Molecular Analysis of FtsZ-Ring Assembly in E. coli cytokinesis

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 βγ-proteobacterial species. Introduction The assembly of the tubulin-like GTPase, FtsZ, at midcell is a conserved, essential feature of bacterial cytokinesis in most species. Polymers of FtsZ form a cytokinetic ring-like structure called the Z-ring that provides a scaffold for the recruitment of other division proteins and PLOSONE | DOI:10.1371/journal.pone. 0153337 April 18, 2016 1 / 24 a11111 OPEN ACCESS Citation: Huang K-H, Mychack A, Tchorzewski L, Janakiraman A (2016) Characterization of the FtsZ CTerminal Variable (CTV) Region in Z-Ring Assembly and Interaction with the Z-Ring Stabilizer ZapD in E. coli Cytokinesis. PLoS ONE 11(4): e0153337. doi:10.1371/journal.pone. 0153337 Editor: Eric Cascales, Centre National de la Recherche Scientifique, Aix-Marseille Université, FRANCE Received: December 27, 2015 Accepted: March 28, 2016 Published: April 18, 2016 Copyright: © 2016 Huang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was funded by a National Science Foundation grant (MCB 1158059) to AJ. A National Institute on Minority Health and Health Disparities award (8G12MD007603-29) provides core facility support to the City College of New York. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. plausibly contributes towards the generation of a constriction force on the inner cell membrane [1–5]. In its GTP-bound state, FtsZ assembles into single stranded polymers (protofilaments) [6– 10]. In vitro, FtsZ protofilaments can assemble with various geometries including bundles, sheets or rings but the in vivo relevance of these structures is unclear [11,12]. Super-resolution microscopy of E. coli cells have revealed the Z-ring to consist of short, randomly arranged, overlapping protofilaments that are held together by lateral interactions [13–16]. The FtsZ monomer comprises four domains: an unstructured poorly conserved region of ~10 residues at the extreme N-terminus; a highly conserved globular core containing the GTP binding and hydrolytic functions; a disordered flexible linker that is poorly conserved in length and sequence among species; and the C-terminal conserved peptide (CCTP) which contains two sub-regions: a conserved C-terminal constant region (CTC) and a C-terminal variable region (CTV) (Fig 1) [17–19]. In vitro, the globular core alone is sufficient for formation of FtsZ protofilaments [20,21]. But, recent work has implicated the flexible linker and FtsZ CTV sequences to be key determinants of the end-to-end and lateral interactions of FtsZ in vitro implying a role for these domains in the architecture of FtsZ assemblies in the cell [17,18,22]. Most known stabilizers and destabilizers of FtsZ interact with the CCTP, which serves as a dock for proteins that regulate FtsZ-ring assembly dynamics. Such proteins include the essential FtsZ membrane tethers FtsA and ZipA, positional regulators MinC and SlmA, the conserved protease ClpX, and the Z-ring stabilizer ZapD [23–28]. The structure of CCTP bound to ZipA in E. coli and FtsA in T.maritima have been solved [29,30]. Although the CCTP in each case contains a helical segment starting at a conserved proline, the extended structures are not identical, suggesting that the CCTP is capable of acquiring a variety of structures possibly to enable interactions with varied binding partners [30,31]. In E. coli and related species, Z-ring assembly is thought to initiate through the formation of an unstable proto-ring that consists of FtsZ, FtsA, and ZipA [32]. In addition to attaching FtsZ filaments to the membrane, both FtsA and ZipA contribute to the integrity of the Z-ring, ZipA by increasing lateral associations among FtsZ protofilaments [21,33,34]. The Z-ring is subsequently stabilized through interactions with several FtsZ-ring associated proteins (Zaps): ZapA, ZapB, ZapC, and ZapD. The Zaps localize to midcell early during cytokinesis and exhibit functional overlap in affecting the assembly dynamics of FtsZ [25,28,35–41]. Although deletion of a single zap gene shows only modest defects in division and Z-ring morphology in WT cells, the phenotypes are exacerbated in cells lacking two or more Zap proteins indicating their important contributions to the architecture and function of the Z-ring. Notably, though the Zaps are functionally redundant they are not homologous proteins, and only ZapA is widely conserved [35]. ZapA interacts directly with FtsZ and promotes both longitudinal and lateral interactions of FtsZ polymers in vitro with a concomitant reduction in FtsZ GTPase activity [37,38,42,43]. Both ZapA and ZapB, which is recruited to the Z-ring by ZapA, are implicated in condensing FtsZ polymers into a tight-pitched ring at midcell, and influencing cell constriction [44–47]. The structure of ZapC has recently been solved by two groups independently, and evidence suggests that it employs an extensive binding surface to interact with FtsZ [48–50]. Less is known about the specific contributions of ZapD to Z-ring architecture and function. Towards our long-term goal of characterizing the modulatory roles of the Zap proteins in Z-ring dynamics, we sought to understand their functional overlap in stabilizing Z-ring assembly in E. coli and related species. The net-charge of amino acid residues in the FtsZ CTV has been shown to affect FtsZ lateral interactions in vitro independent of modulatory proteins [22]. Since the net-charge of the FtsZ CTV varies from species to species, we sought to test the hypothesis, first suggested by Buske and Levin, that modulatory proteins may act by E. coli FtsZ-ZapD Interaction PLOS ONE | DOI:10.1371/journal.pone. 0153337 April 18, 2016 2 / 24 Competing Interests: The authors have declared that no competing interests exist.