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

Area of Science:

• Bacterial Toxinology
• Structural Biology
• Molecular Microbiology

Background:

• Cytotoxic necrotizing factors (CNFs) are bacterial exotoxins that activate host Rho GTPases, impacting cellular processes.
• CNFs are secreted, bind receptors, undergo endocytosis, and translocate a catalytic domain into the host cell cytosol.
• A detailed three-dimensional structure of CNFs, crucial for understanding their mechanism, was previously unavailable.

Purpose of the Study:

• To determine the crystal structure of the full-length Yersinia pseudotuberculosis CNF_Y.
• To elucidate the structural basis for CNF export, translocation, and catalytic activity.
• To explore the potential of CNF domains as protein delivery tools.

Main Methods:

• X-ray crystallography was employed to determine the three-dimensional structure of full-length CNF_Y.
• Structural and functional analyses were integrated to understand domain roles.
• The crystal structure of the CNF_Y D4-5 fragment was determined.

Main Results:

• The crystal structure of full-length CNF_Y revealed five distinct domains (D1-D5).
• Domains D1-3 function as an export and translocation module for the catalytic unit (D4-5) and reporter proteins.
• In the D4-5 fragment, domain D4 repositioned to interact with D5, relieving steric hindrance and enhancing catalytic deamidation activity.

Conclusions:

• The determined structure provides mechanistic insights into CNF_Y function.
• CNF_Y domains D1-3 are essential for delivering the catalytic D4-5 unit.
• CNF translocation modules represent a promising platform for broad-specificity protein delivery applications.