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Article Cite This: ACS Omega 2019, 4, 11987−11992
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Investigating the Deoxyribonuclease Activity of CRM197 with SiteDirected Mutagenesis Nathalie Bravo-Bautista,† Hieu Hoang,‡ Anusha Joshi,§ Jennifer Travis,† Melissa Wooten,§ and Nathan J. Wymer*,† †
Department of Chemistry and Biochemistry, ‡Department of Biological and Biomedical Sciences, and §Department of Pharmaceutical Sciences, North Carolina Central University, Durham, North Carolina 27707, United States
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ABSTRACT: The protein cross-reactive material 197 (CRM197) is known to catalyze the hydrolytic cleavage of DNA (DNase activity). A suspected metalbinding site (S109, T111, and E112) and suspected DNA-binding motif (T89, K90, and V91) were predicted within the CRM197 protein X-ray crystal structure (4AE0) using METSITE and DNABindProt, respectively. Between these two predicted sites is a groove (K103, E116, T120, E122, F123, and R126) that may assist in DNase activity. Alanine scanning was performed at these sites to determine which amino acids might be important for DNase activity. These mutations individually or in combination either maintained or increased the overall DNase activity compared to the unmodified CRM197. Mutation at the suspected metal-binding site showed similar fluctuations to the overall DNase activity whether the DNase assays were run with Mg2+ and Ca2+ or Mn2+. However, many of the mutations within the suspected DNA-binding motif saw significant differences depending on which metal was used. Only some of the improvements in DNase activity could be attributed to improved folding of the mutants compared to the unmodified CRM197. This study should provide a basis for further mutagenesis studies to remove the DNase activity of CRM197.
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INTRODUCTION Cross-reactive material 197 (CRM197) is a nontoxic mutant of diphtheria toxin (DT) that is used as a carrier protein in several conjugate vaccines currently available and within human clinical trials.1 DT targets the heparin-binding EGFlike receptor (HB-EGF) on the surface of antigen-presenting cells (APC) by using its receptor-binding domain (R-domain) to trigger endocytosis.2 Once within the vesicle and the pH drops, the transmembrane domain (T-domain) undergoes an allosteric conformational change to allow the catalytic domain (C-domain) to be released into the APC’s cytoplasm.3 The released C-domain then uses its NAD+-diphthamide ADPribosyltransferase (ADPRT) activity to inhibit eukaryotic elongation factor 2 (eEF2) and impede protein translation.2 A single mutation (G52E) within the catalytic domain of CRM197 prevents NAD+ binding and is responsible for the loss of the ADPRT cytotoxicity.4 CRM197 has gained in popularity for creating conjugate vaccines for infants because CRM197 utilizes an unmodified Rdomain to actively transport antigens into an APC.1 This active transport of the antigen into the APC begins a T-celldependent immune response, which is active in infants shortly after birth, thus offering immunological protection earlier in an infant’s life.1 While the ADPRT activity of DT is well known, Wisnieski and co-workers demonstrated that DT also possesses deoxyribonuclease (DNase) activity.5,6 This DNase activity is © 2019 American Chemical Society
located within the catalytic domain of the DT, is distinct from the NAD binding site, and is not affected by the G52E mutation found within CRM197.6 As such, CRM197 continues to possess this inherited DNase activity. Wisnieski and co-workers were able to show that this DNase activity can be cytotoxic to mammalian cells.6 The DNase activity is unneeded for vaccine efficacy, so removing this activity could improve the overall safety profile of CRM197-based conjugate vaccines. This initial study uses alanine scanning within a predicted metal-binding site and DNA-binding motif of the CRM197 structure.7 Alanine scanning can offer an estimate on the importance of the individual amino acids to a protein’s folding efficiency or catalytic activity.7 This technique should offer additional clues as to the location of the DNase catalytic site within CRM197.
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RESULTS AND DISCUSSION The CRM197 is known to use either Mg2+ and Ca2+ or Mn2+ for DNase activity.6 This metal−cofactor dependence suggests that identifying a metal-binding site within the C-domain might provide a starting point for mutagenesis studies. The CRM197 X-ray crystal structure (4AE0)4 was submitted to the Received: February 13, 2019 Accepted: May 13, 2019 Published: July 10, 2019 11987
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online metal-binding site prediction server METSITE.8 METSITE predicted three amino acids (S109, T111, and E112) that might function as a metal-binding site. These residues are located within the C-domain of the CRM197, as predicted by a previous study6 (Figure 1).
