ZapA uses a two-pronged mechanism to facilitate Z ring formation in Escherichia coli

Abstract

The tubulin-like protein FtsZ assembles into the Z ring that leads to the assembly and activation of the division machinery in most bacteria. ZapA, a widely conserved protein that interacts with FtsZ, plays a pivotal role in organizing FtsZ filaments into a coherent Z ring. Previous studies revealed that ZapA forms a dumbbell-like tetramer that binds cooperatively to FtsZ filaments and aligns them in parallel, leading to the straightening and organization of FtsZ filament bundles. However, how ZapA interacts with FtsZ remains obscure. Here, we reveal that ZapA uses a two-pronged mechanism to interact with FtsZ to facilitate Z ring formation in Escherichia coli. We find that mutations affecting surface-exposed residues at the junction between adjacent FtsZ subunits in a filament as well as in an N-terminal motif of FtsZ weaken its interaction with ZapA in vivo and in vitro, indicating that ZapA binds to these regions of FtsZ. Consistent with this, ZapA prefers FtsZ polymers over monomeric FtsZ molecules and site-specific crosslinking confirmed that the dimer head domain of ZapA is in contact with the junction of FtsZ subunits. As a result, disruption of the putative interaction interfaces between FtsZ and ZapA abolishes the midcell localization of ZapA. Taken together, our results suggest that ZapA tetramers grab the N-terminal tails of FtsZ and bind to the junctions between FtsZ subunits in the filament to straighten and crosslink FtsZ filaments into the Z ring.

Keywords

bacterial cell division / Z ring / FtsZ / ZapA / Z ring organization

References

[1] Bi E, Lutkenhaus J. FtsZ ring structure associated with division in Escherichia coli. Nature. 1991; 354: 161–164.

[2] Yang X, Lyu Z, Miguel A, McQuillen R, Huang KC, Xiao J. GTPase activity-coupled treadmilling of the bacterial tubulin FtsZ organizes septal cell wall synthesis. Science. 2017; 355: 744–747.

[3] Bisson-Filho AW, Hsu YP, Squyres GR, Kuru E, Wu F, Jukes C, et al. Treadmilling by FtsZ filaments drives peptidoglycan synthesis and bacterial cell division. Science. 2017; 355: 739–743.

[4] McQuillen R, Xiao J. Insights into the structure, function, and dynamics of the bacterial cytokinetic FtsZ-ring. Annu Rev Biophys. 2020; 49: 309–341.

[5] Cameron TA, Margolin W. Insights into the assembly and regulation of the bacterial divisome. Nat Rev Microbiol. 2024; 22: 33–45.

[6] Thanedar S, Margolin W. FtsZ exhibits rapid movement and oscillation waves in helix-like patterns in Escherichia coli. Curr Biol. 2004; 14: 1167–1173.

[7] Squyres GR, Holmes MJ, Barger SR, Pennycook BR, Ryan J, Yan VT, et al. Single-molecule imaging reveals that Z-ring condensation is essential for cell division in Bacillus subtilis. Nat Microbiol. 2021; 6: 553–562.

[8] Whitley KD, Jukes C, Tregidgo N, Karinou E, Almada P, Cesbron Y, et al. FtsZ treadmilling is essential for Z-ring condensation and septal constriction initiation in Bacillus subtilis cell division. Nat Commun. 2021; 12: 2448.

[9] Fu G, Huang T, Buss J, Coltharp C, Hensel Z, Xiao J. In vivo structure of the E. coli FtsZ-ring revealed by photoactivated localization microscopy (PALM). PLoS One. 2010; 5:e12682.

[10] Strauss MP, Liew ATF, Turnbull L, Whitchurch CB, Monahan LG, Harry EJ. 3D-SIM super resolution microscopy reveals a bead-like arrangement for FtsZ and the division machinery: implications for triggering cytokinesis. PLoS Biol. 2012; 10:e1001389.

[11] Biteen JS, Goley ED, Shapiro L, Moerner WE. Three-dimensional super-resolution imaging of the midplane protein FtsZ in live Caulobacter crescentus cells using astigmatism. Chemphyschem. 2012; 13: 1007–1012.

