Spatial Structure of the C-Terminal Domain of Bacillus cereus Hemolysin II is Stabilized in the Composition of the Full-Size Toxin

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Abstract

Hemolysin II (HlyII) is one of the key pathogenic factors of Bacillus cereus, a pore-forming toxin with a spatial structure of the β-barrel type, possessing a C-terminal extension of 94 amino acid residues, designated as the C-terminal domain of HlyII (HlyIICTD). In this work, site-directed mutagenesis of amino acid residues lying on the surface of the HlyIICTD protein globule was carried out. The work has been demonstrated that the C-terminal domain can simultaneously exist in several structural isoforms. The move of the three-dimensional structure of HlyIICTD into a stable form as a part of water-soluble full-length toxin monomer is observed. Recombinant proteins and their mutant forms using producing strain Escherichia coli BL21 (DE3) were obtained. Their interaction with monoclonal antibodies HlyIIC-16 and HlyIIC-23 by enzyme immunoassay was studied. To define the epitopes of the phage display of HlyIIC-16 and HlyIIC-23, site-directed mutagenesis, gene cloning of individual parts of the HlyIICTD molecule, three-dimensional modeling of HlyIICTD fused to SlyD using the AlphaFold program were used. It was shown that monoclonal antibodies obtained against HlyIICTD interacted with intact HlyIICTD much more effectively than with the full-length toxin and the chimeric protein – HlyIICTD fused with SlyD. Antibodies HlyIIC-16 and HlyIIC-23 effectively inhibited each other's interaction with immobilized HlyIICTD in an enzyme-linked immunosorbent assay, indicating the proximity of their epitopes on the surface of the HlyIICTD molecule. Phage display, site-directed mutagenesis, and gene cloning of individual parts of the HlyIICTD molecule were used to determine the epitopes of HlyIIC-16 and HlyIIC-23. Spatial modeling of HlyIICTD fused to SlyD using the AlphaFold program suggested the location of the HlyIIC-16 and HlyIIC-23 epitopes on the Gly341–Gly364 region of the HlyII protein. It was demonstrated that the C-terminal domain can simultaneously exist in several structural states (isoforms). In the water-soluble form of the full-length toxin monomer, a transition of the spatial structure of HlyIICTD to a stable form is observed.

About the authors

N. V Rudenko

Branch of the Federal State Budgetary Institution of Science, State Scientific Center of the Russian Federation, Institute of Bioorganic Chemistry named after Academicians M.M. Shemyakin and Yu.A. Ovchinnikova Russian Academy of Sciences (Branch of the State Research Center IBCh RAS)

Email: nrudkova@mail.ru
Pushchino, Russia

B. S Melnik

Branch of the Federal State Budgetary Institution of Science, State Scientific Center of the Russian Federation, Institute of Bioorganic Chemistry named after Academicians M.M. Shemyakin and Yu.A. Ovchinnikova Russian Academy of Sciences (Branch of the State Research Center IBCh RAS); Institute of Protein Research of the Russian Academy of Sciences (IPR RAS)

Pushchino, Russia; Pushchino Russia

A. P Karatovskaya

Branch of the Federal State Budgetary Institution of Science, State Scientific Center of the Russian Federation, Institute of Bioorganic Chemistry named after Academicians M.M. Shemyakin and Yu.A. Ovchinnikova Russian Academy of Sciences (Branch of the State Research Center IBCh RAS)

Pushchino, Russia

A. S Nagel

G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms of the Russian Academy of Sciences (IBFM RAS) “Federal Research Center “Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”

Pushchino, Russia

Zh. I Andreeva-Kovalevskaya

G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms of the Russian Academy of Sciences (IBFM RAS) “Federal Research Center “Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”

Pushchino, Russia

A. V Zamyatina

Branch of the Federal State Budgetary Institution of Science, State Scientific Center of the Russian Federation, Institute of Bioorganic Chemistry named after Academicians M.M. Shemyakin and Yu.A. Ovchinnikova Russian Academy of Sciences (Branch of the State Research Center IBCh RAS)

Pushchino, Russia

O. S Vetrova

Branch of the Federal State Budgetary Institution of Science, State Scientific Center of the Russian Federation, Institute of Bioorganic Chemistry named after Academicians M.M. Shemyakin and Yu.A. Ovchinnikova Russian Academy of Sciences (Branch of the State Research Center IBCh RAS)

Pushchino, Russia

A. V Siunov

G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms of the Russian Academy of Sciences (IBFM RAS) “Federal Research Center “Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”

Pushchino, Russia

F. A Brovko

Branch of the Federal State Budgetary Institution of Science, State Scientific Center of the Russian Federation, Institute of Bioorganic Chemistry named after Academicians M.M. Shemyakin and Yu.A. Ovchinnikova Russian Academy of Sciences (Branch of the State Research Center IBCh RAS)

Pushchino Russia

A. S Solonin

G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms of the Russian Academy of Sciences (IBFM RAS) “Federal Research Center “Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”

Pushchino, Russia

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