FtsA forms actin-like protofilaments

Abstract

FtsA is an early component of the Z-ring, the structure that divides most bacteria, formed by tubulin-like FtsZ. FtsA belongs to the actin family of proteins, showing an unusual subdomain architecture. Here we reconstitute the tethering of FtsZ to the membrane via FtsA's C-terminal amphipathic helix in vitro using Thermotoga maritima proteins. A crystal structure of the FtsA:FtsZ interaction reveals 16 amino acids of the FtsZ tail bound to subdomain 2B of FtsA. The same structure and a second crystal form of FtsA reveal that FtsA forms actin-like protofilaments with a repeat of 48 Å. The identical repeat is observed when FtsA is polymerized using a lipid monolayer surface and FtsAs from three organisms form polymers in cells when overexpressed, as observed by electron cryotomography. Mutants that disrupt polymerization also show an elongated cell division phenotype in a temperature-sensitive FtsA background, demonstrating the importance of filament formation for FtsA's function in the Z-ring.

Figure 1

In vitro reconstitution of the FtsZ/FtsA/membrane complex using T. maritima proteins. (A) Schematic representation of T. maritima proteins used. Dotted violet squares represent enlarged views of the C-terminal regions, which are crucial for the experimental design. (B) FtsA-FtsZ co-pelleting assay. S: supernatant, P: pellet. The C-terminal eight residues of FtsZ are necessary to interact with FtsA. (C) The C-terminal 16 residues of FtsZ are sufficient to interact with FtsA as shown in a ParM-Z-FtsA co-pelleting assay. A hybrid protein consisting of R1 ParM and the last 16 residues of FtsZ binds to FtsA. (D) Full-length FtsA binds to the membrane via its C-terminal amphipathic helix and tethers FtsZ to the membrane as shown by a liposome co-sedimentation assay. FtsZ does not have affinity for lipids itself.

Figure 2

Crystal structure and solution NMR studies show that FtsZ binds to FtsA’s surface between helices H6 and H8 within subdomain 2B. (A) Crystallographic asymmetric unit containing two FtsA molecules, each with the C-terminal 16-residue FtsZ peptide bound (left). FtsA has been colour-coded according to the conserved actin family of proteins subdomain architecture. The peptide (purple) is bound on the surface of subdomain 2B (green). ATP is shown as spheres. (B) Solution NMR studies identifying amide resonances perturbed upon addition of the FtsZ peptide (red). These results support the crystal structure as well as previous in vivo mutations by Pichoff-Lutkenhaus (2007) (yellow). Residues highlighted by both in vivo and NMR studies are in blue. (C) A stereo representation of the FtsA–FtsZ interacting site. The peptide is depicted in purple, and 2B subdomain in green. Three salt bridges have been identified: FtsA(Arg301)–FtsZ(Asp338), FtsA(Glu304)–FtsZ(Arg344) and FtsA(Lys293)–FtsZ(Leu351) (C-terminal carboxyl group).

Figure 3

Crystallographic and EM analysis reveal that FtsA, in spite of its unusual subdomain architecture, is able to form actin-like protofilaments. (A) FtsA, in the presence of ATPγS, crystallized as a continuous polymer (crystallographic unit cell edge a is aligned with the longitudinal axis of the protofilament). The packing remarkably resembles that of MreB, which has the canonical actin-like fold (subdomains 1A, 1B, 2A, 2B) and forms actin-like, straight protofilaments. The longitudinal spacings are 51.1 and 48.0 Å for MreB and FtsA, respectively. (B) A more detailed view of FtsA and MreB dimers. Monomeric subunits are rimmed in red. FtsA and MreB adopt a similar subdomain architecture; however, the 1B subdomain of MreB (yellow) is missing in FtsA and instead subdomain 1C in FtsA is located on the other side of the molecule. It therefore appears that FtsA is a subdomain variation of the actin fold that still enables the formation of canonical protofilaments. (C) FtsA polymerizes on a lipid monolayer forming long filaments, which often form doublets. (D) Occasionally, FtsA forms 2D sheets on the lipid monolayer. Fourier transformation of a sheet (white dotted square) reveals the same longitudinal spacing of about 48 Å, which matches the spacing present in the crystal structure.

Figure 4

FtsA polymers can be visualized in living cells by fluorescence microscopy and electron cryotomography. (A) N-terminal mCherry fusions to EcFtsA and BsFtsA (C-terminal amphipathic helix removed) as well as to four BsFtsA mutants have been overexpressed in E. coli using a T7 expression system. Membranes were stained with FM1-43. The three top panels show distinct, polymeric structures and the three bottom panels show that some mutations introduced within the polymerization interface prevent polymerization, leading to diffuse localization. The locations of these mutations are shown in Supplementary Figure 7A. All images using SIM. For conventional EPI images and more examples please consult Supplementary Figure 6. (B) Untagged E. coli, B. subtilis and TmFtsA proteins were overexpressed in E. coli cells and imaged by electron cryotomography. EcFtsA without the amphipathic helix (top left) forms long, straight bundles in the middle of the cell (navy blue dotted line represents cross-section’s orientation). Full-length TmFtsA (top right) causes membrane distortion and forming of lipid tubes (turquoise) that are coated with protein polymers (red) in the cell membrane’s proximity (see Supplementary movie 1). The bottom panels show either full-length BsFtsA (right) or BsFtsA truncated for the amphipathic helix (left). As expected, they trigger membrane distortion and straight filaments, respectively. White dotted areas are shown enlarged in the insets.

Figure 5

Replacing thermo-sensitive FtsA with either polymerization-deficient mutants or a non-functional version has dramatic effects on cell division in B. subtilis. (A) DIC images of genetically engineered B. subtilis strains showing that non-polymerizing FtsAs (K145A, M147E, I278K) do not fully complement the thermo-sensitive spoIIN279(ts) allele. As a control, non-functional FtsA (Q87Stop) shows a very severe cell division defect. S46F, which was found to still polymerize (Figure 4A), shows no effect on cell division. (B) A chart showing the average cell lengths of strains bearing tested FtsA variants. The wild type was 5.89±1.74 μm and S46F exhibits similar values. Cells of the Q87Stop variant were 13.40±8.30 μm, and the three polymerization-deficient mutants were as follows: K145A 8.29±4.36 μm, M147E 9.93±6.6 μm and I278K 9.04±4.86 μm. (C) Fractions of successful cell divisions were calculated referring to the wild-type FtsA variant.

Figure 6

FtsA is a subdomain variation of the actin fold that still enables the formation of similar protofilaments. (A) The 1C subdomain is present on the other side of a monomeric subunit in respect to 1B subdomain of MreB. (see also Figure 3A and B). (B) Both the 1C subdomain of FtsA and the 1B subdomain of MreB consist of an α-alpha helix and a three-stranded β-sheet, but the topology is different. For 1C it is S5-H2-S6-S7 and for 1B it is S4-S5-H1-S6. (C) Architecture of the FtsA/FtsZ membrane-bound complex, as analysed here for T. maritima. (D) The Z-ring made of polymerized tubulin-like FtsZ may be attached to the membrane by an ‘A-ring’, in turn made of polymerized actin-like FtsA or short stretches of FtsA polymers, based on the relative number of FtsZ and FtsA molecules in cells.