The bacterial flagellar motor is a self-assembling supramolecular nanodevice.Its spontaneous biosynthesis is initiated by the insertion of the MS ring protein FliF into the inner membrane, followed by attachment of the switch protein FliG. Assembly of this multiprotein complex is tightly regulated to avoid nonspecific aggregation, but the molecular mechanisms governing flagellar assembly are unclear. Here, we present the crystal structure of the cytoplasmic domain of FliF complexed with the Nterminal domain of FliG (FliF C -FliG N ) from the bacterium Helicobacter pylori. Within this complex, FliF C interacted with FliG N through extensive hydrophobic contacts similar to those observed in the FliF C -FliG N structure from the thermophile Thermotoga maritima, indicating conservation of the FliF C -FliG N interaction across bacterial species. Analysis of the crystal lattice revealed that the heterodimeric complex packs as a linear superhelix via stacking of the armadillo-repeat-like motifs (ARM) of FliG N . Notably, this linear helix was similar to that observed for the assembly of the FliG middle domain. We validated the in vivo relevance of the FliG N stacking by complementation studies in Escherichia coli. Furthermore, structural comparison with apo FliG from the thermophile Aquifex aeolicus indicated that FliF regulates the conformational transition of FliG and exposes the complementary ARM-like motifs of FliG N , containing conserved hydrophobic residues. FliF apparently both provides a template for FliG polymerization and spatiotemporally controls subunit interactions within FliG. Our findings reveal that a small protein fold can serve as a versatile building block to assemble into a multiprotein machinery of distinct shapes for specific functions.The bacterial flagellar motor is a self-assembled reversible rotary nanodevice. This dynamic rotary motor of about 40 nm in diameter is composed of rings of protein oligomers: the L ring (outer membrane), P ring (peptidoglycan layer), MS ring (inner membrane) and C ring (cytoplasm) (1,2). The L and P rings are believed to act as a bushing through which a central rotating rod can pass. The MS and C rings contribute to the rotor part of the flagellar motor. Torque is generated by a membrane-bound stator, which converts electrochemical potential into mechanical force that acts on the rotor. The synthesis of the flagellum begins at the rotor, which is composed of the MS and C ring protein subassemblies ( Figure 1). Specifically, the assembly of the MS ring protein FliF prompts the incorporation of the switch protein FliG and subsequently the proteins FliM and FliN to form the cytoplasmic motor switch complex. The MS ring plays an important role as it interacts both with the rod where the flagellum is anchored and with the C ring where torque Crystal structure of FliF-FliG complex from H. pylori generation and rotation switching take place.In Salmonella typhimurium and Escherichia coli, there are about 26 copies of FliF in the MS ring and 26 copies of FliG, 34 c...

Bacterial flagella are rotary nano-machines 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 (FliY C ) 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 FliY C -FliN C complex revealed a saddle-shape structure homologous to the FliN-FliN dimer of Thermotoga maritima, consistent with a FliY-FliN heterodimer forming the functional unit. Analysis of the FliY C -FliN C interface indicated that oppositely charged residues specific to each protein drive heterodimer formation. Moreover, both FliY C -FliM C and FliY C -FliN C 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.Bacterial flagella are rotary nano-machines that contribute to bacterial fitness in a variety of settings, including mammalian and plant colonization (1,2). Although the basic function of flagella as a motor organelle is conserved, substantial variation exists among microbes in the components used to build and operate key aspects of the flagella. For example, we now know that there are diverse motor structures from cryo-electron tomography studies (3,4), and that bacterial motors consist of FliG, FliM and either FliN or FliY or the combination of both FliN and FliY (5) The flagellar motor switch complex, also called the C-ring, is found at the base of each flagellum and resides within the cytoplasm (6). It plays an important role in flagellar assembly, torque generation, and rotational switching. Numerous studies have dissected the composition, arrangement, and structure of the switch proteins, with a focus on those from Escherichia coli and Salmonella typhimurium that possess FliG, FliM and FliN. The motor C-rings of these bacteria contain 26 copies of FliG, 34 copies of FliM and >110 copies of . Most of these structures were determined using proteins from other organisms, and their assembly models have been recently proposed (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Electron microscop...

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Functional flagella formation is a widespread virulence factor that plays a critical role in survival and host colonization. Flagellar synthesis is a complex and highly coordinated process. The assembly of the axial structure beyond the cell membrane is mediated by export chaperone proteins that transport their cognate substrates to the export gate complex. The export chaperone FliS interacts with flagellin, the basic component used to construct the filament. Unlike enterobacteria, the gastric pathogen Helicobacter pylori produces two different flagellins, FlaA and FlaB, which exhibit distinct spatial localization patterns in the filament. Previously, we demonstrated a molecular interaction between FliS and an uncharacterized protein, HP1076, in H. pylori. Here, we present the crystal structure of FliS in complex with both the C-terminal D0 domain of FlaB and HP1076. Although this ternary complex reveals that FliS interacts with flagellin using a conserved binding mode demonstrated previously in Aquifex aeolicus, Bacillus subtilis, and Salmonella enterica serovar Typhimurium, the helical conformation of FlaB in this complex was different. Moreover, HP1076 and the D1 domain of flagellin share structural similarity and interact with the same binding interface on FliS. This observation was further validated through competitive pull-down assays and kinetic binding analyses. Interestingly, we did not observe any detrimental flagellation or motility phenotypes in an hp1076-null strain. Our localization studies suggest that HP1076 is a membraneassociated protein with a cellular localization independent of FliS. As HP1076 is uniquely expressed in H. pylori and related species, we propose that this protein may contribute to the divergence of the flagellar system, although its relationship with FliS remains incompletely elucidated.

Drug discovery is a crucial part of human healthcare and has dramatically benefited human lifespan and life quality in recent centuries, however, it is usually time- and effort-consuming. Structural biology has been demonstrated as a powerful tool to accelerate drug development. Among different techniques, cryo-electron microscopy (cryo-EM) is emerging as the mainstream of structure determination of biomacromolecules in the past decade and has received increasing attention from the pharmaceutical industry. Although cryo-EM still has limitations in resolution, speed and throughput, a growing number of innovative drugs are being developed with the help of cryo-EM. Here, we aim to provide an overview of how cryo-EM techniques are applied to facilitate drug discovery. The development and typical workflow of cryo-EM technique will be briefly introduced, followed by its specific applications in structure-based drug design, fragment-based drug discovery, proteolysis targeting chimeras, antibody drug development and drug repurposing. Besides cryo-EM, drug discovery innovation usually involves other state-of-the-art techniques such as artificial intelligence (AI), which is increasingly active in diverse areas. The combination of cryo-EM and AI provides an opportunity to minimize limitations of cryo-EM such as automation, throughput and interpretation of medium-resolution maps, and tends to be the new direction of future development of cryo-EM. The rapid development of cryo-EM will make it as an indispensable part of modern drug discovery.