[12] Yang X, McQuillen R, Lyu Z, Phillips-Mason P, De La Cruz A, McCausland JW, et al. A two-track model for the spatiotemporal coordination of bacterial septal cell wall synthesis revealed by single-molecule imaging of FtsW. Nat Microbiol. 2021; 6: 584–593.

[13] Mahone CR, Payne IP, Lyu Z, McCausland JW, Barrows JM, Xiao J, et al. Integration of cell wall synthesis and chromosome segregation during cell division in Caulobacter. J Cell Biol. 2024; 223:e202211026.

[14] Osawa M, Anderson DE, Erickson HP. Reconstitution of contractile FtsZ rings in liposomes. Science. 2008; 320: 792–794.

[15] Loose M, Mitchison TJ. The bacterial cell division proteins FtsA and FtsZ self-organize into dynamic cytoskeletal patterns. Nat Cell Biol. 2014; 16: 38–46.

[16] Ramirez-Diaz DA, García-Soriano DA, Raso A, Mücksch J, Feingold M, Rivas G, et al. Treadmilling analysis reveals new insights into dynamic FtsZ ring architecture. PLoS Biol. 2018; 16:e2004845.

[17] Kohyama S, Merino-Salomón A, Schwille P. In vitro assembly, positioning and contraction of a division ring in minimal cells. Nat Commun. 2022; 13: 6098.

[18] Huang KH, Durand-Heredia J, Janakiraman A. FtsZ ring stability: of bundles, tubules, crosslinks, and curves. J Bacteriol. 2013; 195: 1859–1868.

[19] Gong H, Yan D, Cui Y, Li Y, Yang J, Yang W, et al. The divisome is a self-enhancing machine in Escherichia coli and Caulobacter crescentus. Nat Commun. 2024; 15: 8198.

[20] Gueiros-Filho FJ, Losick R. A widely conserved bacterial cell division protein that promotes assembly of the tubulin-like protein FtsZ. Genes Dev. 2002; 16: 2544–2556.

[21] Buss J, Coltharp C, Huang T, Pohlmeyer C, Wang SC, Hatem C, et al. In vivo organization of the FtsZ-ring by ZapA and ZapB revealed by quantitative super-resolution microscopy. Mol Microbiol. 2013; 89: 1099–1120.

[22] Durand-Heredia JM, Yu HH, De Carlo S, Lesser CF, Janakiraman A. Identification and characterization of ZapC, a stabilizer of the FtsZ ring in Escherichia coli. J Bacteriol. 2011; 193: 1405–1413.

[23] Durand-Heredia J, Rivkin E, Fan G, Morales J, Janakiraman A. Identification of ZapD as a cell division factor that promotes the assembly of FtsZ in Escherichia coli. J Bacteriol. 2012; 194: 3189–3198.

[24] Hale CA, Shiomi D, Liu B, Bernhardt TG, Margolin W, Niki H, et al. Identification of Escherichia coli ZapC (YcbW) as a component of the division apparatus that binds and bundles FtsZ polymers. J Bacteriol. 2011; 193: 1393–1404.

[25] Marteyn BS, Karimova G, Fenton AK, Gazi AD, West N, Touqui L, et al. ZapE is a novel cell division protein interacting with FtsZ and modulating the Z-ring dynamics. mBio. 2014; 5:e00022-14.

[26] Ebersbach G, Galli E, Møller-Jensen J, Löwe J, Gerdes K. Novel coiled-coil cell division factor ZapB stimulates Z ring assembly and cell division. Mol Microbiol. 2008; 68: 720–735.

[27] Galli E, Gerdes K. Spatial resolution of two bacterial cell division proteins: ZapA recruits ZapB to the inner face of the Z-ring. Mol Microbiol. 2010; 76: 1514–1526.

[28] Woldemeskel SA, McQuillen R, Hessel AM, Xiao J, Goley ED. A conserved coiled-coil protein pair focuses the cytokinetic Z-ring in Caulobacter crescentus. Mol Microbiol. 2017; 105: 721–740.

[29] Perez AJ, Villicana JB, Tsui HCT, Danforth ML, Benedet M, Massidda O, et al. FtsZ-ring regulation and cell division are mediated by essential EzrA and accessory proteins ZapA and ZapJ in Streptococcus pneumoniae. Front Microbiol. 2021; 12:780864.

