Elucidation of bezlotoxumab binding specificity to toxin B in Clostridioides difficile.

C. difficile or Clostridioides difficile infection (CDI) is currently one of the major causes of epidemics worldwide. Toxin B from Clostridioides difficile toxin B (TcdB) infection is the main target protein inhibiting CDI recurrence. Clinical research suggested that bezlotoxumab's (Bez) efficiency is significantly reduced in neutralizing the B2 strain compared to the B1 strain. The monoclonal antibody (mAb) functions by binding to the epitope 1 and 2 regions in the combined repetitive oligopeptide (CROP) domain. Some binding residues are distinctively different between B1 and B2 strains. In this work, we aimed to elucidate and compare insights into the interaction of toxins B1 and B2 in complex with Bez by using all-atom molecular dynamics (MD) simulations and binding free energy calculations. The predicted ΔGbinding values suggested that the antibody (Ab) could bind to toxin B1 significantly better than B2, supported by higher salt bridge and hydrogen bonding (H-bonding) interactions, as well as the number of contact residues between the two focused proteins. The toxin B1 residues important for binding with Bez were E1878, T1901, E1902, F1905, N1941, V1946, N2031, T2032, E2033, V2076, V2077, and E2092. The lower susceptibility of Bez towards toxin B2 was primarily due to a change of residue E2033 from glutamate to alanine (A2033) and the loss of E1878 and E1902 contributions, as determined by the intermolecular interaction changes from the dynamic residue interaction network (dRIN) analysis. The obtained data strengthen our understanding of Bez/toxin B binding.

[1]  T. Rungrotmongkol,et al.  Discovery of JAK2/3 Inhibitors from Quinoxalinone-Containing Compounds , 2022, ACS omega.

[2]  T. Rungrotmongkol,et al.  Pharmacophore-Based Virtual Screening and Experimental Validation of Pyrazolone-Derived Inhibitors toward Janus Kinases , 2022, ACS omega.

[3]  R. Jin,et al.  Receptor binding mechanisms of Clostridioides difficile toxin B and implications for therapeutics development , 2021, The FEBS journal.

[4]  D. Lacy,et al.  Clostridioides difficile toxins: mechanisms of action and antitoxin therapeutics , 2021, Nature Reviews Microbiology.

[5]  Lan Huang,et al.  Structural basis for CSPG4 as a receptor for TcdB and a therapeutic target in Clostridioides difficile infection , 2021, Nature Communications.

[6]  Mohini Yadav,et al.  Dynamic residue interaction network analysis of the oseltamivir binding site of N1 neuraminidase and its H274Y mutation site conferring drug resistance in influenza A virus , 2021, PeerJ.

[7]  T. Rungrotmongkol,et al.  Source of oseltamivir resistance due to single E276D, R292K, and double E276D/R292K mutations in H10N4 influenza neuraminidase , 2021 .

[8]  L. Bry,et al.  Phylogenomics of 8,839 Clostridioides difficile genomes reveals recombination-driven evolution and diversification of toxin A and B , 2020, bioRxiv.

[9]  Antibiotic resistance threats in the United States, 2019 , 2019 .

[10]  T. Rungrotmongkol,et al.  Atomistic mechanisms underlying the activation of the G protein-coupled sweet receptor heterodimer by sugar alcohol recognition , 2019, Scientific Reports.

[11]  J. Jakana,et al.  Selection and characterization of ultrahigh potency designed ankyrin repeat protein inhibitors of C. difficile toxin B , 2019, PLoS biology.

[12]  T. Rungrotmongkol,et al.  Low susceptibility of asunaprevir towards R155K and D168A point mutations in HCV NS3/4A protease: A molecular dynamics simulation. , 2019, Journal of molecular graphics & modelling.

[13]  Simon C. Potter,et al.  The EMBL-EBI search and sequence analysis tools APIs in 2019 , 2019, Nucleic Acids Res..

[14]  Lam K. Huynh,et al.  A theoretical study on the molecular encapsulation of luteolin and pinocembrin with various derivatized beta-cyclodextrins , 2019, Journal of Molecular Structure.

[15]  Alberto J. M. Martin,et al.  RIP-MD: a tool to study residue interaction networks in protein molecular dynamics , 2018, PeerJ.

[16]  Pemra Ozbek,et al.  gRINN: a tool for calculation of residue interaction energies and protein energy network analysis of molecular dynamics simulations , 2018, Nucleic Acids Res..

[17]  Radka Svobodová Vařeková,et al.  PDBsum: Structural summaries of PDB entries , 2017, Protein science : a publication of the Protein Society.