using an agarose gel-based DNase activity assay (Figure 2). For the Mg2+ and Ca2+ reactions, the S109A and E112Q mutations significantly increased the DNase activity compared to the unmodified CRM197, while the T111A mutation showed little change (Figure 3). As additional mutations were included within the CRM197, DNase activity started to revert back to levels similar to that of the unmodified CRM197. The CRM197 X-ray crystal structure (4AE0)4 was also submitted to the online DNA-binding motif prediction server DNABindProt.9 DNABindProt predicted a DNA-binding motif (T89, K90, and V91) within the CRM197 structure. A small groove connects this predicted DNA-binding motif to the predicted metal-binding site (Figure 1). This groove may help facilitate the DNase activity within CRM197. Alanine scanning was performed on several amino acids along this groove (K103A, E116A, T120A, E122A, F123A, and R126A) (Figure 4). These mutations were prepared individually as well as in combination. The assay results for the Mg2+ and Ca2+ reactions (Figure 3) showed that the individual mutants had significantly increased DNase activity. The double and triple mutants showed reduced DNase activities compared to the individual mutants but were still generally higher than the unmodified CRM197. The E116A/E122A mutant did display DNase activity similar to the unmodified CRM197. The DNase activity of some of the CRM197 mutants did appear to change depending on which metal was used in the assay. The K103A, E116A, F123A, E116A/R126A, and E116A/E122A/R126A mutants increased their DNase activity with Mg2+ and Ca2+, while the R126A and E116A/E122A mutants accelerated with Mn2+. The remaining mutations within the suspected DNA-binding motif as well as all of the
Figure 1. X-ray crystal structure of the C-domain of the CRM197.4 METSITE predicted a metal-binding domain at S109, T111, and E112.8 DNABindProt predicted a DNA-binding motif at Lys90.9 The G52E mutation of CRM197 occurs within the NAD+-binding site and significantly reduces the cytotoxic ADPRT activity that occurs within the DT.
The amino acids forming the predicted metal-binding site were mutated individually (S109A, T111A, and E112Q) as well as in combination. After expression in Escherichia coli and purification, the DNase activity of each mutant was measured
Figure 2. Agarose gel electrophoresis results from DNase assay for mutants with Mg2+ and Ca2+ at 32 °C. Gel A: Metal-binding site mutants. Gel B: DNA-binding motif mutants. Marker: GeneRuler Express (ThermoFisher). Samples: (1) CRM197; (2) S109A; (3) T111A; (4) E112Q; (5) S109A/T111A; (6) S109A/E112Q; (7) T111A/E112Q; (8) S109A/T111A/E112Q; (9) negative control; (10) K103A; (11) E116A; (12) T120A; (13) E122A; (14) F123A; (15) R126A; (16) K103A/E122A; (17) E116A/E122A; (18) E116A/R126A; (19) E122A/R126A; (20) E116A/E122A/R126A. 11988
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Figure 3. Effects of (A) Mg2+ and Ca2+ and (B) Mn2+ on DNase activity for each mutant relative to the negative control.
of the mutations did not follow this tendency. Mutants S109A/ T111A/E112Q, K103A/E122A, E116A/R126A, and F123A all had similar percentages of folded protein compared to CRM197 but also had increased DNase activity. In particular, CRM197 and the F123A mutant both contained only ∼50% properly folded proteins. Yet, the F123A contained some of the highest DNase activity observed in this study. This study sets out to explore the DNase activity of CRM197 using a combination of protein structural prediction and alanine scanning to determine where the DNase active site might be located. The improvements in folding and overall DNase activity of the mutants were unexpected and cannot be easily explained at this time. However, this initial screening data does provide a roadmap for full saturation mutagenesis studies in the future. The goal of the overall project is to create a CRM197 mutant that would eliminate its DNase activity.
mutations within the suspected metal-binding site appeared to react similarly to either metal. Expression of properly folded and active CRM197 within E. coli is difficult due to the presence of two disulfide bonds.10−12 The CRM197 and mutants were purified from inclusion bodies by lysing the cells in the presence of 6 M guanidine so as to denature all of bacterial proteins, following a similar procedure as described by Malito and co-workers.4 The samples were then allowed to refold during the affinity chromatography purification step. The increased DNase activity of the mutants might be caused by an improved folding efficiency during purification. To test this hypothesis, freshly prepared CRM197 and mutant samples were analyzed with size exclusion chromatography (SEC) (Figure 5). The overall trend of the SEC data showed that improved folding of the mutant increased overall DNase activity (Figure 6). However, several 11989
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Figure 4. X-ray crystal structure of the C-domain of the CRM197.4 The amino acid sites selected for mutagenesis are indicated. Lys90 is located immediately behind Arg126.
Figure 6. Fraction of CRM197 mutant sample that contained properly folded protein as determined by SEC compared to overall DNase activity. A low quantity of large DNA fragments remaining indicates higher overall DNase activity.
