Septin C-Terminal Domain Interactions: Implications for Filament Stability and Assembly

Ivo de Almeida Marques, Napoleão Fonseca Valadares, Wanius Garcia, Julio Cesar Pissuti Damalio, Joci Neuby Alves Macedo, Ana Paula Ulian de Araújo, Carlos Alfonso Botello, José Manuel Andreu, Richard Charles Garratt

Research output: Contribution to journalArticlepeer-review

Abstract

Septins form a conserved family of filament forming GTP binding proteins found in a wide range of eukaryotic cells. They share a common structural architecture consisting of an N-terminal domain, a central GTP binding domain and a C-terminal domain, which is often predicted to adopt a coiled-coil conformation, at least in part. The crystal structure of the human SEPT2/SEPT6/SEPT7 heterocomplex has revealed the importance of the GTP binding domain in filament formation, but surprisingly no electron density was observed for the C-terminal domains and their function remains obscure. The dearth of structural information concerning the C-terminal region has motivated the present study in which the putative C-terminal domains of human SEPT2, SEPT6 and SEPT7 were expressed in E. coli and purified to homogeneity. The thermal stability and secondary structure content of the domains were studied by circular dichroism spectroscopy, and homo- and hetero-interactions were investigated by size exclusion chromatography, chemical cross-linking, analytical ultracentrifugation and surface plasmon resonance. Our results show that SEPT6-C and SEPT7-C are able to form both homo- and heterodimers with a high α-helical content in solution. The heterodimer is elongated and considerably more stable than the homodimers, with a K D of 15.8 nM. On the other hand, the homodimer SEPT2-C has a much lower affinity, with a K D of 4 μM, and a moderate α-helical content. Our findings present the first direct experimental evidence toward better understanding the biophysical properties and coiled-coil pairings of such domains and their potential role in filament assembly and stability.

Original languageEnglish (US)
Pages (from-to)317-328
Number of pages12
JournalCell Biochemistry and Biophysics
Volume62
Issue number2
DOIs
StatePublished - Mar 2012
Externally publishedYes

Keywords

  • Coiled-coil
  • Homo- and hetero-interactions
  • Human septin complex
  • Linear hexameric complex
  • SPR
  • Thermal unfolding

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

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