research projects and providing a rich academic environment. Alan Weiner, Dominic Chung, and Ling Lin Liu (Department of Biochemistry, University of Washington) generously provided laboratory space for the undergraduates involved in this work. We thank Drs. Patricia Legler and Brock Siegel for insightful comments that helped refine the manuscript.

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