[30] Krupka M, Rowlett VW, Morado D, Vitrac H, Schoenemann K, Liu J, et al. Escherichia coli FtsA forms lipid-bound minirings that antagonize lateral interactions between FtsZ protofilaments. Nat Commun. 2017; 8:15957.

[31] Krupka M, Sobrinos-Sanguino M, Jiménez M, Rivas G, Margolin W. Escherichia coli ZipA organizes FtsZ polymers into dynamic ring-like protofilament structures. mBio. 2018; 9:e01008-18.

[32] Espéli O, Borne R, Dupaigne P, Thiel A, Gigant E, Mercier R, et al. A MatP-divisome interaction coordinates chromosome segregation with cell division in E. coli. EMBO J. 2012; 31: 3198–3211.

[33] Bailey MW, Bisicchia P, Warren BT, Sherratt DJ, Männik J. Evidence for divisome localization mechanisms independent of the Min system and SlmA in Escherichia coli. PLoS Genet. 2014; 10:e1004504.

[34] Buss J, Coltharp C, Shtengel G, Yang X, Hess H, Xiao J. A multi-layered protein network stabilizes the Escherichia coli FtsZ-ring and modulates constriction dynamics. PLoS Genet. 2015; 11:e1005128.

[35] Mercier R, Petit MA, Schbath S, Robin S, El Karoui M, Boccard F, et al. The MatP/matS site-specific system organizes the terminus region of the E. coli chromosome into a macrodomain. Cell. 2008; 135: 475–485.

[36] Buss JA, Peters NT, Xiao J, Bernhardt TG. ZapA and ZapB form an FtsZ-independent structure at midcell. Mol Microbiol. 2017; 104: 652–663.

[37] Du S, Lutkenhaus J. The N-succinyl-l,l-diaminopimelic acid desuccinylase DapE acts through ZapB to promote septum formation in Escherichia coli. Mol Microbiol. 2017; 105: 326–345.

[38] Ozaki S, Jenal U, Katayama T. Novel divisome-associated protein spatially coupling the Z-ring with the chromosomal replication terminus in Caulobacter crescentus. mBio. 2020; 11:e00487-20.

[39] Ozaki S, Wakasugi Y, Katayama T. Z-ring-associated proteins regulate clustering of the replication terminus-binding protein ZapT in Caulobacter crescentus. mBio. 2021; 12:e02196-20.

[40] Low HH, Moncrieffe MC, Löwe J. The crystal structure of ZapA and its modulation of FtsZ polymerisation. J Mol Biol. 2004; 341: 839–852.

[41] Roach EJ, Kimber MS, Khursigara CM. Crystal structure and site-directed mutational analysis reveals key residues involved in Escherichia coli ZapA function. J Biol Chem. 2014; 289: 23276–23286.

[42] Pacheco-Gómez R, Cheng X, Hicks MR, Smith CJI, Roper DI, Addinall S, et al. Tetramerization of ZapA is required for FtsZ bundling. Biochem J. 2013; 449: 795–802.

[43] Meiresonne NY, den Blaauwen T. The in vitro non-tetramerizing ZapA(I83E) mutant is unable to recruit ZapB to the division plane in vivo in Escherichia coli. Int J Mol Sci. 2020; 21: 3130.

[44] Roseboom W, Nazir MG, Meiresonne NY, Mohammadi T, Verheul J, Buncherd H, et al. Mapping the contact sites of the Escherichia coli division-initiating proteins FtsZ and ZapA by BAMG cross-linking and site-directed mutagenesis. Int J Mol Sci. 2018; 19: 2928.

[45] Small E, Marrington R, Rodger A, Scott DJ, Sloan K, Roper D, et al. FtsZ polymer-bundling by the Escherichia coli ZapA orthologue, YgfE, involves a conformational change in bound GTP. J Mol Biol. 2007; 369: 210–221.

[46] Caldas P, López-Pelegrín M, Pearce DJG, Budanur NB, Brugués J, Loose M. Cooperative ordering of treadmilling filaments in cytoskeletal networks of FtsZ and its crosslinker ZapA. Nat Commun. 2019; 10: 5744.

[47] Vanhille-Campos C, Whitley KD, Radler P, Loose M, Holden S, Šarić A. Self-organization of mortal filaments and its role in bacterial division ring formation. Nat Phys. 2024; 20: 1670–1678.

