title: Septin C-terminal domain interactions: implications for filament stability and assembly.
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.
authors: Centro de Biotecnologia Molecular Estrutural, Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador são-carlense 400, São Carlos, SP 13560-970, Brazil.
authors: Ivo
authors: I
authors: de Almeida Marques
authors: Napoleão Fonseca
authors: NF
authors: Valadares
authors: Wanius
authors: W
authors: Garcia
authors: Julio Cesar Pissuti
authors: JC
authors: Damalio
authors: Joci Neuby Alves
authors: JN
authors: Macedo
authors: Ana Paula Ulian
authors: AP
authors: de Araújo
authors: Carlos Alfonso
authors: CA
authors: Botello
authors: José Manuel
authors: JM
authors: Andreu
authors: Richard Charles
authors: RC
authors: Garratt
mesh_terms: D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D006801:Humans; D025941:Protein Interaction Mapping; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D058112:Septins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D011994:Recombinant Proteins; C000602252:SEPTIN2 protein, human; C436485:SEPTIN6 protein, human; C113158:SEPTIN7 protein, human; D058112:Septins