Binding specificity of type three secretion system effector NleH2 to multi-cargo chaperone CesT and their...

Area of Science:

• Microbiology
• Molecular Biology
• Structural Biology

Background:

• Gram-negative pathogens employ type three secretion systems (T3SS) to deliver effector proteins into host cells.
• Specialized chaperones, like CesT, are crucial for stabilizing and translocating these effectors.
• CesT is a unique chaperone capable of interacting with approximately ten different effector proteins.

Purpose of the Study:

• To investigate the specific interaction between the CesT chaperone and the NleH2 effector protein.
• To elucidate the role of NleH2 as a potential kinase responsible for CesT phosphorylation.
• To characterize the binding domains and sites involved in CesT-NleH2 complex formation.

Main Methods:

• Identification and characterization of the NleH2 chaperone-binding domain (CBD).
• Mapping of specific interaction sites between CesT and NleH2 using structural and biochemical approaches.
• Comparative analysis of CesT binding with effectors (NleH2, Tir) and a regulator (CsrA).
• Kinase activity assays to assess NleH2's phosphorylation of CesT.

Main Results:

• The N- and C-terminal residues of NleH2's CBD bind to the dimeric interface of CesT.
• Effectors bind to the β-sheet core and dimer interface of CesT, distinct from CsrA binding sites.
• NleH2 retains kinase activity within the CesT complex, phosphorylating itself and CesT at Tyr153.

Conclusions:

• NleH2 acts as a kinase, phosphorylating CesT at Tyr153 within the T3SS effector translocation complex.
• The distinct binding interfaces for effectors and regulators on CesT highlight specificity in chaperone interactions.
• This work deepens the understanding of CesT's role in coordinating effector protein delivery via T3SS.