Mass spectrometry-complemented molecular modeling predicts the interaction interface for a camelid single-domain antibody targeting the Plasmodium falciparum circumsporozoite protein's C-terminal domain.

Background: Bioanalytical methods that enable rapid and high-detail characterization of binding specificities and strengths of protein complexes with low sample consumption are highly desired. The interaction between a camelid single domain antibody (sdAbCSP1) and its target antigen (PfCSP-Cext) was selected as a model system to provide proof-of-principle for the here described methodology. Research design and methods: The structure of the sdAbCSP1 - PfCSP-Cext complex was modeled using AlphaFold2. The recombinantly expressed proteins, sdAbCSP1, PfCSP-Cext, and the sdAbCSP1 - PfCSP-Cext complex, were subjected to limited proteolysis and mass spectrometric peptide analysis. ITEM MS (Intact Transition Epitope Mapping Mass Spectrometry) and ITC (Isothermal Titration Calorimetry) were applied to determine stoichiometry and binding strength. Results: The paratope of sdAbCSP1 mainly consists of its CDR3 (aa100-118). PfCSP-Cext's epitope is assembled from its α-helix (aa40-52) and opposing loop (aa83-90). PfCSP-Cext's GluC cleavage sites E46 and E58 were shielded by complex formation, confirming the predicted epitope. Likewise, sdAbCSP1's tryptic cleavage sites R105 and R108 were shielded by complex formation, confirming the predicted paratope. ITEM MS determined the 1:1 stoichiometry and the high complex binding strength, exemplified by the gas phase dissociation reaction enthalpy of 50.2 kJ/mol. The in-solution complex dissociation constant is 5 × 10-10 M. Conclusions: Combining AlphaFold2 modeling with mass spectrometry/limited proteolysis generated a trustworthy model for the sdAbCSP1 - PfCSP-Cext complex interaction interface.

5-Aminothiazoles Reveal a New Ligand-Binding Site on Prolyl Oligopeptidase Which is Important for Modulation of Its Protein-Protein Interaction-Derived Functions.

A series of novel 5-aminothiazole-based ligands for prolyl oligopeptidase (PREP) comprise selective, potent modulators of the protein-protein interaction (PPI)-mediated functions of PREP, although they are only weak inhibitors of the proteolytic activity of PREP. The disconnected structure-activity relationships are significantly more pronounced for the 5-aminothiazole-based ligands than for the earlier published 5-aminooxazole-based ligands. Furthermore, the stability of the 5-aminothiazole scaffold allowed exploration of wider substitution patterns than that was possible with the 5-aminooxazole scaffold. The intriguing structure-activity relationships for the modulation of the proteolytic activity and PPI-derived functions of PREP were elaborated by presenting a new binding site for PPI modulating PREP ligands, which was initially discovered using molecular modeling and later confirmed through point mutation studies. Our results suggest that this new binding site on PREP is clearly more important than the active site of PREP for the modulation of its PPI-mediated functions.