Crystal structure of full-length cytotoxic necrotizing factor CNF_Yreveals molecular building blocks for intoxication

Abstract

Cytotoxic necrotizing factors (CNFs) are single-chain exotoxins. They are secreted by several bacterial pathogens to modulate cytokinetic/oncogenic and inflammatory processes through activation host cell Rho-GTPases, but their secretion-translocation mechanism still remains an enigma. Here, we determined the crystal structure of fulllength Yersinia pseudotuberculosis CNF Y , revealing five separate domains (D1-D5) of which D1-D3 act as translocation module for the catalytic unit (D4-5) and for other fused reporter proteins. By integrating structural and functional data, we suggest a model in which the α-helical D1 domain constitutes a membrane-spanning translocation unit. This unit promotes bacterial export and exposes the host cell recognition sites of D2. Receptor binding then triggers endosomal uptake, release and structural reorientation of the catalytic unit implicating D3. Sequence comparison also suggests that this translocation mechanism is used by many other bacterial proteins and could be employed as universal drug delivery tool.Structure-function analysis of CNF Y

Insertion-trigger residues differentially modulate endosomal escape by cytotoxic necrotizing factor toxins

“…As mentioned before, the structural topology of the putative HLH region in the crystal structure of CNFy ( 16 ) did not match its proposed role as a pH-trigger for the “dagger” membrane-insertion model ( 24 ). To explore this further in light of our identification of two additional pH-sensing acidic residues that discriminate CNFy and CNF3, we mapped the electrostatic surface for this region (residues 344–423) in the structure of CNFy.…”

“…However, although these four acidic residues were confirmed to be important for cargo delivery of CNF1 ( 24 ), the crystal structure of CNFy ( 16 ) did not reveal the predicted HLH structure, and it was proposed that since the structure was determined under neutral conditions, perhaps the region changes its conformation once it is in an acidic environment. Other studies have revealed that there are differences among the CNF toxins with regard to efficiency ( 14 ) and pH dependency ( 29 ) of cargo delivery.…”

“…A recent crystal structure of CNFy revealed five structural domains ( 16 ). The cellular receptor-binding domain is located near the N-terminus (CNFy residues 23–134).…”

“…CNF1 and CNFy reportedly have an additional binding region in the C-terminus: CNF1 residues 709 to 730 bind to Lu/BCAM adhesion molecule ( 20 ), and CNFy residues 772 to 779 bind to heparan sulfates ( 21 ). Based on previously predicted functional domain organization of the CNF family ( 22 , 23 , 24 ), the N-terminal membrane translocation module, comprised of domains D1 and D3 (CNFy residues 135–530), facilitates endosomal escape of the C-terminal cargo, which includes domain D4 of unknown function (CNFy residues 530–700) and the catalytic Rho-deamidase domain D5 (CNFy residues 718–1014) ( 16 ), which is also consistent with the structure of the catalytic domain of CNF1 ( 25 ).…”

The gatekeeper of Yersinia type III secretion is under RNA thermometer control

“…Several of them are located upstream of genes critical for various aspects of bacterial virulence. A recently documented example is the cnfY thermometer controlling translation of the cytotoxic necrotizing factor that enhances inflammation and Yop delivery by activation of Rho GTPases in the host [39][40][41]. In contrast to Yops, the CnfY toxin is not secreted by the T3SS but delivered to host cells via outer membrane vesicles [42].…”