The Crystal Structure of ZapA and its Modulation of FtsZ Polymerisation

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Abstract

FtsZ is part of a mid-cell cytokinetic structure termed the Z-ring that recruits a hierarchy of fission related proteins early in the bacterial cell cycle. The widely conserved ZapA has been shown to interact with FtsZ, to drive its polymerisation and to promote FtsZ filament bundling thereby contributing to the spatio-temporal tuning of the Z-ring. Here, we show the crystal structure of ZapA (11.6 kDa) from Pseudomonas aeruginosa at 2.8 Å resolution. The electron density reveals two dimers associating via an extensive C-terminal coiled-coil protrusion to form an elongated anti-parallel tetramer. In solution, ZapA exists in a dimer–tetramer equilibrium that is strongly correlated with concentration. An increase in concentration promotes formation of the higher oligomeric state. The dimer is postulated to be the predominant physiological species although the tetramer could become significant if, as FtsZ is integrated into the Z-ring and is cross-linked, the local concentration of the dimer becomes sufficiently high. We also show that ZapA binds FtsZ with an approximate 1 : 1 molar stoichiometry and that this interaction provokes dramatic FtsZ polymerisation and inter-filament association as well as yielding filaments, single or bundled, more stable and resistant to collapse. Whilst in vitro dynamics of FtsZ are well characterised, its in vivo arrangement within the ultra-structural architecture of the Z-ring is yet to be determined despite being fundamental to cell division. The ZapA dimer has single 2-fold symmetry whilst the bipolar tetramer displays triple 2-fold symmetry. Given the symmetry of these ZapA oligomers and the polar nature of FtsZ filaments, the structure of ZapA carries novel implications for the inherent architecture of the Z-ring in vivo.

Introduction

The discovery that the FtsZ GTPase formed a circumferential cytokinetic scaffold at the mid-cell yielded a potential molecular mechanism for bacterial cell division and its regulation.1 Subsequent immunofluorescence analysis demonstrated the prominence of this structure early in the cell cycle,2., 3. whilst GFP confirmed the three-dimensional nature of this “Z-ring”.4., 5. Visualisation of FtsZ at atomic resolution6 revealed a structure with strong homology to eukaryotic tubulin7., 8. that naturally alluded to a prokaryotic origin for this key eukaryotic structural element. Further similarity between these two evolutionarily distant domains emerged amongst other components of the prokaryotic cytoskeletal machinery, for example between MreB and eukaryotic actin,9., 10. and more recently Crescentin and eukaryotic intermediate filaments,11 which reinforced the theory that these are examples of true homology rather than convergence. FtsZ is remarkably conserved amongst the Eubacteria and Archaea with those lacking a Z-ring being an exception.12., 13. Its existence has also been shown in chloroplasts14 and more recently in mitochondria.15., 16.

It is thought that the precise selection of the division site, usually at the mid-cell, is controlled by localisation of the Z-ring through several mechanisms of which the Min system is the best characterised.17., 18. A hierarchy of cytoplasmic and membrane proteins, both intrinsic and extrinsic, are subsequently recruited to the Z-ring and have diverse roles18., 19., 20. that include anchoring the ring to the cell membrane, invagination and constriction, septum formation and peptidoglycan synthesis to yield two separate daughter cells. In vitro, FtsZ is extremely versatile, forming protofilaments, thick filaments, sheets,21., 22. helical tubes23., 24. and asters.25 This ability to form an array of dynamic polymers is dependent on GTP hydrolysis26., 27. and strong inter-molecular longitudinal contacts. During polymerisation, the FtsZ T7 loop extends into the nucleotide-binding active site of a neighbouring subunit and likely mediates the polymerisation-dependent GTPase activation mechanism.22., 28. Weaker lateral contacts are also involved in defining polymer character,29 perhaps through the S3 strand21., 22. or the N-terminal helix, but these are much less well understood. Despite such in vitro knowledge, the inherent morphology of the Z-ring in vivo still remains obscured.

There are a host of accessory proteins involved in regulating FtsZ polymerisation dynamics and which together contribute to the exquisite spatio-temporal tuning of the Z-ring. Acting negatively, the Min system involves accumulation of division inhibitors MinC and MinD at the cell poles via non-universally conserved mechanisms that are dependent on topological specificity factors, namely MinE in Escherichia coli30., 31. or DivIVA in Bacillus subtilis.32., 33. EzrA is another known FtsZ destabilising agent whose deletion in B. subtilis both provokes the formation of multiple Z-rings at polar and medial locations, and decreases the critical concentration of FtsZ required for ring formation.34 Also acting negatively, the SOS cell division inhibitor SulA in E. coli binds directly to FtsZ,35., 36., 37. prevents Z-ring assembly and consequently inhibits cell division.