[18]  Jiahui Chen,et al.  Improvements to the APBS biomolecular solvation software suite , 2017, Protein science : a publication of the Protein Society.

[19]  David K. Brown,et al.  MD-TASK: a software suite for analyzing molecular dynamics trajectories , 2017, Bioinform..

[20]  C. Simmerling,et al.  ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB. , 2015, Journal of chemical theory and computation.

[21]  Michael J E Sternberg,et al.  The Phyre2 web portal for protein modeling, prediction and analysis , 2015, Nature Protocols.

[22]  Matteo Tiberti,et al.  PyInteraph: A Framework for the Analysis of Interaction Networks in Structural Ensembles of Proteins , 2014, J. Chem. Inf. Model..

[23]  Thanyada Rungrotmongkol,et al.  Key Binding and Susceptibility of NS3/4A Serine Protease Inhibitors against Hepatitis C Virus , 2014, J. Chem. Inf. Model..

[24]  Saraswathi Vishveshwara,et al.  An automated approach to network features of protein structure ensembles , 2013, Protein science : a publication of the Protein Society.

[25]  Youyong Li,et al.  Assessing the performance of MM/PBSA and MM/GBSA methods. 3. The impact of force fields and ligand charge models. , 2013, The journal of physical chemistry. B.

[26]  Daniel R Roe,et al.  PTRAJ and CPPTRAJ: Software for Processing and Analysis of Molecular Dynamics Trajectory Data. , 2013, Journal of chemical theory and computation.

[27]  S. Tzipori,et al.  Antibody against TcdB, but not TcdA, prevents development of gastrointestinal and systemic Clostridium difficile disease. , 2013, The Journal of infectious diseases.

[28]  Shixia Wang,et al.  Protective antibody responses against Clostridium difficile elicited by a DNA vaccine expressing the enzymatic domain of toxin B , 2013, Human vaccines & immunotherapeutics.

[29]  Fumio Hirata,et al.  Placevent: An algorithm for prediction of explicit solvent atom distribution—Application to HIV‐1 protease and F‐ATP synthase , 2012, J. Comput. Chem..

[30]  Matteo Tiberti,et al.  xPyder: A PyMOL Plugin To Analyze Coupled Residues and Their Networks in Protein Structures , 2012, J. Chem. Inf. Model..

[31]  Tingjun Hou,et al.  Assessing the performance of the molecular mechanics/Poisson Boltzmann surface area and molecular mechanics/generalized Born surface area methods. II. The accuracy of ranking poses generated from docking , 2011, J. Comput. Chem..

[32]  Tingjun Hou,et al.  Assessing the Performance of the MM/PBSA and MM/GBSA Methods. 1. The Accuracy of Binding Free Energy Calculations Based on Molecular Dynamics Simulations , 2011, J. Chem. Inf. Model..

[33]  D. Ho,et al.  A DNA vaccine targeting the receptor-binding domain of Clostridium difficile toxin A. , 2009, Vaccine.

[34]  Imran Siddiqi,et al.  Solvated Interaction Energy (SIE) for Scoring Protein-Ligand Binding Affinities, 1. Exploring the Parameter Space , 2007, J. Chem. Inf. Model..

[35]  D. Case,et al.  Exploring protein native states and large‐scale conformational changes with a modified generalized born model , 2004, Proteins.

[36]  P. Shannon,et al.  Cytoscape: a software environment for integrated models of biomolecular interaction networks. , 2003, Genome research.

[37]  L. Nilsson,et al.  Structure and Dynamics of the TIP3P, SPC, and SPC/E Water Models at 298 K , 2001 .

[38]  C. von Eichel-Streiber,et al.  The C-terminal ligand-binding domain of Clostridium difficile toxin A (TcdA) abrogates TcdA-specific binding to cells and prevents mouse lethality. , 1997, FEMS microbiology letters.

[39]  J. Ponder,et al.  The NMR solution structure of intestinal fatty acid-binding protein complexed with palmitate: application of a novel distance geometry algorithm. , 1996, Journal of molecular biology.

[40]  T. Darden,et al.  The effect of long‐range electrostatic interactions in simulations of macromolecular crystals: A comparison of the Ewald and truncated list methods , 1993 .

[41]  H. Berendsen,et al.  Molecular dynamics with coupling to an external bath , 1984 .

[42]  G. Ciccotti,et al.  Numerical Integration of the Cartesian Equations of Motion of a System with Constraints: Molecular Dynamics of n-Alkanes , 1977 .

[43]  OUP accepted manuscript , 2022, Nucleic Acids Research.

[44]  Roman A. Laskowski,et al.  PDBsum: summaries and analyses of PDB structures , 2001, Nucleic Acids Res..