Oligomerization of the FliF Domains Suggests a Coordinated Assembly of the Bacterial Flagellum MS Ring

Abstract

The bacterial flagellum is a complex, self-assembling macromolecular machine that powers bacterial motility. It plays diverse roles in bacterial virulence, including aiding in colonization and dissemination during infection. The flagellum consists of a filamentous structure protruding from the cell, and of the basal body, a large assembly that spans the cell envelope. The basal body is comprised of over 20 different proteins forming several concentric ring structures, termed the M- S- L- P- and C-rings, respectively. In particular, the MS rings are formed by a single protein FliF, which consists of two trans-membrane helices anchoring it to the inner membrane and surrounding a large periplasmic domain. Assembly of the MS ring, through oligomerization of FliF, is one of the first steps of basal body assembly. Previous computational analysis had shown that the periplasmic region of FliF consists of three structurally similar domains, termed Ring-Building Motif (RBM)1, RBM2, and RBM3. The structure of the MS-ring has been reported recently, and unexpectedly shown that these three domains adopt different symmetries, with RBM3 having a 34-mer stoichiometry, while RBM2 adopts two distinct positions in the complex, including a 23-mer ring. This observation raises some important question on the assembly of the MS ring, and the formation of this symmetry mismatch within a single protein. In this study, we analyze the oligomerization of the individual RBM domains in isolation, in the Salmonella enterica serovar Typhimurium FliF ortholog. We demonstrate that the periplasmic domain of FliF assembles into the MS ring, in the absence of the trans-membrane helices. We also report that the RBM2 and RBM3 domains oligomerize into ring structures, but not RBM1. Intriguingly, we observe that a construct encompassing RBM1 and RBM2 is monomeric, suggesting that RBM1 interacts with RBM2, and inhibits its oligomerization. However, this inhibition is lifted by the addition of RBM3. Collectively, this data suggest a mechanism for the controlled assembly of the MS ring.

Keywords: Salmonella; bacteria; cryo-EM; flagellum; structure.

Figure 1

Salmonella typhimurium FliF domain organization. (A) Schematic representation of the domain composition and their boundaries in FliF. (B) Summary of the constructs, encompassing distinct FliF regions, used in this study. (C) Structure of FliF (PDB: 6SNC, 7CIK), in cartoon representation, placed in the corresponding region of the bacterial flagellum basal body electron density map.

Figure 2

Oligomerization of the FliF domains. (A) Size exclusion chromatography (SEC) UV trace of constructs encompassing the entire periplasmic regions of S. typhimurium FliF and Helicobacter pylori FliF. (B) SEC UV trace of constructs encompassing the individual domains of S. typhimurium FliF (C–E) Negative stain analysis of (C) RBM1-RBM2-RBM3 (FliF_50–438), (D) RBM2 (FliF_124–229), and (E) RBM3 (FliF_231–438). RBM1-RBM2-RBM3 and RBM2 show mostly side views, while RBM3 mainly displays top views.

Figure 3

FliF RBM2 and RBM3 stoichiometry. (A) Cryo-electron micrograph and (B) selected 2D classes of RBM1-RBM2-RBM3 (FliF_50–438). (C) A 2D class from a side view of RBM1-RBM2-RBM3 is shown, overlayed to a cartoon representation of the 33-mer FliF structure (PDBID: 6SD1). The majority of particles is side view, and matches the architecture of intact FliF. (D) Cryo-electron micrograph, and (E) selected 2D classes of RBM2 (FliF_124–229). Particles show top views and some filaments that consist of aggregation of single RBM2 disks. (F) A 2D class of a RBM2 top view is shown, overlayed to a cartoon model of the 23-mer RBM2 inner ring structure (from PDBID: 7BK0).

Figure 4

Ring-Building Motif (RBM)3 induces oligomerization of the RBM1-RBM2 construct. (A) Schematic representation of the experiment. RBM1-RBM2 (FliF_50–229) was mixed with RBM3 (FliF_231–438) oligomers and imaged by negative stain EM. (B) Negative stain electron micrograph of the RBM3 and RBM1-RBM2 mixture. Ring-like structures, representative ofRBM3alone, are visible, as well as tubular structures that are likely composed of stacks RBM1-RBM2-RBM3 rings.

Figure 5

Structural representation of the proposed MS-ring assembly process. Cartoon representations of each domain, colored as in Figure 1, are used. (A) As it gets exported to the membrane via the Sec pathway, FliF is monomeric, (B) The presence of adjacent FliF molecules allows RBM3 to drive the oligomerization process, establishing the 34-mer stoichiometry. The contacts between RBM1 and RBM2 prevent RBM2 to oligomerize. (C) At completion of RBM3 oligomerization, RBM1 is displaced from RBM2, allowing RBM2 domains from different FliF molecules to come into contact and assemble, forming a 23-mer. The remaining 11 RBM2 domains sit outside this ring. (D) In the final stage, the MS-ring reaches the final correct conformation, with asymmetry mismatch between RBM2 and RBM3.