Structure and Mutational Analyses of Escherichia coli ZapD Reveal Charged Residues Involved in FtsZ Filament Bundling

Abstract

Bacterial cell division is an essential and highly coordinated process. It requires the polymerization of the tubulin homologue FtsZ to form a dynamic ring (Z-ring) at midcell. Z-ring formation relies on a group of FtsZ-associated proteins (Zap) for stability throughout the process of division. In Escherichia coli, there are currently five Zap proteins (ZapA through ZapE), of which four (ZapA, ZapB, ZapC, and ZapD) are small soluble proteins that act to bind and bundle FtsZ filaments. In particular, ZapD forms a functional dimer and interacts with the C-terminal tail of FtsZ, but little is known about its structure and mechanism of action. Here, we present the crystal structure of Escherichia coli ZapD and show it forms a symmetrical dimer with centrally located ␣-helices flanked by ␤-sheet domains. Based on the structure of ZapD and its chemical cross-linking to FtsZ, we targeted nine charged ZapD residues for modification by site-directed mutagenesis. Using in vitro FtsZ sedimentation assays, we show that residues R56, R221, and R225 are important for bundling FtsZ filaments, while transmission electron microscopy revealed that altering these residues results in different FtsZ bundle morphology compared to those of filaments bundled with wild-type ZapD. ZapD residue R116 also showed altered FtsZ bundle morphology but levels of FtsZ bundling similar to that of wild-type ZapD. Together, these results reveal that ZapD residues R116, R221, and R225 likely participate in forming a positively charged binding pocket that is critical for bundling FtsZ filaments. IMPORTANCEZ-ring assembly underpins the formation of the essential cell division complex known as the divisome and is required for recruitment of downstream cell division proteins. ZapD is one of several proteins in E. coli that associates with the Z-ring to promote FtsZ bundling and aids in the overall fitness of the division process. In the present study, we describe the dimeric structure of E. coli ZapD and identify residues that are critical for FtsZ bundling. Together, these results advance our understanding about the formation and dynamics of the Z-ring prior to bacterial cell division. Bacterial cell division is an essential and complex process that requires the coordinated assembly of a multiprotein molecular machine termed the divisome. The divisome is responsible for constriction of the inner and outer membranes, synthesis of septal peptidoglycan, and subsequent septum formation. In Escherichia coli, divisome proteins are recruited in a hierarchical manner and can be divided into three main groups based on their order of assembly: (i) the proto-ring, (ii) early divisome proteins, and (iii) late divisome proteins (1, 2). The successful assembly of the divisome depends on the initial formation of the Z-ring, which is comprised of the 40-kDa bacterial tubulin homologue FtsZ. FtsZ assembles into filaments in a GTP-dependent manner and a headto-tail fashion (3-6). The filaments are then tethered to the membrane, forming the Z-ring. They act as the...

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

“…This could also be the case for positive regulators of FtsZ assembly, such as ZapD. Indeed, this type of binding model could explain data from Roach et al (37) that showed cross-linking between ZapD and FtsZ core residues. Alternatively, the results from these cross-linking experiments could be due to nonspecific contacts between ZapD and FtsZ resulting from ZapD induced bundling and aggregation of FtsZ molecules.…”

“…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).…”

“…The ZapD protein contains a helical domain and a ␤-strand domain and is dimeric (37,38). Two studies reporting on putative FtsZ-binding residues generated conflicting results, and hence the FtsZ binding pocket in ZapD remains unknown (37,38).…”

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

“…To unveil overall structural similarities with other known structures from PDB, a search using the DALI program was performed (Holm and Rosenstrom, 2010). It showed several structurally similar proteins including (i) cell division protein ZapD from E. coli (5DKO, Z-score 30) (Roach et al, 2016), (ii) the DUF1342 protein (unknown function) from V. parahaemolyticus (2OEZ, Z-score 27), and (iii) Integron cassette protein from V. paracholerae (3JRT, Z-score 8.2) (Sureshan et al, 2013). When we compared our StZapD refined structure with the high Z-score structures (PDB codes 5DKO, 2OEZ, and 3JRT), the r.m.s.…”

“…Zaps, which are FtsZ positive regulators, include ZapA, ZapB, ZapC, and ZapD and they are structurally characterized (Ebersbach et al, 2008; Low et al, 2004; Ortiz et al, 2015; Roach et al, 2016). ZapA is well known as a positive regulator of Z-ring formation, which can bind to FtsZ at the globular core directly (Galli and Gerdes, 2012).…”