Structural insights into the assembly mechanism of the flagellar MS-ring with three different symmetries

Abstract

ABSTRACTThe flagellar basal body MS-ring, formed by 34 FliF subunits, is the core of the flagellar motor as well as the base for flagellar assembly. The MS-ring is also a housing for the flagellar protein export gate complex that is required for construction of the flagellum on the cell surface. A large periplasmic region of FliF contains three ring-building motifs named RBM1, RBM2, and RBM3. RBM3 forms the S-ring and β-collar with C34 symmetry. RBM2 forms the inner core ring of the M-ring with C23 symmetry surrounded by 11 cog-like structures formed by RBM1 and RBM2. However, it remains unknown how FliF assembles to generate these three different symmetries within the MS-ring. Here, we report the two cryoEM structures of the MS-ring formed by FliF co-expressed with FliG and transmembrane export gate proteins. Structural comparison of 33-mer and 34-mer MS-rings revealed that a subtle change in the conformation of RBM3 produces the different rotational symmetries. Combination of cryoEM structural and mutational analyses of the MS-ring with C33 symmetry provides evidence that the well-conserved DQxGxxL motif within a flexible loop connecting RBM2 and RBM3 allows RBM2 to take two different orientations relative to RBM3 to form not only 11 cog-like structures just outside the inner core ring with C22 symmetry but also an appropriately sized central pore in the inner core ring to accommodate the export gate complex.IMPORTANCEThe flagellar MS-ring is the core of the flagellar motor and serves not only as an initial template for flagellar assembly but also as a base to accommodate the flagellar protein export complex. The MS-ring is formed by 34 subunits of FliF with two transmembrane helices and a large periplasmic region containing ring-building motifs, RBM1, RBM2, and RBM3. FliF adopts two different conformations in the MS-ring to generate three different rotational symmetries, C34, C23, and C11. However, how FliF assembles to produce these three symmetries remains a mystery. Combination of cryoEM structural and mutational analyses has provided evidence that the well-conserved DQxGxxL motif within a hinge loop connecting RBM2 and RBM3 allows RBM2 to take two different orientations relative to RBM3, allowing 23 RBM2 domains of 34 subunits to form the inner core ring with a properly sized central pore to accommodate the flagellar protein export complex.