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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - MIPZ (Functional characterization of the cell division inhibitor MipZ)

Teaser

Summary

The mechanisms controlling the proper temporal and spatial regulation of cell division are a fundamental issue in cell biology. Cell division in most bacteria is initiated with the localization of the protein FtsZ in the correct place. FtsZ will form a polymer (called the Z-ring) around the membrane and will then serve as a scaffold for many other proteins that will be responsible for the formation of the septum that separates the two daughter cells. In bacteria, the minCD system and nucleoid occlusion factors are the most studied proteins regulating the positioning of the septa that divides the cell, since they are present in two of the most studied model organisms, Escherichia coli and Bacillus subtilis. However, many other new systems have been discovered in different bacteria in the past years, illustrating the diversity present, even for such an essential mechanism as cell division positioning. Thus, it has become clear that in order to properly understand how a particular mechanism works, especially deciphering cell division in bacteria is key to find new targets for the development of antimicrobials, we need to broaden our study to non-classical organisms.
In Caulobacter crescentus, an alpha-proteobacteria, the MipZ protein alone is able to localize FtsZ, the core protein of the divisome, both in time and space. MipZ inhibits FtsZ polymerization and localizes at the poles of the cell forming a gradient towards the cell center, leaving the latter as the only space where FtsZ can form the Z-ring. In order to create its characteristic gradient localization, MipZ needs to interact with the protein ParB at the poles and with the chromosomal DNA in the cell. ParB is a component of the DNA segregation machinery and recognizes a cluster of sites (parS) in the origin-proximal region of the chromosome. Since the molecular mechanisms supporting the interaction of MipZ with these elements are unknown, the overall objective in the MIPZ project has been the characterization in detail of the MipZ functioning, especially its relationship with ParB and FtsZ.
Regarding the relationship of MipZ with ParB, we could map the interaction interface to the C-terminal region of MipZ, and identify the exact residues involved. The binding region is composed mainly of positively charged amino acids, suggesting that it might bind to a negatively charged region on ParB through electrostatic interactions.
In addition, we characterized the MipZ-FtsZ binding interface and studied in vitro the molecular mechanism underlying the regulation of FtsZ polymerization by MipZ. Based on all the experimental data obtained, we have been able to create a model of the inhibitory activity of MipZ, in which it acts as a minus-end capper and severer. Although its affinity for FtsZ is not very high, a MipZ dimer can cap two FtsZ monomers/polymers and prevent them to incorporate into the forming filament. MipZ can produce a conformational change in FtsZ, which could stimulate the depolymerization process, by binding in the C-terminal region of the core of the protein, close to the T7-loop, which is necessary for FtsZ polymerization.

Work performed

Study of the interaction between MipZ and ParB
In order to study this interaction, we purified both proteins and performed a Biolayer Interferometry assay in vitro. We confirmed that four of the previously identified residues are important for the interaction. As an additional proof, we conducted Hydrogen-Deuterium Exchange to map the binding interface in the MipZ surface. A region located in the C-terminal end of the protein turned out to be involved in the binding. The four amino acids selected before were inside this region, confirming the biochemical and in vivo assays.

Study of the interaction between MipZ and FtsZ
We characterized the interaction surfaces in both MipZ and FtsZ by Hydrogen-Deuterium Exchange. We found that dimeric MipZ is able to bind two independent FtsZ molecules by its lateral sides. MipZ binds FtsZ in its core C-terminal region, very close to the T7-loop. We also found that after MipZ binding there is a conformational change in the N-terminal region of FtsZ, which may reduce the FtsZ-FtsZ affinity. Many FtsZ inhibitors bind to the conserved C-terminal peptide of FtsZ. Therefore, we purified a mutant protein of FtsZ lacking this peptide and studied its effect in the regulation by sedimentation assay and GTP hydrolysis assay. We found that MipZ is able to inhibit this FtsZ variant to the same degree as the wild-type protein. We therefore concluded that this region is not essential for MipZ-mediated inhibition of FtsZ polymerization. We showed that MipZ is able to bind to the monomeric, polymeric, GTP- and GDP-bound forms of FtsZ using Biolayer Interferometry. We estimated an affinity constant for the interaction of monomeric FtsZ with dimeric MipZ in the micromolar range. We performed in vitro experiments of MipZ inhibition of FtsZ polymerization in the presence of non-hydrolyzable analogs of GTP and found that the FtsZ GTPase activity is not important for its regulation by MipZ. We used in vitro Fluorescence Correlation Spectroscopy to follow FtsZ polymerization and depolymerization in the presence of MipZ in real time. We found that FtsZ polymers are not broken to monomers but to small oligomers, meaning that the FtsZ polymerization rate must be always higher than the depolymerization rate of MipZ to FtsZ. We could also establish the effect of protein concentration on the MipZ inhibitory activity which is related to the type of inhibitory mechanism. The results obtained were disseminated by presentations at 3 international conferences, and a manuscript in preparation will be sent to a peer-reviewed journal.

Final results

Although eukaryotic cytoskeletal proteins have been extensively studied, little is known about how FtsZ is regulated at the molecular level. The model created during the MIPZ project represents one of the very few FtsZ-positioning systems that have been characterized at the molecular level, and the first one that does not belong to the classical model organisms for cell division study, E. coli or Bacillus subtilis, helping to broaden the knowledge about other systems. In order to characterize it, we used a combination of state-of-the-art techniques that have not been used in similar systems before, such as Hydrogen-Deuterium Exchange that allowed us to unravel the severing mechanism of MipZ, being the first time this mechanism has been described in a bacterial polymeric protein. We also performed Fluorescence Correlation Spectroscopy for the first time to follow polymerization of a prokaryotic cytoskeleton protein in real time.