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.

Autor:	Opuni KFM; Department of Pharmaceutical Chemistry, School of Pharmacy, College of Health Science, University of Ghana, P.O. Box LG43, Legon, Ghana., Ruß M; Proteome Center Rostock, University Medicine Rostock and University of Rostock, Schillingallee 69, 18057 Rostock, Germany., Geens R; Laboratory of Medical Biochemistry, Faculty of Pharmaceutical, Biomedical, and Veterinary Sciences, University of Antwerp, Universiteitsplein 1, Wilrijk, 2610 Antwerp, Belgium., Vocht L; Laboratory of Medical Biochemistry, Faculty of Pharmaceutical, Biomedical, and Veterinary Sciences, University of Antwerp, Universiteitsplein 1, Wilrijk, 2610 Antwerp, Belgium., Wielendaele PV; Laboratory of Medical Biochemistry, Faculty of Pharmaceutical, Biomedical, and Veterinary Sciences, University of Antwerp, Universiteitsplein 1, Wilrijk, 2610 Antwerp, Belgium., Debuy C; Laboratory of Medical Biochemistry, Faculty of Pharmaceutical, Biomedical, and Veterinary Sciences, University of Antwerp, Universiteitsplein 1, Wilrijk, 2610 Antwerp, Belgium., Sterckx YG; Laboratory of Medical Biochemistry, Faculty of Pharmaceutical, Biomedical, and Veterinary Sciences, University of Antwerp, Universiteitsplein 1, Wilrijk, 2610 Antwerp, Belgium., Glocker MO; Proteome Center Rostock, University Medicine Rostock and University of Rostock, Schillingallee 69, 18057 Rostock, Germany.
Jazyk:	angličtina
Zdroj:	Computational and structural biotechnology journal [Comput Struct Biotechnol J] 2024 Aug 28; Vol. 23, pp. 3300-3314. Date of Electronic Publication: 2024 Aug 28 (Print Publication: 2024).
DOI:	10.1016/j.csbj.2024.08.023
Abstrakt:	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. Competing Interests: The authors declare no conflicts of interest. (© 2024 The Authors.)
Databáze:	MEDLINE
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