The flexible linker of the secreted FliK ruler is required for export switching of the flagellar protein export apparatus

Miki Kinoshita,Yumi Inoue,Tohru Minamino,Keiichi Namba,Seina Tanaka,Shin-Ichi Aizawa

doi:10.1038/s41598-020-57782-5

Abstract

The hook length of the flagellum is controlled to about 55 nm in Salmonella. The flagellar type III protein export apparatus secretes FliK to determine hook length during hook assembly and changes its substrate specificity from the hook protein to the filament protein when the hook length has reached about 55 nm. Salmonella FliK consists of an N-terminal domain (FliKN, residues 1–207), a C-terminal domain (FliKC, residues 268–405) and a flexible linker (FliKL, residues 208–267) connecting these two domains. FliKN is a ruler to measure hook length. FliKC binds to a transmembrane export gate protein FlhB to undergo the export switching. FliKL not only acts as part of the ruler but also contributes to this switching event, but it remains unknown how. Here we report that FliKL is required for efficient interaction of FliKC with FlhB. Deletions in FliKL not only shortened hook length according to the size of deletions but also caused a loose length control. Deletion of residues 206–265 significantly reduced the binding affinity of FliKC for FlhB, thereby producing much longer hooks. We propose that an appropriate length of FliKL is required for efficient interaction of FliKC with FlhB.

Highlights

FliK ruler is required for export switching of the flagellar protein export apparatus Miki Kinoshita[1], Seina Tanaka2,Yumi Inoue[1,5], Keiichi Namba 1,3,4, Shin-Ichi Aizawa2 & Tohru Minamino 1*
To clarify the role of residues 206–235 of FliKL in the hook length control, we constructed a series of mutant variants of FliK with sequential 5-amino-acid deletions within a region of residues 206–235, namely FliK(∆206–210), FliK(∆211–215), FliK(∆216–220), (∆221–225), (∆226–230) and FliK(∆231–235) (Table 1)
The core domain of FliKC is conserved among FliK/SctP family[53] and possesses a fold similar to the C-terminal domain of SctP of the injectisome of Pseudomonas aeruginosa[54]

Summary

Introduction

FliK ruler is required for export switching of the flagellar protein export apparatus Miki Kinoshita[1], Seina Tanaka2,Yumi Inoue[1,5], Keiichi Namba 1,3,4, Shin-Ichi Aizawa2 & Tohru Minamino 1*. The flagellar type III protein export apparatus secretes FliK to determine hook length during hook assembly and changes its substrate specificity from the hook protein to the filament protein when the hook length has reached about 55 nm. When hook length has reached about 55 nm, the flagellar type III protein export apparatus changes its substrate specificity, thereby terminating hook assembly and initiating filament assembly[9,10]. If certain mutations in FliK, FlhB or FlhA inhibits the export switching of the flagellar type III protein export apparatus, unusually elongated hooks called polyhooks are generated. Conformational changes of FlhAC and FlhBC are required for the substrate specificity switching of the flagellar protein export apparatus upon hook completion[18,28,33]. Flk interferes with premature switching of the protein export apparatus during hook-basal body assembly[13,14,34]

Methods

Results

Conclusion