Structural properties of FliH, an ATPase regulatory component of the Salmonella type III flagellar export apparatus.
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.
Minamino
Tohru
T
Protonic NanoMachine Project, ERATO, JST, 1-7 Hikaridai, Seika, Kyoto, Japan.
González-Pedrajo
Bertha
B
Oosawa
Kenji
K
Namba
Keiichi
K
Macnab
Robert M
RM
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D001692:Biological Transport; D002850:Chromatography, Gel; D019281:Dimerization; D005407:Flagella / Q000737:chemistry*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D006180:Proton-Translocating ATPases; D012475:Salmonella / Q000737:chemistry*; D017384:Sequence Deletion; D014357:Trypsin / Q000378:metabolism
D001426:Bacterial Proteins; D021122:Protein Subunits; D011506:Proteins; C069250:fliH protein, Bacteria; C069251:fliI protein, bacteria; D014357:Trypsin; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases