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

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.

Fig. 1. Identification of a tetravalent peptide that potently inhibits Stx by targeting the B-subunit pentamer.

a Structure of the tetravalent peptides synthesized on a cellulose membrane is shown. The density of tetravalent peptides on the membrane and the spacer length was optimized for the screening using Stx as follows. The density was set to 100% by using Fmoc-β-Ala-OH without Boc-β-Ala-OH for the first peptide synthesis cycle, and the number of aminohexanoic acid residues (U; spacer length) was set to one (upper panel). Met or Ala at position three or five of MMA-tet, respectively, was replaced by the indicated amino acid (βA; beta-Ala). The tetravalent peptides synthesized on a membrane were blotted with ¹²⁵I-Stx1a (left panel). The screening was performed three times (Supplementary Fig. 1). Eight Stx1a-binding motifs (shaded in the right panel) were identified. b Structure of the tetravalent peptide and the identified motifs are shown. c MMβA-tet most efficiently inhibited the cytotoxicity of Stx1a and Stx2a. Vero cells were treated with Stx for 72 h in the presence of each peptide. Data are presented as a percentage of the control value (mean ± standard error (SE), n = 3). d AlphaScreen assay to examine the binding between MMβA-tet and the B-subunit or its mutant. Data are presented as signal intensity (mean ± SE, n = 3). ***P < 0.001, **P < 0.01, *P < 0.05 (compared with wild type by Dunnett’s test).

Fig. 2. MMβA-mono binds to the catalytic A-subunit, but not to the B-subunit pentamer, to inhibit Stx.

a MMβA-mono efficiently inhibited the cytotoxicity of Stx1a and Stx2a. Data are presented as a percentage of the control value (mean ± SE, n = 3). b AlphaScreen assay to examine the binding between MMβA-tet/MMβA-mono and the A-subunit (upper panels) or the B-subunit pentamer (lower panels). Data are presented as a percentage of the maximum binding value (mean ± SE, n = 7). c Enzyme-linked immunoassay to examine the binding between MMβA-tet/MMβA-mono and the B-subunit pentamer. Data are presented as an Optical Density at 490 nm (OD₄₉₀) (mean ± SE, n = 3). ***P < 0.001, **P < 0.01, *P < 0.05 by Student’s t test.

Fig. 3. Structural analysis of the interaction between MMβA-mono and the Stx2a A-subunit.

a Overall structure of Stx2a holotoxin in complex with MMβA-mono. Stx2a is shown as a charge distribution surface model. The surface of the Stx2a A-subunit is colored by charge (blue, positive; red, negative). MMβA-mono is shown as a stick model. A close-up view shows the catalytic cavity of the Stx2a A-subunit, which is where MMβA-mono binds. b Structural view of the binding between the A-subunit and MMβA-mono. Interacting residues are shown as stick models, hydrogen bonds are shown as broken lines, and water molecules are shown as spheres. c AlphaScreen assay to examine the binding between the Stx2a A-subunit and MMβA-mono with amino acid substitution(s). Data are presented as a percentage of the maximum binding value (mean ± SE, n = 3). ***P < 0.001, **P < 0.01, *P < 0.05 (compared with MMβA-mono by Dunnett’s test).