Molecular Basis of Motor Switch Complex from Helicobacter pylori

Bacterial chemotaxis is the directional movement of bacterium towards favourable environment. In flagellated bacteria, chemotaxis is achieved by controlling the frequency of alternating clockwise-counterclockwise switching. Center of this control is the interaction between signaling molecule CheY and switch protein complex (SPC) located at the cytosolic part of flagellum. SPC is a ring-shaped macromolecular complex composed of ∼26 copies of FliG, ∼34 copies of FliM, and over 100 copies of FliN in E. coli . Each component plays distinctive roles in flagellar assembly, export, torque generation and flagella switching. The structures and functions of chemotaxis and switch proteins have been extensively studied in E. coli and S. typhimurium, however, the molecular basis governing their assembly and the switching process remains controversial.;All gastric Helicobacter species possess a prominent feature of flagella-driven motility that is essential for colonization and infection. Interestingly, the chemotactic system of H. pylori is marked by the presence of multiple response regulators: CheY1, oneCheY-like-containing CheA protein (CheAY2), and three CheV proteins. Besides, H. pylori harbors an additional SPC member FliY which bears a CheC-phosphatase-like domain fused with a FliN-like domain. Deletions of FliG, FliM, FliN and FliY and FliN/FliY led to nonflagellate, suggesting that all four switch proteins are required for flagellation and FliY is a structural component of SPC.;The organization and functions of SPC in H. pylori are not well understood. This study aims to characterize the structures and functions of chemotaxis protein CheY1 and switch proteins from H. pylori. Here we report the crystal structures of CheY1, FliM middle domain (FliMM) and FliG middle and C-terminal domain (FliGMC). These proteins share high structural homology to their counterpart in E. coli or T. maritima. The interactions among the switch proteins, specifically CheY-FliM, FliM-FliG, FliG-FliF were verified, suggest they function similarly as in other bacteria.;Structural comparison of CheY1 with BeF3--bound CheY and fluorescence quenching experiments reveal the importance of Thr84 in the phosphotransfer reaction. Complementation assays using various nonchemotactic E. coli mutants demonstrated that CheY1 displays differential association with the flagellar motor in E. coli. Structural rearrangement of helix 5 and the C-terminal loop in CheYI provide a different interaction surface for FliM. On the other hand, interaction of the CheA-P2 domain with CheY1, but not with CheY2/CheV proteins, underlines the preferential recognition of CheY1 by CheA in the phosphotransfer reaction.;Structure of FliMM shows the position of a flexible loop close to the FliG binding site that was previously unresolved in T. maritima FliMM (TmFliMM). Mutagenesis studies supported that residues 139YDQ141 on the loop are important for FliG interaction.;Two crystal structures of FliGMC were resolved each showing distinct domain orientations from previously-solved structures. Structural comparisons highlight the flexibility of a conserved 245MFXF 248 motif connecting the three helices ARM and the charged-ridge-bearing subdomains of FliG C-terminal domain. Remarkably, rotational freedom of M245 psi and F246 phi prompted a ∼180° rotation of the charge ridge. We identified a highly conserved Asn216 that is in close proximity to the backbone of 245MF246. Studies of swarming and swimming behavior of E. coli showed that mutation of Asn216 to Asp, Ala and Val leads to CW bias while mutation to His did not affect switching. Furthermore, conformational flexibility of the FliG C-termina1 sub domains coordindated by the MFXF loop in solution was verified by intramolecular cysteine crosslinking. We hypothesized that the 180° rotation of charge ridge prompted by intrinsic flexibility of MFXF motif explained the symmetrical rotation during motor switching event.;FliY contains two discrete domains and likely carries unique function in motility. Here, we demonstrated that FliY C-termina1 domain (FliY C) complemented flagellation of DeltafliY mutant but showed impairment in motility. FliYC binds to FliN and the complex interacts with FliH, suggesting that FliYC is the minimal domain required for protein export. On the other hand, FliYN is necessary to full motility function, although the specific role of FliY N remains to be elucidated.;In summary, the structural and functional data obtained will provide insights to dissect the mechanistic details of the coupling between chemotaxis and SPC in flagellar rotation and switching in H. pylori.