Characterization of the FtsZ C-Terminal Variable (CTV) Region in Z-Ring Assembly and Interaction with the Z-Ring Stabilizer ZapD in E. coli Cytokinesis

Abstract

Polymerization of a ring-like cytoskeletal structure, the Z-ring, at midcell is a highly conserved feature in virtually all bacteria. The Z-ring is composed of short protofilaments of the tubulin homolog FtsZ, randomly arranged and held together through lateral interactions. In vitro, lateral associations between FtsZ protofilaments are stabilized by crowding agents, high concentrations of divalent cations, or in some cases, low pH. In vivo, the last 4–10 amino acid residues at the C-terminus of FtsZ (the C-terminal variable region, CTV) have been implicated in mediating lateral associations between FtsZ protofilaments through charge shielding. Multiple Z-ring associated proteins (Zaps), also promote lateral interactions between FtsZ protofilaments to stabilize the FtsZ ring in vivo. Here we characterize the complementary role/s of the CTV of E. coli FtsZ and the FtsZ-ring stabilizing protein ZapD, in FtsZ assembly. We show that the net charge of the FtsZ CTV not only affects FtsZ protofilament bundling, confirming earlier observations, but likely also the length of the FtsZ protofilaments in vitro. The CTV residues also have important consequences for Z-ring assembly and interaction with ZapD in the cell. ZapD requires the FtsZ CTV region for interaction with FtsZ in vitro and for localization to midcell in vivo. Our data suggest a mechanism in which the CTV residues, particularly K380, facilitate a conformation for the conserved carboxy-terminal residues in FtsZ, that lie immediately N-terminal to the CTV, to enable optimal contact with ZapD. Further, phylogenetic analyses suggest a correlation between the nature of FtsZ CTV residues and the presence of ZapD in the β- γ-proteobacterial species.

Structure of the Z Ring-associated Protein, ZapD, Bound to the C-terminal Domain of the Tubulin-like Protein, FtsZ, Suggests Mechanism of Z Ring Stabilization through FtsZ Cross-linking

“…Electron density is not visible for the Gln-381 side chain, consistent with the fact that only its peptide backbone makes contacts with ZapD. FtsZ CTV residues 380 -381 make key contacts with ZapD, which explains the in vivo data showing that these residues are required for ZapD to localize with FtsZ during cell division (45). Bio-layer interferometry analyses also showed that truncating FtsZ to residue 379 reduced ZapD binding from 4 M to Ͼ2 mM (38).…”

“…Protein Purification-FtsZ, ZapD, and ZapD mutants were purified as described previously and used in light scattering assays and electron microscopy experiments (20,45).…”

Conserved Dynamics of Chloroplast Cytoskeletal FtsZ Proteins Across Photosynthetic Lineages

“…The positively charged CTV of BsFtsZ, but not the neutral CTV of EcFtsZ, enhanced protofilament bundling, probably via charge-shielding interactions between otherwise negatively charged protofilaments (Buske and Levin, 2012). Electrostatic forces due partly to the CTV are likely to contribute to protofilament interactions in vivo and hence to Z-ring stability (Buske and Levin, 2012;Huang et al, 2016). All the chloroplast FtsZ proteins used in our study, apart from GsFtsZB that lacks the C-terminal region altogether (Fig.…”

Structural and Biochemical Studies Reveal a Putative FtsZ Recognition Site on the Z-ring Stabilizer ZapD

“…3B). In recent study, the importance of K380 of FtsZ in the CTV region was reported (Huang et al, 2016). Thus, we performed another BLItz assay between EcFtsZ (367-383) K380D mutant and EcZapD and measured its binding affinity (K d = 4.7 ± 0.5 μM) (Supplementary Fig.…”

“…Loops are drawn in gray. Right panel indicates domain architectures of Ec-ZapD and EcFtsZ (Huang et al, 2016). EcFtsZ constructs used in biochemical analysis are shown in bold line.…”