Structural Dynamics of the Functional Nonameric Type III Translocase Export Gate.

Area of Science:

• Microbiology
• Molecular Biology
• Structural Biology

Background:

• Type III protein secretion is crucial for Gram-negative pathogens, utilizing a complex injectisome machinery.
• The SctV protein forms a key component of the inner membrane translocase, acting as a receptor for secreted proteins.
• Understanding SctV assembly, structure, and dynamics is vital for deciphering bacterial virulence mechanisms.

Purpose of the Study:

• To investigate the assembly, function, structure, and dynamics of the SctV export gate subunit from enteropathogenic E. coli (EPEC).
• To characterize the oligomeric state and structural features of SctV involved in protein secretion.
• To elucidate the dynamic mechanisms of SctV during chaperone binding and substrate translocation.

Main Methods:

• Biochemical extraction and oligomerization studies to determine SctV assembly.
• Negative staining electron microscopy and cryo-electron microscopy (cryo-EM) for structural determination of SctV complexes.
• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe protein dynamics and conformational changes.

Main Results:

• SctV forms stable, detergent-extracted homo-nonamers (SctV9) in E. coli strains.
• Structural analysis revealed a tripartite particle with a C-domain ring exhibiting a dynamic 'pinching' motion involving flexible sub-domains.
• HDX-MS identified dynamic hinges and a hyper-flexible sub-domain in SctV, crucial for chaperone binding and conformational changes.

Conclusions:

• Membrane-embedded SctV9 is essential for recognizing diverse chaperone/protein pairs, mediating type III secretion.
• The intrinsic dynamics of SctV protomers, modulated by chaperones and the ATPase, are critical for allosteric regulation of the nonameric ring during secretion.
• This study provides novel insights into the structural basis and dynamic regulation of the type III secretion system's export gate.