Three SpoA-domain proteins interact in the creation of the flagellar type III secretion system in Helicobacter pylori

Kwok Ho Lam,Chaolun Xue,Kailei Sun,Huawei Zhang,Wendy Wai Ling Lam,Zeyu Zhu,Juliana Tsz Yan Ng,William E Sause,Paphavee Lertsethtakarn,Kwok Fai Lau,Karen M Ottemann,Shannon Wing Ngor Au

doi:10.1074/jbc.ra118.002263

Kwok Ho Lam, Chaolun Xue + Show 10 more

Open Access

https://doi.org/10.1074/jbc.ra118.002263

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Abstract

Bacterial flagella are rotary nanomachines that contribute to bacterial fitness in many settings, including host colonization. The flagellar motor relies on the multiprotein flagellar motor-switch complex to govern flagellum formation and rotational direction. Different bacteria exhibit great diversity in their flagellar motors. One such variation is exemplified by the motor-switch apparatus of the gastric pathogen Helicobacter pylori, which carries an extra switch protein, FliY, along with the more typical FliG, FliM, and FliN proteins. All switch proteins are needed for normal flagellation and motility in H. pylori, but the molecular mechanism of their assembly is unknown. To fill this gap, we examined the interactions among these proteins. We found that the C-terminal SpoA domain of FliY (FliYC) is critical to flagellation and forms heterodimeric complexes with the FliN and FliM SpoA domains, which are β-sheet domains of type III secretion system proteins. Surprisingly, unlike in other flagellar switch systems, neither FliY nor FliN self-associated. The crystal structure of the FliYC-FliNC complex revealed a saddle-shaped structure homologous to the FliN-FliN dimer of Thermotoga maritima, consistent with a FliY-FliN heterodimer forming the functional unit. Analysis of the FliYC-FliNC interface indicated that oppositely charged residues specific to each protein drive heterodimer formation. Moreover, both FliYC-FliMC and FliYC-FliNC associated with the flagellar regulatory protein FliH, explaining their important roles in flagellation. We conclude that H. pylori uses a FliY-FliN heterodimer instead of a homodimer and creates a switch complex with SpoA domains derived from three distinct proteins.

Highlights

Bacterial flagella are rotary nanomachines that contribute to bacterial fitness in many settings, including host colonization
We found that purified FliYC–FliMC was pulled down by GSTFliH (Fig. 6C). These results suggest that both FliYC–FliNC and FliYC–FliMC in H. pylori functioned as a protein-docking platform for FliH
Val262FliY and Met96FliN faced the outside of the pockets, Val262FliY, but not Met96FliN, interfered with the binding. This outcome could be due to the specific orientation of the FliH peptide on FliY–FliN, which still must be resolved by their cocrystal structure. The presence of both FliY and FliN as switch proteins is a common feature within the ⑀-proteobacteria, and their coexistence could be associated with the adaptation of the motility system in these species [5]

Summary

Introduction

Bacterial flagella are rotary nanomachines that contribute to bacterial fitness in many settings, including host colonization. Different bacteria exhibit great diversity in their flagellar motors One such variation is exemplified by the motorswitch apparatus of the gastric pathogen Helicobacter pylori, which carries an extra switch protein, FliY, along with the more typical FliG, FliM, and FliN proteins. The motor C-rings of these bacteria contain ϳ26 copies of FliG, ϳ34 copies of FliM, and Ͼ110 copies of FliN [7,8,9] Most of these structures were determined using proteins from other organisms, and their assembly models have been recently proposed (10 –22). FliN forms a distinctive donut-shaped structure at the base of the switch complex and is critical for protein export via association with the flagellar type III secretion apparatus [11]

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