Computational protocol: Selection of Nanobodies that Block the Enzymatic and Cytotoxic Activities of the Binary Clostridium Difficile Toxin CDT

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Protocol publication

[…] Molecular modeling of the binding epitopes of three VHHs to CDTa (pdb code 2wn7) were performed using BioLuminate (version 1.7, Schrodinger, New York, NY). Homology models were built with the antibody prediction module in BioLuminate using framework templates obtained from the curated antibody database. CDR loops were grafted from the database based on a combination of structure clustering, sequence similarity and stem residue geometry matching. BioLuminate automatically preserved the extra disulfide bond in nanobodies l+8 and l+18 that connects CDR2 and CDR3 if the template also had one in the aligned position. In other cases, the formation of the disulfide bond was forced manually before de novo loop prediction. Four models were built for l+8, six models for l-14, and five models for l+18. All of these models were used for docking to CDTa using Piper. Prior to docking, NAD+ was removed from CDTa using the Protein Preparation Wizard. An attractive potential (scaling the vdW attractive term by 1.1) was applied to the NAD+-binding site, which helped to restrain the sampling to this site. 70000 rotations were sampled and the top 30 poses were returned from each docking job. The following criteria were used to prune the docking poses: (a) docking poses with low surface complementarity (<0.3) were discarded; (b) VHHs binding with non-CDR regions were discarded; (c) l-14 and l+18 bind partially overlapping epitopes; (d) l+8 does not block or is not blocked by l-14 or l+18; (e) all VHHs block binding of NAD+ and/or actin. The most plausible docking poses that passed the above criteria were: the #16 pose from the 2nd homology model of l+8, the #4 pose from the 1st homology model of l-14, and the #1 pose from the 2nd homology model of l+18 (). The templates used for building these models were: (1) l+8: framework – 4JVP, CDR1 - 4KDT, CDR2 – 4JVP, CDR3 – 1MAM; (2) l-14: framework – 3STB, CDR1 – 3VG9, CDR2 – 2XA3, CDR3 – 2AJ3; (3) l+18: framework – 3STB, CDR1 – 1NFD, CDR2 – 4NBZ, CDR3 – de novo. […]

Pipeline specifications

Software tools BioLuminate, PIPER, Protein Preparation Wizard
Applications Drug design, Protein interaction analysis
Organisms Clostridioides difficile, Homo sapiens, Lama glama
Chemicals Adenosine Diphosphate