Structural and Functional Analyses Reveal Insights into the Molecular Properties of the Escherichia coli Z Ring Stabilizing Protein, ZapC

Abstract

In Escherichia coli cell division is driven by the tubulin-like GTPase, FtsZ, which forms the cytokinetic Z-ring. The Z-ring serves as a dynamic platform for the assembly of the multiprotein divisome, which catalyzes membrane cleavage to create equal daughter cells. Several proteins effect FtsZ assembly, thereby providing spatiotemporal control over cell division. One important class of FtsZ interacting/regulatory proteins is the Z-ring-associated proteins, Zaps, which typically modulate Z-ring formation by increasing lateral interactions between FtsZ protofilaments. Strikingly, these Zap proteins show no discernable sequence similarity, suggesting that they likely harbor distinct structures and mechanisms. The 19.8-kDa ZapC in particular shows no homology to any known protein. To gain insight into ZapC function, we determined its structure to 2.15 Å and performed genetic and biochemical studies. ZapC is a monomer composed of two domains, an N-terminal ␣/␤ region and a C-terminal twisted ␤ barrel-like domain. The structure contains two pockets, one on each domain. The N-domain pocket is lined with residues previously implicated to be important for ZapC function as an FtsZ bundler. The adjacent C-domain pocket contains a hydrophobic center surrounded by conserved basic residues. Mutagenesis analyses indicate that this pocket is critical for FtsZ binding. An extensive FtsZ binding surface is consistent with the fact that, unlike many FtsZ regulators, ZapC binds the large FtsZ globular core rather than C-terminal tail, and the presence of two adjacent pockets suggests possible mechanisms for ZapC-mediated FtsZ bundling.In Escherichia coli, the bacterial tubulin-like GTPase, FtsZ, drives cell division. Unlike the microtubule structures formed by tubulin, FtsZ forms protofilaments that combine to create a ring-like structure at midcell called the Z-ring, which mediates cell division (1-5). FtsZ is an ancient and highly conserved protein that is responsible for cell division in most bacteria as well as many archaea, all chloroplasts, and the mitochondria of primitive eukaryotes (4). FtsZ is made up of five main domains: an N-terminal region, which is largely disordered, a globular core that contains the nucleotide binding site and the T7 synergy loop needed for nucleotide hydrolysis, a long linker of variable sequence and length (ϳ40 -257 residues), a short ϳ15-residue C-terminal tail (CTT) 3 that contains a highly conserved set of residues critical for the docking of several FtsZ interacting proteins, and a more recently defined C-terminal variable (CTV) region (4, 6). Linear protofilaments are created by GTP binding to the globular domains of FtsZ between different protomers (1,7,8). The dynamic assembly and disassembly of protofilaments resulting from cycles of GTP binding and hydrolysis leads to Z-ring remodeling that is thought to contribute to the constrictive force required for cell division (4, 9, 10).In E. coli the intracellular levels of FtsZ remain essentially the same throughout the cell cycle and exceed...

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

“…ZapDFtsZ CTD structure is largely consistent with and explains previous biochemical data (35,38). However, to further probe the structural model, we next made mutations in ZapD residues that the structure indicated are key for CTD binding.…”

“…Like ZapA, both ZapC and ZapD interact directly with FtsZ and also promote FtsZ protofilament assembly (20,27,28). Structures have recently been obtained for all the Zap proteins and show that, interestingly, despite their overlapping functions, they are structurally distinct (22,26,(35)(36)(37)(38). ZapA and ZapB are coiled-coil proteins that form dimeric or tetrameric structures; ZapB consists almost entirely of an extended coiled coil, whereas ZapA contains N-terminal globular domains in addition to its coiled-coil region (22,26).…”

“…Binding studies revealed that the ZapA globular domains mediate FtsZ binding (26). ZapC is composed of an N-terminal ␣/␤ region and a C-terminal twisted ␤-barrel-like domain (35,36). Residues located within two surface-exposed pockets of ZapC appear to be involved in FtsZ binding (35).…”

“…ZapC is composed of an N-terminal ␣/␤ region and a C-terminal twisted ␤-barrel-like domain (35,36). Residues located within two surface-exposed pockets of ZapC appear to be involved in FtsZ binding (35). Multiple studies showed that ZapC binds the FtsZ GTPase core (27,28,35).…”

“…Residues located within two surface-exposed pockets of ZapC appear to be involved in FtsZ binding (35). Multiple studies showed that ZapC binds the FtsZ GTPase core (27,28,35). However, one analysis suggested that ZapC might bind the FtsZ CTD, albeit with low affinity (36).…”