Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Extraepitopic Amino Acid Substitutions Define a Change in the Accessibility of Conformational Epitopes of the Bacillus cereus HlyII C-Terminal Domain

Natalia V. Rudenko
* ORCID logo ,
Alexey S. Nagel
,
Bogdan S. Melnik
ORCID logo ,
Anna P. Karatovskaya
,
Olesya Vetrova
,
Anna V. Zamyatina
,
Zhanna I. Andreeva-Kovalevskaya
,
Alexander V. Siunov
ORCID logo ,
Mikhail G. Shlyapnikov
,
Fedor A. Brovko
,
Alexander S. Solonin
Version 1 : Received: 11 October 2023 / Approved: 11 October 2023 / Online: 12 October 2023 (04:37:50 CEST)

A peer-reviewed article of this Preprint also exists.

Rudenko, N.V.; Nagel, A.S.; Melnik, B.S.; Karatovskaya, A.P.; Vetrova, O.S.; Zamyatina, A.V.; Andreeva-Kovalevskaya, Z.I.; Siunov, A.V.; Shlyapnikov, M.G.; Brovko, F.A.; Solonin, A.S. Utilizing Extraepitopic Amino Acid Substitutions to Define Changes in the Accessibility of Conformational Epitopes of the Bacillus cereus HlyII C-Terminal Domain. Int. J. Mol. Sci. 2023, 24, 16437. Rudenko, N.V.; Nagel, A.S.; Melnik, B.S.; Karatovskaya, A.P.; Vetrova, O.S.; Zamyatina, A.V.; Andreeva-Kovalevskaya, Z.I.; Siunov, A.V.; Shlyapnikov, M.G.; Brovko, F.A.; Solonin, A.S. Utilizing Extraepitopic Amino Acid Substitutions to Define Changes in the Accessibility of Conformational Epitopes of the Bacillus cereus HlyII C-Terminal Domain. Int. J. Mol. Sci. 2023, 24, 16437.

Abstract

Hemolysin II (HlyII) – one of the pathogenic factors of Bacillus cereus, a pore-forming β-barrel toxin – possesses a C-terminal extension of 94 amino acid residues, designated as the C-terminal domain of HlyII (HlyIICTD), which plays an important role in the functioning of the toxin. Our previous work described a monoclonal antibody (HlyIIC-20), capable of strain-specific inhibi-tion of hemolysis caused by HlyII, and demonstrated the dependence of the efficiency of hemol-ysis on the presence of proline at position 324 in HlyII outside the conformational antigenic de-terminant. In this work, we studied 16 mutant forms of HlyIICTD. Each of the mutations, ob-tained by multiple site-directed mutagenesis leading to the replacement of amino acid residues lying on the surface of the 3D structure of HlyIICTD, led to a decrease in the interaction of HlyIIC-20 with the mutant form of the protein. Changes in epitope structure confirm the high conformational mobility of HlyIICTD required for the functioning of HlyII. Comparison of the effect of the introduced mutations on the effectiveness of interaction between HlyIICTD and HlyIIC-20 and a control antibody recognizing a non-overlapping epitope enabled identifying the amino acid residues N339, K340 included in the conformational antigenic determinant rec-ognized by HlyIIC-20.

Keywords

pore-forming toxin; monoclonal antibodies; epitope; site-directed mutagenesis, enzyme immunoassay, phage display; modeling of three-dimensional structure

Subject

Biology and Life Sciences, Biochemistry and Molecular Biology

Copyright: This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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