Identification of a peptide motif that potently inhibits two functionally distinct subunits of Shiga toxin

Toshiya Senda,Miki Senda,Akiko Okuta,Atsuo Miyazawa,Eiko Shimizu,Miho Watanabe-Takahashi,Masakazu Tamada,Kiyotaka Nishikawa,Masahiro Hibino

doi:10.1038/s42003-021-02068-3

Abstract

Shiga toxin (Stx) is a major virulence factor of enterohemorrhagic Escherichia coli, which causes fatal systemic complications. Here, we identified a tetravalent peptide that inhibited Stx by targeting its receptor-binding, B-subunit pentamer through a multivalent interaction. A monomeric peptide with the same motif, however, did not bind to the B-subunit pentamer. Instead, the monomer inhibited cytotoxicity with remarkable potency by binding to the catalytic A-subunit. An X-ray crystal structure analysis to 1.6 Å resolution revealed that the monomeric peptide fully occupied the catalytic cavity, interacting with Glu167 and Arg170, both of which are essential for catalytic activity. Thus, the peptide motif demonstrated potent inhibition of two functionally distinct subunits of Stx.

Highlights

Shiga toxin (Stx) is a major virulence factor of enterohemorrhagic Escherichia coli, which causes fatal systemic complications
We identified a tetravalent peptide MMA-tet that efficiently inhibits Stx1a and Stx2a by screening a library of tetravalent peptides for highaffinity binding to the B-subunit pentamer[20,21,22]
The density of tetravalent peptides on the membrane and the spacer length was optimized for the screening using Stx as shown previously[23,24]

Summary

Introduction

Shiga toxin (Stx) is a major virulence factor of enterohemorrhagic Escherichia coli, which causes fatal systemic complications. We identified a tetravalent peptide that inhibited Stx by targeting its receptor-binding, B-subunit pentamer through a multivalent interaction. The peptide motif demonstrated potent inhibition of two functionally distinct subunits of Stx. 1234567890():,; Shiga toxin (Stx) is a major virulence factor of enterohemorrhagic Escherichia coli, which causes bloody diarrhea, hemorrhagic colitis, and sometimes fatal systemic complications, such as acute encephalopathy and hemolyticuremic syndrome[1,2,3,4,5,6]. Screening of the library led to the identification of tetravalent peptides that bind to the B-subunit pentamer and inhibit its toxicity in vitro and in vivo[20,21,22] One of these peptides, MMA-tet (containing the following motif, Met-Met-Ala-Arg-Arg-Arg-Arg), inhibits the major Stx family members (e.g., Stx1a and Stx2a), as well as other highly virulent Stx[2] subtypes, including Stx2d and Stx2c23. Crystallographic analysis showed that the monomeric peptide fully occupied the catalytic cavity of the A-subunit

Methods

Results

Conclusion