Periodic Reporting for period 1 - MIPZ (Functional characterization of the cell division inhibitor MipZ)
Período documentado: 2015-08-03 hasta 2017-08-02
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.
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.