Domain organization and function of Salmonella FliK, a flagellar hook-length control protein.
Salmonella hook-length control protein FliK, which consists of 405 amino acid residues, switches substrate specificity of the type III flagellar protein export apparatus from rod/ hook-type to filament-type by causing a conformational change in the cytoplasmic domain of FlhB (FlhB(C)) upon completion of the hook assembly. An N-terminal region of FliK contains an export signal, and a highly conserved C-terminal region consisting of amino acid residues 265-405 (FliK((265-405))) is directly involved in the switching of FlhB(C). Here, we have investigated the structural properties of FliK. Gel filtration chromatography, multi-angle light scattering and analytical ultracentrifugation showed that FliK is monomeric in solution and has an elongated shape. Limited proteolysis showed that FliK consists of two domains, the N-terminal (FliK(N)) and C-terminal domains (FliK(C)), and that the first 203 and the last 35 amino acid residues are partially unfolded and subjected to proteolysis. Both FliK(N) and FliK(C) are more globular than full-length FliK, suggesting that these domains are connected in tandem. Overproduced His-FliK((199-405)) failed to switch export specificity of the export apparatus. Affinity blotting revealed that FlhB(C) binds to FliK and FliK((1-147)), but not to FliK((265-405)). Based on these results, we propose that FliK(N) within the central channel of the hook-basal body during the export of FliK is the sensor and transmitter of hook completion information and that the binding interaction of FliK(C) to FlhB(C) is structurally regulated by FliK(N) so as to occur only when the hook has reached a preset length. The conformational flexibility of FliK(C) may play a role in interfering with switching at an inappropriate point of flagellar assembly.
Minamino
Tohru
T
Dynamic NanoMachine Project, ICORP, JST, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan. tohru@npn.jst.go.jp
Saijo-Hamano
Yumiko
Y
Furukawa
Yukio
Y
González-Pedrajo
Bertha
B
Macnab
Robert M
RM
Namba
Keiichi
K
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000502:physiology*; D001692:Biological Transport; D002850:Chromatography, Gel; D003593:Cytoplasm / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005407:Flagella / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012475:Salmonella / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism; D014461:Ultracentrifugation
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C099487:FliK protein, Salmonella; D014357:Trypsin