Making a DNase-free CRM197 could allow for the creation of safer conjugate vaccines in the future.
ampicillin. A single colony was picked and cultured overnight at 37 °C/200 rpm in 2 mL of LB media supplemented with ampicillin. A 125 mL baffled flask containing 25 mL of Turbo broth (AthenaES) was then inoculated with a 1% (v/v) seed from the overnight culture. The flask was incubated at 37 °C/ 200 rpm until log phase, which is typically 3 h. The flask was then cooled to 16 °C and allowed to shake at 200 rpm for approximately 30 min. The cultures were induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and allowed to incubate overnight at 16 °C/200 rpm. The cells were then harvested with centrifugation (4000g, 10 min), and the medium was decanted. The cells were resuspended in 10 mL of 6 M guanidine/50 mM Tris buffer (pH 8). The resuspended cells were lysed with sonication for 1 min (typically 5 W). The cell debris was pelleted by centrifugation (5000g, 15 min). The
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MATERIALS AND METHODS Expression and Purification of Wild-Type and Mutant CRM197 Proteins. The CRM197 gene (UniProt P00588) was synthesized by ATUM, Inc. (Newark, CA) and cloned into the pET19b(+) expression plasmid (MilliporeSigma) between the NdeI and BamHI restriction sites using standard PCR and ligation techniques.13 The pET19b(+) plasmid adds an Nterminal 10×His purification tag onto the expressed protein. Park and co-workers showed that the presence of this purification tag did not disrupt CRM197 folding or activity.11 The constructed plasmid was transformed into BL21 (DE3) cells (Lucigen) and plated onto LB media supplemented with
Figure 5. Fraction of properly folded CRM197 protein within each sample as determined by the integrations for folded and aggregated peaks within size exclusion chromatography (SEC). 11990
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cell lysate was applied to 1 mL of HisLink purification resin (Promega) and allowed to drip with gravity flow. The resin was washed with 20 column volumes of wash buffer (50 mM Tris (pH 7.6)/500 mM NaCl/10 mM imidazole). The protein was eluted from the resin with three column volumes of elution buffer (50 mM Tris (pH 7.6)/500 mM NaCl/500 mM imidazole). The purified protein was then dialyzed against 2 L of buffer (20 mM Tris (pH 7.5)/100 mM NaCl) overnight at 4 °C with stirring and two buffer changes. The purified protein was then checked with SDS-PAGE. Each of the mutant CRM197 proteins was expressed and purified as described above. Each mutant used a separate column and purification resin so as to not cross-contaminate. A negative control was also prepared using the above protocol, except the pET19b plasmid did not contain the CRM197 gene. This negative control would account for any contaminating native DNase enzymes that might be unexpectedly copurified with the CRM197 proteins. Mutagenesis. The mutants were prepared using a QuikChange mutagenesis kit (Agilent Technologies) following the standard protocol (see Supporting Information). A K125A mutation was prepared but this protein could not be expressed. DNase Assay. The protein concentration for each protein was assayed by mixing 100 μL of purified protein solution with 900 μL of 6 M urea and measuring A280 (ε = 54,500 Μ−1). For the DNase assay, 2.5 μg of protein was mixed with 2.5 μg of pUC19 plasmid (EcoRI-linearized) in buffer (20 mM Tris (pH 7.5)/2.5 mM MgSO4/2.5 mM CaCl2 or 20 mM Tris (pH 7.5)/2.5 mM MgSO4/5 mM MnCl2) and diluted to a final volume of 20 μL. The reaction was incubated within a PCR machine at 32 or 37 °C for 1 h (metal-binding site mutants) or 3 h (DNA-binding site mutants). The samples were then run on a 1% agarose gel with ethidium bromide staining. The image of the gel was analyzed with ImageJ (version 1.51, NIH).14 For each sample, the image intensity of DNA between 5 and 1.5 kb was divided by the image intensity of the DNA between 5 kb and 100 bp. Each replication set was normalized from its negative control sample. The reactions were performed in triplicate for each temperature and metal. Size Exclusion Chromatography (SEC). Freshly prepared CRM197 and mutant samples were analyzed using a Bio SEC-5 column (300 mm × 4.6 mm; 150 Å; Agilent) fitted to an Agilent 1200 HPLC. The mobile phase used 100 mM Na2HPO4 (pH 7.2)/150 mM NaCl buffer, and each injection was run at 0.3 mL/min for 40 min. A UV/Vis detector was used at 214 nm. Twenty microliters of each CRM197 and mutant sample or 10 μL of SEC protein standard (15−600 kDa; MilliporeSigma) was injected per run. The fraction of peak integration for folded protein (∼55 kDa) compared to total CRM197 protein (aggregated (∼260 kDa) and folded) was used to calculate the overall folded fraction for each sample. Little to no CRM197 dimer was observed in any of the samples, so it was not included in the calculations. Each CRM197 or mutant sample was run in triplicate, while the SEC protein standard was run six times.
