Crystal structure of bacterial cytotoxic necrotizing factor CNFY reveals molecular building blocks for intoxication

Cytotoxic necrotizing factors (CNFs) are bacterial single‐chain exotoxins that modulate cytokinetic/oncogenic and inflammatory processes through activation of host cell Rho GTPases. To achieve this, they are secreted, bind surface receptors to induce endocytosis and translocate a catalytic unit into the cytosol to intoxicate host cells. A three‐dimensional structure that provides insight into the underlying mechanisms is still lacking. Here, we determined the crystal structure of full‐length Yersinia pseudotuberculosis CNF Y . CNF Y consists of five domains (D1–D5), and by integrating structural and functional data, we demonstrate that D1–3 act as export and translocation module for the catalytic unit (D4–5) and for a fused β‐lactamase reporter protein. We further found that D4, which possesses structural similarity to ADP‐ribosyl transferases, but had no equivalent catalytic activity, changed its position to interact extensively with D5 in the crystal structure of the free D4–5 fragment. This liberates D5 from a semi‐blocked conformation in full‐length CNF Y , leading to higher deamidation activity. Finally, we identify CNF translocation modules in several uncharacterized fusion proteins, which suggests their usability as a broad‐specificity protein delivery tool. SYNOPSIS Single‐chain bacterial cytotoxic necrotizing factor (CNF) toxins constitutively activate small Rho GTPases in host cells, leading to actin rearrangements and multinucleation. Here, crystal structures and accompanying cell biological experiments using CNFY from Yersinia pseudotuberculosis reveal the molecular basis of CNFY secretion, host cell binding, and translocation into the host cell cytosol. The CNFY structure reveals five domains, D1–5, with distinct functions in cell entry and RhoA activation. Domains D1–3 constitute a minimal translocation unit that can be employed as broad‐specificity protein delivery tool. A structure of the enzymatically‐active fragment D4–5 reveals structural rearrangements that accompany its release into the host cell cytosol. D4 constitutes a DUF4765 domain and displays structural similarity to ADP‐ribosyl transferases. Graphical Abstract Structure‐function analyses of the full‐length Yersinia pseudotuberculosis toxin CNFY offer insights into individual domain contributions to stepwise receptor binding, endocytosis, and translocation into the host cell cytosol.