Septin C-terminal domain interactions: implications for filament stability and assembly

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Septins are conserved filament-forming GTP-binding proteins crucial for eukaryotic cell structure.
• While the GTP-binding domain's role in filament formation is known, the C-terminal domains' functions are poorly understood due to limited structural data.

Purpose of the Study:

• To investigate the biophysical properties and interactions of the C-terminal domains of human SEPT2, SEPT6, and SEPT7.
• To elucidate the potential role of these C-terminal domains in septin filament assembly and stability.

Main Methods:

• Expression and purification of human SEPT2, SEPT6, and SEPT7 C-terminal domains (SEPT2-C, SEPT6-C, SEPT7-C) in E. coli.
• Circular dichroism spectroscopy to assess thermal stability and secondary structure.
• Size exclusion chromatography, chemical cross-linking, analytical ultracentrifugation, and surface plasmon resonance to study homo- and hetero-interactions.

Main Results:

• SEPT6-C and SEPT7-C form stable homo- and heterodimers with high alpha-helical content.
• The SEPT6-C/SEPT7-C heterodimer is elongated and significantly more stable (K(D) = 15.8 nM) than homodimers.
• SEPT2-C forms a homodimer with lower affinity (K(D) = 4 μM) and moderate alpha-helical content.

Conclusions:

• Provides the first experimental evidence on the biophysical properties of septin C-terminal domains.
• Highlights distinct dimerization behaviors and stabilities among SEPT2-C, SEPT6-C, and SEPT7-C.
• Suggests a potential role for C-terminal domain interactions in the assembly and stability of septin filaments.