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Sequences of the DNA primers used for mutagenesis of the CRM197 gene (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Nathan J. Wymer: 0000-0001-6908-399X Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors would like to thank Somnath Mukhopadhyay, Kevin Williams, and the BRITE Institute (NCCU) for their advice and the use of their equipment. The images of the protein structures were prepared by using Chimera.15 Funding for this research was provided by North Carolina Central University (Durham, NC).
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REFERENCES
(1) Shinefield, H. R. Overview of the development and current use of CRM197 conjugate vaccines for pediatric use. Vaccine 2010, 28, 4335−9. (2) Bröker, M.; Costantino, P.; DeTora, L.; McIntosh, E. D.; Rappuoli, R. Biochemical and biological characteristics of crossreacting material 197 (CRM197), a non-toxic mutant of diphtheria toxin: Use as a conjugation protein in vaccines and other potential clinical applications. Biologicals 2011, 39, 195−204. (3) Ladokhin, A. S.; Vargas-Uribe, M.; Rodnin, M.; Ghatak, C.; Sharma, O. Cellular Entry of the Diphtheria Toxin Does Not Require the Formation of the Open-Channel State by Its Translocation Domain. Toxins 2017, 9, 299. (4) Malito, E.; Bursulaya, B.; Chen, C.; Surdo, P. L.; Picchianti, M.; Balducci, E.; Biancucci, M.; Brock, A.; Berti, F.; Bottomley, M. J.; Nissum, M.; Costantino, P.; Rappuoli, R.; Spraggon, G. Structural Basis for Lack of Toxicity of the Diphtheria Toxin Mutant CRM197. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 5229−5234. (5) Bruce, C.; Baldwin, R. L.; Lessnick, S. L.; Wisnieski, B. J. Diphtheria toxin and its ADP-ribosyltransferase-defective homologue CRM197 possess deoxyribonuclease activity. Proc. Natl. Acad. Sci. U. S. A. 1990, 87, 2995−8. (6) Lee, J. W.; Nakamura, L. T.; Chang, M. P.; Wisnieski, B. J. Mechanistic aspects of the deoxyribonuclease activity of diphtheria toxin. Biochim. Biophys. Acta 2005, 1747, 121−31. (7) Morrison, K. L.; Weiss, G. A. Combinatorial Alanine-Scanning. Curr. Opin. Chem. Biol. 2001, 5, 302−7. (8) Sodhi, J. S.; Bryson, K.; McGuffin, L. J.; Ward, J. J.; Wernisch, L.; Jones, D. T. Predicting metal-binding site residues in low-resolution structural models. J. Mol. Biol. 2004, 342, 307−320. (9) Ozbek, P.; Soner, S.; Erman, B.; Haliloglu, T. DNABINDPROT: fluctuation-based predictor of DNA-binding residues within a network of interacting residues. Nucleic Acids Res. 2010, 38, W417−23. (10) Goffin, P.; Dewerchin, M.; De Rop, P.; Blais, N.; Dehottay, P. High-yield production of recombinant CRM197, a non-toxic mutant of diphtheria toxin, in the periplasm ofEscherichia coli. Biotechnol. J. 2017, 12, 1700168. (11) Park, A.-R.; Jang, S.-W.; Kim, J.-S.; Park, Y.-G.; Koo, B.-S.; Lee, H.-C. Efficient recovery of recombinant CRM197 expressed as inclusion bodies in E.coli. PLoS One 2018, 13, No. e0201060. (12) Hickey, J. M.; Toprani, V. M.; Kaur, K.; Mishra, R. P. N.; Goel, A.; Oganesyan, N.; Lees, A.; Sitrin, R.; Joshi, S. B.; Volkin, D. B. Analytical Comparability Assessments of 5 Recombinant CRM197 Proteins From Different Manufacturers and Expression Systems. J. Pharm. Sci. 2018, 107, 1806−1819. (13) Green, M. R.; Sambrook, J. Molecular Cloning, 4th ed.; Cold Springs Harbor Laboratory Press: 2012.
ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.9b00418. 11991
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(14) Abràmoff, M. D.; Magalhães, P. J.; Ram, S. J. Image Processing with ImageJ. Biophotonic International 2004, 11, 36−42. (15) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 2004, 25, 1605−12.
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