RESUMO
Mammalian septins comprise a family of 13 genes that encode GTP-binding proteins. Specific combinations of septins can hetero-oligomerize and form filaments in vivo and in vitro, by mechanisms that are not understood. Using fluorescence resonance energy transfer, size exclusion chromatography, and multi-angle light scattering techniques, we have characterized the conformation of a complex of filamentous human septins, Sept2, Sept6, and Sept7. We now show that Sept6 and Sept7 interact through a parallel coiled-coil, and that Sept2 interacts with Sept6 through their C-terminal domains. We have also been able to produce soluble, stable individual septins that behave as rod-like monomers and dimers. Taken together, these observations suggest that polymerized filaments could be comprised of laterally arranged septin core subunits.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ligação ao GTP/química , Monoéster Fosfórico Hidrolases/química , Animais , Células COS , Chlorocebus aethiops , Proteínas do Citoesqueleto , Dimerização , Transferência Ressonante de Energia de Fluorescência/métodos , Células HeLa , Humanos , Polímeros/química , Conformação Proteica , Estrutura Terciária de Proteína , Septinas , Espectrometria de FluorescênciaRESUMO
The crystal structure of the E. coli RecA protein was solved more than 10 years ago, but it has provided limited insight into the mechanism of homologous genetic recombination. Using electron microscopy, we have reconstructed five different states of RecA-DNA filaments. The C-terminal lobe of the RecA protein is modulated by the state of the distantly bound nucleotide, and this allosteric coupling can explain how mutations and truncations of this C-terminal lobe enhance RecA's activity. A model generated from these reconstructions shows that the nucleotide binding core is substantially rotated from its position in the RecA crystal filament, resulting in ATP binding between subunits. This simple rotation can explain the large cooperativity in ATP hydrolysis observed for RecA-DNA filaments.
Assuntos
Trifosfato de Adenosina/metabolismo , Recombinases Rec A/química , Sítios de Ligação , Escherichia coli/química , Escherichia coli/metabolismo , Microscopia Eletrônica , Modelos Moleculares , Conformação Proteica , Recombinases Rec A/metabolismo , Recombinases Rec A/ultraestruturaRESUMO
There are 10 known mammalian septin genes, some of which produce multiple splice variants. The current nomenclature for the genes and gene products is very confusing, with several different names having been given to the same gene product and distinct names given to splice variants of the same gene. Moreover, some names are based on those of yeast or Drosophila septins that are not the closest homologues. Therefore, we suggest that the mammalian septin field adopt a common nomenclature system, based on that adopted by the Mouse Genomic Nomenclature Committee and accepted by the Human Genome Organization Gene Nomenclature Committee. The human and mouse septin genes will be named SEPT1-SEPT10 and Sept1-Sept10, respectively. Splice variants will be designated by an underscore followed by a lowercase "v" and a number, e.g., SEPT4_v1.
