Structure and activation mechanism of the Makes caterpillars floppy 1 toxin

Abstract

The bacterial Makes caterpillars floppy 1 (Mcf1) toxin promotes apoptosis in insects, leading to loss of body turgor and death. The molecular mechanism underlying Mcf1 intoxication is poorly understood. Here, we present the cryo-EM structure of Mcf1 from Photorhabdus luminescens, revealing a seahorse-like shape with a head and tail. While the three head domains contain two effectors, as well as an activator-binding domain (ABD) and an autoprotease, the tail consists of two putative translocation and three putative receptor-binding domains. Rearrangement of the tail moves the C-terminus away from the ABD and allows binding of the host cell ADP-ribosylation factor 3, inducing conformational changes that position the cleavage site closer to the protease. This distinct activation mechanism that is based on a hook-loop interaction results in three autocleavage reactions and the release of two toxic effectors. Unexpectedly, the BH3-like domain containing ABD is not an active effector. Our findings allow us to understand key steps of Mcf1 intoxication at the molecular level.

Fig. 1. The structure of Mcf1.

Composite cryo-EM density (a) and atomic model (b) of Mcf1. Schematic representation of the secondary structure (c) and domain architecture (d). e Density and coordination of the head-stabilizing element. f Surface at the interface of the three domains of the Mcf1 head (N-terminal effector domain, protease effector domain and activator-binding domain) colored by electrostatic Coulomb potential from −10 kcal mol⁻¹ (red) to +10 kcal mol⁻¹ (blue). g Close-up view of the neck region, connecting the head and tail of Mcf1 (regulatory C-terminus - magenta). NED N-terminal effector domain, ABD activator-binding domain, PED protease effector domain, RBD receptor-binding domain, TD translocation domain, TH transmembrane helices, HSE head-stabilizing element.

Fig. 2. Autoproteolytic cleavage of Mcf1.

a Western blot analysis of Sf9 cells intoxicated with wild-type (WT) or C1397A Mcf1 variants. The proteins were Myc-tagged at the N- (N-Myc) or C-terminus (C-Myc). b Mapping of the expected cleavage products on the Mcf1 structure. Approximate molecular weights of the fragments are indicated. c SDS-PAGE and western blots (WB) of the Arf3-dependent autoproteolysis of N-terminally His-tagged (N-His) and C-terminally FLAG-tagged (C-FLAG) Mcf1 in vitro. d Autoproteolysis of C-terminally truncated (Δ15C) Mcf1 variants in vitro. Source data are provided as a Source Data file. The cleavage experiments and their corresponding western blot analysis were performed twice. Abbreviations: see Fig. 1.

Fig. 3. Toxicity of Mcf1 effectors.

a Structure of the three head domains and comparison with structural homologs of two of them^,. b, c, d Growth phenotype assay with S. cerevisiae expressing Mcf1 fragments under galactose promoter in the experimental conditions with low (glucose) or high (galactose) Mcf1 fragment expression. The protein expression and the level of ADP-ribosylation was analyzed by western blot of cells grown on galactose-containing media with anti-Myc, anti-Mcf1, ADP-ribose binding reagent (anti-ADP-ribose) and anti-ribosomal protein S9 (RPS9) antibodies. Blf1 Burkholderia lethal factor 1, NED N-terminal effector domain, ABD activator-binding domain, PED protease effector domain, ART ADP-ribosyltransferase. Source data are provided as a Source Data file. Western blot analysis was performed twice.

Fig. 4. Mechanism of activation of Mcf1.

Different 3D class averages (a) and corresponding molecular models (b) of the Mcf1_{C1397A Δ15C} and Mcf1_{C1397A Δ15C}-Arf3 complex datasets. Schematic illustration of the mechanism of Mcf1 activation from the bottom (c) and front (f). Upon Arf binding, helices of the ABD are strained, pulling the linker towards the active site, while being guided by the hook-loop interaction. d The cleavable linker is localized in 25 Å distance from the catalytic center of the protease effector domain (PED). e Structure of the hook (R957) – loop interaction of Mcf1. Abbreviations: see Fig. 1.

Fig. 5. Mechanism of action of Mcf1 toxin.

Abbreviations: see Fig. 1.