Dissection of a human septin: definition and characterization of distinct domains within human SEPT4.
The septins are a conserved family of guanosine-5'-triphosphate (GTP)-binding proteins. In mammals they are involved in a variety of cellular processes, such as cytokinesis, exocytosis, and vesicle trafficking. Specifically, SEPT4 has also been shown to be expressed in both human colorectal cancer and malignant melanoma, as well as being involved in neurodegenerative disorders. However, many of the details of the modes of action of septins in general remain unclear, and little is known of their detailed molecular architecture. Here, we define explicitly and characterize the domains of human SEPT4. Regions corresponding to the N-terminal, GTPase, and C-terminal domains as well as the latter two together were successfully expressed in Escherichia coli in soluble form and purified by affinity and size-exclusion chromatographies. The purified domains were analyzed by circular dichroism spectroscopy, fluorescence spectroscopy, dynamic light scattering, and small-angle X-ray scattering, as well as with bioinformatics tools. Of the three major domains that comprise SEPT4, the N-terminal domain contains little regular secondary structure and may be intrinsically unstructured. The central GTPase domain is a mixed alpha/beta structure, probably based on an open beta sheet. As defined here, it is catalytically active and forms stable homodimers in vitro. The C-terminal domain also forms homodimers and can be divided into two regions, the second of which is alpha-helical and consistent with a coiled-coil structure. These studies should provide a useful basis for future biophysical studies of SEPT4, including the structural basis for their involvement in diseases such as cancer and neurodegenerative disorders.
Garcia
Wanius
W
Centro de Biotecnologia Molecular e Estrutural (CBME), Instituto de Física de São Carlos (IFSC), Universidade de São Paulo (USP), Av. Trabalhador São Carlense 400, centro, Box 369, São Carlos, SP, 13560-970, Brazil. wanius@if.sc.usp.br
de Araújo
Ana Paula Ulian
AP
Neto
Mario de Oliveira
Mde O
Ballestero
Michel R M
MR
Polikarpov
Igor
I
Tanaka
Manami
M
Tanaka
Tomoo
T
Garratt
Richard Charles
RC
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D058112:Septins; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
D003598:Cytoskeletal Proteins; D020558:GTP Phosphohydrolases; C492027:SEPTIN4 protein, human; D058112:Septins