The zinc ions stabilize the three-dimensional structure and are required for the binding of staphylococcal enterotoxin-like protein P (SEIP) with MHC-II receptors

Irina Shulcheva; Margarita Shchannikova; Bogdan Melnik; Ksenia Fursova; Svetlana Semushina; Anna Zamyatina; Vladimir Oleinikov; Fedor Brovko

doi:10.1016/j.pep.2022.106098

The zinc ions stabilize the three-dimensional structure and are required for the binding of staphylococcal enterotoxin-like protein P (SEIP) with MHC-II receptors

Protein Expr Purif. 2022 Sep:197:106098. doi: 10.1016/j.pep.2022.106098. Epub 2022 May 2.

Authors

Irina Shulcheva¹, Margarita Shchannikova², Bogdan Melnik³, Ksenia Fursova², Svetlana Semushina⁴, Anna Zamyatina⁴, Vladimir Oleinikov⁵, Fedor Brovko²

Affiliations

¹ Ernst All-Russian Research Institute of Animal Husbandry, Dubrovitsy, Moscow Region, 142132, Russia; Pushchino Branch, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Pushchino, Moscow Region, 142290, Russia. Electronic address: loskutova-i@mail.ru.
² Ernst All-Russian Research Institute of Animal Husbandry, Dubrovitsy, Moscow Region, 142132, Russia; Pushchino Branch, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Pushchino, Moscow Region, 142290, Russia.
³ Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia.
⁴ Pushchino Branch, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Pushchino, Moscow Region, 142290, Russia.
⁵ Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Ulitsa Miklukho-Maklaya, 16/10 Moscow, 117997, Russia.

PMID: 35513232
DOI: 10.1016/j.pep.2022.106098

Abstract

Staphylococcus aureus is a common human and animal pathogen. These bacteria have various pathogenicity factors, including enterotoxin-like proteins. SElP (staphylococcal enterotoxin-like protein P) has potential zinc ion-binding sites and is able to interact with major histocompatibility complex class II (MHCII) and T-cell receptor (TCR). A method for the expression and isolation of the enterotoxin-like protein of Staphylococcus aureus (SElP) was developed. The expression was carried out in E. coli cells, and the protein was isolated by affinity chromatography on a NiNTA column. The endotoxins were separated by affinity chromatography on Affi-Prep® polymyxin. It was shown by gel filtration that the resulting protein had a monomeric form. The protein in zinc-bound and zinc-free forms was characterized by protein melting using fluorescence method and it was shown that zinc stabilizes the spatial structure of SElP. The functional activity of SElP was investigated by the ability to interact with the histocompatibility antigen class II receptor (MHC-II) exposed on the B cell line Raji by flow cytofluorometry. The zinc-bound and zinc-free forms were shown to differ in their interaction with MHC-II. The localization of the zinc-binding site was confirmed by the introduction of the H225 and D227 mutations. The mutant protein was characterized by melting, and its propensity to form aggregates was shown.

Keywords: Enterotoxin-like protein; Protein stability; Recombinant protein; Staphylococcus; Superantigen; Zinc.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Animals
Binding Sites
Enterotoxins* / genetics
Escherichia coli / genetics
Escherichia coli / metabolism
Histocompatibility Antigens Class II / chemistry
Ions
Receptors, Antigen, T-Cell
Staphylococcus aureus / metabolism
Superantigens* / genetics
Superantigens* / metabolism
Zinc / chemistry

Substances

Enterotoxins
Histocompatibility Antigens Class II
Ions
Receptors, Antigen, T-Cell
Superantigens
Zinc