[48] Galli E, Gerdes K. FtsZ-ZapA-ZapB interactome of Escherichia coli. J Bacteriol. 2012; 194: 292–302.

[49] Shen B, Lutkenhaus J. The conserved C-terminal tail of FtsZ is required for the septal localization and division inhibitory activity of MinC(C)/MinD. Mol Microbiol. 2009; 72: 410–424.

[50] Du S, Lutkenhaus J. SlmA antagonism of FtsZ assembly employs a two-pronged mechanism like MinCD. PLoS Genet. 2014; 10:e1004460.

[51] Schumacher MA, Ohashi T, Corbin L, Erickson HP. High-resolution crystal structures of Escherichia coli FtsZ bound to GDP and GTP. Acta Crystallogr F Struct Biol Commun. 2020; 76: 94–102.

[52] Fujita J, Amesaka H, Yoshizawa T, Hibino K, Kamimura N, Kuroda N, et al. Structures of a FtsZ single protofilament and a double-helical tube in complex with a monobody. Nat Commun. 2023; 14: 4073.

[53] Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA. 2000; 97: 6640–6645.

[54] Ingerman E, Nunnari J. A continuous, regenerative coupled GTPase assay for dynamin-related proteins. Methods Enzymol. 2005; 404: 611–619.

[55] Karimova G, Pidoux J, Ullmann A, Ladant D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc Natl Acad Sci USA. 1998; 95: 5752–5756.

[56] Li Y, Hsin J, Zhao L, Cheng Y, Shang W, Huang KC, et al. FtsZ protofilaments use a hinge-opening mechanism for constrictive force generation. Science. 2013; 341: 392–395.

[57] Du S, Pichoff S, Kruse K, Lutkenhaus J. FtsZ filaments have the opposite kinetic polarity of microtubules. Proc Natl Acad Sci USA. 2018; 115: 10768–10773.

[58] Abramson J, Adler J, Dunger J, Evans R, Green T, Pritzel A, et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature. 2024; 630: 493–500.

[59] Dajkovic A, Pichoff S, Lutkenhaus J, Wirtz D. Cross-linking FtsZ polymers into coherent Z rings. Mol Microbiol. 2010; 78: 651–668.

[60] Fujita J, Hibino K, Kagoshima G, Kamimura N, Kato Y, Uehara R, et al. Structural basis for the interaction between the bacterial cell division proteins FtsZ and ZapA. Nat Commun. 2025; 16: 5985.

[61] Yan Y, Zhang D, Zhou P, Li B, Huang SY. HDOCK: a web server for protein-protein and protein-DNA/RNA docking based on a hybrid strategy. Nucleic Acids Res. 2017; 45: W365–W373.

[62] Yan Y, Tao H, He J, Huang SY. The HDOCK server for integrated protein-protein docking. Nat Protoc. 2020; 15: 1829–1852.

[63] Arumugam S, Chwastek G, Fischer-Friedrich E, Ehrig C, Mönch I, Schwille P. Surface topology engineering of membranes for the mechanical investigation of the tubulin homologue FtsZ. Angew Chem Int Ed. 2012; 51: 11858–11862.

[64] González de Prado Salas P, Hörger I, Martín-García F, Mendieta J, Alonso Á, Encinar M, et al. Torsion and curvature of FtsZ filaments. Soft Matter. 2014; 10: 1977–1986.

[65] Lv D, Li J, Ye S. The assembly switch mechanism of FtsZ filament revealed by all-atom molecular dynamics simulations and coarse-grained models. Front Microbiol. 2021; 12:639883.

[66] Erickson HP. Modeling the physics of FtsZ assembly and force generation. Proc Natl Acad Sci USA. 2009; 106: 9238–9243.

[67] Erickson HP, Anderson DE, Osawa M. FtsZ in bacterial cytokinesis: cytoskeleton and force generator all in one. Microbiol Mol Biol Rev. 2010; 74: 504–528.

[68] Hsin J, Gopinathan A, Huang KC. Nucleotide-dependent conformations of FtsZ dimers and force generation observed through molecular dynamics simulations. Proc Natl Acad Sci USA. 2012; 109: 9432–9437.

[69] Knapp BD, Shi H, Huang KC. Complex state transitions of the bacterial cell division protein FtsZ. Mol Biol Cell. 2024; 35:ar130.

[70] Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012; 9: 676–682.

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