Structural Properties of FliH, an ATPase Regulatory Component of the Salmonella Type III Flagellar Export Apparatus
| Autor: | Tohru Minamino, Bertha González-Pedrajo, Kenji Oosawa, Keiichi Namba, Robert M. Macnab |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
medicine.medical_treatment
Proteolysis ATPase Dimer Size-exclusion chromatography Flagellum Cleavage (embryo) chemistry.chemical_compound Bacterial Proteins Salmonella Structural Biology medicine Trypsin Protein Structure Quaternary Molecular Biology Sequence Deletion Adenosine Triphosphatases Protease medicine.diagnostic_test biology Proteins Biological Transport Protein Structure Tertiary Protein Subunits Proton-Translocating ATPases Biochemistry chemistry Flagella Chromatography Gel biology.protein Dimerization Protein Binding medicine.drug |
| Zdroj: | Journal of Molecular Biology. 322:281-290 |
| ISSN: | 0022-2836 |
| DOI: | 10.1016/s0022-2836(02)00754-4 |
| Popis: | FliH is a regulatory component for FliI, the ATPase that is responsible for driving flagellar protein export in Salmonella. FliH consists of 235 amino acid residues, has a quite elongated shape, exists as a homodimer and together with FliI forms a heterotrimer. Here, we have investigated the structural properties of the FliH homodimer in further detail. Like intact His-tagged FliH homodimer, fragment His-FliH(N2) (consisting of the first 102 amino acid residues of FliH), exhibited anomalous elution behavior in gel filtration chromatography; the same was true of His-FliH(C1) (consisting of amino acid residues 119-235), but to a much lesser degree. Thus the elongated shape of FliH appears to derive primarily from its N-terminal region. A deletion version of N-His-FliH, lacking amino acid residues 101-140, does not dimerize and so we were able to establish the gel filtration properties of an almost full-size monomeric form; it also exhibited anomalous elution behavior. We performed trypsin proteolysis of the FliH homodimer and subjected the cleavage products to gel filtration chromatography. FliH was degraded by trypsin and a contaminating protease into two stable fragments: FliH(Prt1) (missing both the first ten and the last 12 amino acid residues), and FliH(Prt2) (missing both the first ten and the last 63 amino acid residues); however, substantial amounts remained undigested even after 24 hours. Under native conditions, both FliH(Prt1) and FliH(Prt2) co-eluted with undigested His-FliH from the gel filtration column, indicating that the fragments exist as a hybrid dimer with intact FliH. These results suggest that the two subunits within the dimer differ in their proteolytic susceptibility. No heterotrimer was observed by gel filtration chromatography when His-FliI was mixed with either His-FliH/FliH(Prt1) or His-FliH/FliH(Prt2) hybrid dimers. A hybrid dimer of FliH and His-FliHDelta1 (lacking the first ten amino acid residues) retained the ability to form a complex with His-FliI. In contrast, hybrid dimers consisting of FliH and either His-FliH(W223ochre) or His-FliH(V172ochre) failed to complex to His-FliI, demonstrating that the C-terminal region of both FliH monomers within the FliH dimer are required for heterotrimer formation. |
| Databáze: | OpenAIRE |
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