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Protein interactions regulating bacterial cell division: integrating structure and function

Principal Supervisor: Dr David Roper - School of Life Sciences

Co-supervisor: Dr Józef Lewandowski - Department of Chemistry

PhD project title: Protein interactions regulating bacterial cell division: integrating structure and function

University of Registration: Warwick

Project outline:

Bacterial cell division is orchestrated by a group of membrane bound and cytoplasmic proteins, which form a complex known as the Divisome. In order for the cell to divide correcty, the division process must be coordinated with biosynthesis of new cell wall material and separation of daughter chromosomes mediated by specific divisomal protein sub-complexes. Arrest of these events leads to the cessation of normal growth or cell lysis and is of interest therefore from both a basic scientific and translational perspective. At the core of the divisome is the bacterial homologue of tubulin; FtsZ, which polymerises and exerts a cytokinetic force on the cell membrane that leads to cell division. FtsZ contain a GTP-binding domain and in the presence of GTP FtsZ can assemble into protofilaments, two-dimensional sheets, and ring structures and overall FtsZ And tubulin share substantial structural similarities. The GTPase active site is formed at the interface of individual FtsZ monomers, which are located in a head to tail arrangement and GTP hydrolysis is associated with disassembly of higher order structures. Whilst X-ray crystallography has provided high-resolution “snap shot” information on FtsZ and FtsZ modulating proteins, there is relatively little information about how the protein-protein interactions with FtsZ are mediated and how that interaction effects FtsZ polymerisation and function and thus cell division overall. In order to investigate this we need to engage other structural techniques which are better suited to elucidate the molecular events in these dynamic biological molecules in a more integrated approach.

A number of proteins are known to bind to the carboxyl terminal domain of FtsZ, anchoring it to the membrane (ZipA, FtsA) and are known to be important in the overall cell division process. In addition, the FtsZ-associated proteins ZapA to ZapE interact at an early stage of FtsZ-ring assembly. Our previous work has shown that the cell division protein ZapA needs to tetramerise into a “dog bone” shape in order to achieve ordered assembly of FtsZ fibres, but a mutant dimer form of ZapA will still bind to FtsZ. The interaction of ZapA decreases the overall GTPase activity of FtsZ stabilising the formation of FtsZ fibres as seen by light microscopy and measurements of GTP turnover. These experiments in combination with existing X-ray structures strongly implicate the conserved structure of the globular head group of ZapA as the site of interaction with FtsZ. The ZapA protein is widely found in bacterial species and is a relatively small protein which can be expressed in high yield making it ideal for specialised NMR labelling and biochemical studies. When combined with the library of established biochemical assays for FtsZ function as well as in-vivo and in-vitro assay of function and structure, we have a powerful arsenal of experimental techniques assembled (including solution and solid state NMR, EM and other biophysical and biochemical techniques) to address important biological questions including:

  • What is the interaction site between FtsZ and ZapA?
  • How does the binding of ZapA to FtsZ mediate its effect on ftsZ polymerisation.
  • How is this interaction effected by other binding partners including ZipA, ZapB and FtsA?

The outcome of these studies will be a molecular level understanding of the protein-protein interactions that underpin this aspect of the cell division process and contribute to wider appreciation of how the separation bacterial cells is orchestrated. This project will form part of a wider collaboration between the University’s of Warwick and Birmingham with a stated mission of determining the overall structure and function of the bacterial divisome.

BBSRC Strategic Research Priority: Molecules, cells and systems

Techniques that will be undertaken during the project:

Techniques: solution and solid-state NMR; TEM; cryoEM; x-ray crystallography; biochemical assays; potentially SPR and other techniques for characterising binding.

Below we outline briefly how the main biophysical approaches will be used in the context of the postulated questions about divisome.

What is the interaction site between FtsZ and ZapA?

  • Approach: Solution state NMR assignment of ZapA structure and definition and FtsZ-ZapA binding site. Solid state NMR structure of ZapA in complex with FtsZ (whereas solution NMR is a prime tool for studying ZapA in isolation and probing its interactions with other proteins the resulting complexes are too large to be able to determine conformation of ZapA in them; fortunately, the conformation of ZapA in complexes should be accessible through solid state NMR methods).

How does the binding of ZapA to FtsZ mediate its effect on ftsZ polymerisation?

  • Approach: Use the above structure to biochemically and structurally (again solution and solid state NMR, EM) interrogate the interaction, can we rationalise the known ZapA mutants that alter FtsZ interaction? Correlate existing and new ZapA mutants with effects on FtsZ polymerisation and GTPase activity. Measure ZapA-FtsZ affinity. Correlate the

How is this interaction effected by other binding partners including ZipA, ZapB and FtsA?

  • Approach: Use a combination of interaction studies by solution NMR and interaction/structural studies by solid state NMR.

Contact: Dr David Roper, University of Warwick