Structural Properties of FliH, an ATPase Regulatory Component of the Salmonella Type III Flagellar Export Apparatus

Abstract

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-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.

Introduction

Bacteria secrete a number of proteins to the external environment, using a variety of systems that have been grouped into at least four families: types I–IV.¹ The export of bacterial flagellar proteins (which then self-assemble) has characteristics in common with the secretion of various virulence effector proteins by pathogenic bacteria; these processes now carry the common designation of type III export/secretion pathways.2., 3., 4., 5. The component proteins of the flagellar export apparatus and those involved in virulence factor secretion show a high degree of similarity. Also, the ultrastructure of the type III virulence factor secretion apparatus, called the needle complex, resembles that of the hook–basal body complex of the flagellum,6., 7. further supporting an evolutionary relationship between them.

Almost all external flagellar components are translocated across the cytoplasmic membrane into the central channel of the nascent structure by a specialized apparatus, which consists of six integral-membrane proteins (FlhA, FlhB, FliO, FliP, FliQ and FliR) and three cytoplasmic proteins (FliH, FliI and FliJ).8., 9. All of the integral-membrane components are believed to be located in a patch of membrane within an annular pore in the flagellar basal body MS ring. It has been demonstrated experimentally that at least three of these proteins –FlhA, FliP and FliR– are associated with the MS ring.10., 11.

One of the cytoplasmic components, FliH, a protein consisting of 235 amino acid residues, exists as a homodimer and forms a heterotrimer together with FliI, the ATPase which supplies the energy for the translocation of flagellar proteins across the plane of the cytoplasmic membrane.12., 13., 14., 15., 16. FliH inhibits the ATPase activity of FliI, perhaps to prevent it from hydrolyzing ATP until the flagellar export apparatus is competent to link this hydrolysis to the translocation of its substrates into the channel of the growing flagellar structure.¹⁶ FliI that has a R7C/L12P double mutation at its N terminus¹² fails to make a complex with FliH and truncation of the first seven amino acid residues from the N terminus of FliI also prevents it from forming a complex with FliH.¹³ Affinity blotting⁹ and Ni-NTA affinity chromatography¹⁷ have shown that FliH also interacts with another cytoplasmic component, FliJ, which is capable of preventing its export substrates from premature aggregation in the cytoplasm and is thought to be a general chaperone.¹⁸ Finally, FliH interacts with the cytoplasmic domains of FlhA and FlhB.9., 19. We have recently carried out a scanning deletion analysis of the entire 235 amino acid residue sequence of FliH,¹⁷ and shown that it consists of at least three regions: an N-terminal region, a central region between residues 101 and 140 which contains a sequence with a significant probability of α-helical coiled-coil structure, and a C-terminal region (Figure 1). The middle and C-terminal regions of FliH are responsible for dimerization and association with FliI, respectively, whereas the function of the N-terminal region is less well understood; it appears to be involved in the interaction with the general chaperone FliJ, and is certainly important for the function of FliH.

In the present study, to understand the structural properties of FliH in more detail, we have carried out limited proteolysis by trypsin and also examined various engineered fragments, using both gel filtration chromatography and affinity chromatography.