Identification of Novel Nontoxic Mutants of Diphtheria Toxin Unable to ADP-ribosylate EF2 Using Molecular Dynamics Simulations and Free Energy Calculations

Introduction: The Diphtheria vaccine is currently manufactured by chemical inactivation of Diphtheria toxin (DT) following large-scale cultivation of the pathogenic Corynebacterium diphtheriae. The development of non-toxic variants of DT in non-pathogenic recombinant hosts improves the process safety aspects considerably and is therefore desirable. The cytotoxicity of DT was exerted through ADPribosylation of elongation factor 2 (EF2) by the catalytic (C) domain of DT using NAD as the ADP donor. Aim: We, therefore, aim to design DT variants unable to perform ADP-ribosylation of EF2. Methods: We used molecular dynamics simulations (MDS) for docking of the C-domain of DT onto EF2 to identify interacting amino acid fluctuations using the root mean square fluctuation (RMSF). Amino acids of the C domain possessing low fluctuations and hence low flexibility were then specified and used for the mutant design. The C domain and the mutants were docked with NAD and ADENYLYL-3'-5'- PHOSPHO-URIDINE3'-MONOPHOSPHATE (APU) as a C domain inhibitor. G52E showing negligible NAD binding was also included in our study. Free binding energies of the complexes were calculated and used to select the desired mutants. Moreover, contact maps of the C domain and the selected mutants were compared to elucidate their structural differences. The SCRATCH tool was used to estimate their solubility upon recombinant expression in E. coli. ElliPro was further used to determine their B-cell epitopes. Results: Our results indicated that amino acids Y20, V80, V81, V83, and Y149 within the C domain showed low flexibility. The constructed mutants, including Y20E and Y80E were able to bind APU, whereas Y149E was not able to bind it. Both Y20E and Y149E were unable to bind NAD. Thus, these mutants did not have ADP-ribosylation activity and were nontoxic. These were structurally different from the C domain. However, their solubility in E. coli and their conformational B-cell epitopes were similar to those of the C domain. Conclusion: Therefore, Y20E and Y149E are applicable for the vaccine design with DT using a much safer process compared to the commercial process.