In contrast, there are antagonistic proteins that promote FtsZ stability and drive its intra-cellular equilibrium towards filament formation. FtsA is essential for the recruitment of division components via direct interaction with the Z-ring early in the cell cycle.38 In addition, it is required for Z-ring assembly and stabilisation,39 perhaps through membrane association, whilst its ability to dimerise40 suggests a possible secondary role in FtsZ cross-linking. In E. coli, ZipA is as important as FtsA in downstream recruitment41 and has also been shown to interact directly with FtsZ.42 Over-expression of this membrane-associated protein abolishes cell division and suppresses the classic temperature-sensitive ftsZ84 mutant through its ability to bundle and stabilise FtsZ protofilaments.43., 44. Although ZipA may exhibit homology to eukaryotic microtubule-associated proteins (MAPs)44 its existence is restricted within the γ-proteobacteria.

Recently, another FtsZ stabilising agent has been discovered with broad conservation amongst bacterial species including E. coli. The Z-ring associated protein ZapA has been shown to interact with FtsZ and to direct the formation of heavily bundled and branched filamentous networks in vitro.45 Light-scattering assays demonstrate ZapA is capable of driving FtsZ polymerisation independently of nucleotide although polymerisation is shown to be highly accentuated in the presence of GTP. Electron microscopy reveals either filament bundling reminiscent of ZipA–FtsZ synergy or the promotion of ∼25 nm diameter mini rings when associated with either GTP or GDP, respectively.

Despite the extensive conservation of ZapA, Z-ring formation in null mutants is indistinguishable from wild-type with no significant division defect. However, ZapA deletion becomes lethal when combined as a double mutant with either EzrA or DivIVA, or in cells with reduced FtsZ levels.45 ZapA seemingly differs from other known FtsZ stabilising elements, such as FtsA and ZipA, as its deletion yields no obvious abnormality and is non-lethal. However, these molecules are likely indispensable as a result of the downstream division events they orchestrate rather than their stabilising role. Thus, ZapA seems unique in its specific dedication to the modulation of FtsZ polymerisation and regulation of Z-ring dynamics. Here, we have used X-ray crystallography to elucidate the structure of this 11.6 kDa protein and biochemical techniques to further characterise its interaction with FtsZ.

Section snippets

ZapA oligomerisation

The full-length Pseudomonas aeruginosa ZapA, comprising 104 amino acid residues, was crystallised and solved to 2.8 Å resolution in space group P41212. The electron density reveals two ZapA dimers associating via an extensive C-terminal coiled coil protrusion to form an anti-parallel tetramer. This initially appears contrary to previous expectations, as ZapA from B. subtilis was predicted to be dimeric.45 The integrity of the tetramer was confirmed in solution by size-exclusion chromatography (

Discussion

The crystal structure of ZapA shows two dimers that associate to yield an anti-parallel tetramer. Formation of the tetramer could be irrelevant in vivo given the dimer is the dominant species at lower and possibly more physiologically relevant protein concentrations. Irrespective of oligomerisation state, however, it may be predicted that 1 mol of ZapA monomer might bind 0.5 or 1 mol of FtsZ. In accordance, the pelleting assays (Figure 4A and B) indicate that an equal stoichiometry between

Protein expression, purification and crystallisation

Pseudomonas aeruginosa PA01-LAC zapA (accession number ATCC47085D) was amplified using the polymerase chain reaction (PCR) from genomic DNA with the primers 5′-TGACTACCATATGAGCCAGTCGAATACCCTCACCGTGC-3′ and 5′-AGTCTACGGATCCGGCTTCGCCGGCATCGGCCGGATTCG-3’. NdeI and BamHI treated fragments were cloned into pHis-17 vector yielding a 112 amino acid residue protein with a C-terminal extension of eight residues (GSHHHHHH). Transformed C41(DE3)58 cells were grown at 37 °C in 2× TY medium containing 100

Acknowledgements

We thank Linda A. Amos for helpful discussions; Sew Peak-chew for N-terminal sequencing (MRC Laboratory of Molecular Biology, Cambridge, UK); and Frank Sobott and Carol V. Robinson for mass spectrometry (Department of Chemistry, Cambridge University, UK).

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