Add time:08/25/2019         Source:sciencedirect.com

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-FliHN2 (consisting of the first 102 amino acid residues of FliH), exhibited anomalous elution behavior in gel filtration chromatography; the same was true of His-FliHC1 (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: FliHPrt1 (missing both the first ten and the last 12 amino acid residues), and FliHPrt2 (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 FliHPrt1 and FliHPrt2 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/FliHPrt1 or His-FliH/FliHPrt2 hybrid dimers. A hybrid dimer of FliH and His-FliHΔ1 (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.

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