Preferred Degradation of the Cell Division Protein FtsZ by ADEP-activated ClpP Protease and its use in understanding Divisome Progression

Abstract

Cell division is a vital process for bacteria and therefore requires the precise coordination in terms of timing and spacing. FtsZ polymerizes in a nucleotide-dependent manner into protofilaments that form higher ordered structures via lateral interactions, thereby assembling the so called Z ring at the future division site. Here, the Z ring serves as a scaffold for other cell division proteins to adhere to, together assembling the so-called divisome. During the past years, cell division has gained considerable attention as it represents a promising target for antibiotic interference. Since FtsZ is highly conserved among bacteria and acts as a central player during cell division, the protein emerged to be of particular interest. In this context, cellular FtsZ is rapidly depleted when cells were treated with acyldepsipeptide antibiotics (ADEP). Importantly, ADEP does not directly bind to FtsZ but displays an unusual mechanism. ADEPs bind to the bacterial ClpP peptidase thereby inhibiting all natural functions of ClpP and furthermore activating it for the degradation of nascent peptides at the ribosome and, interestingly, for the untimely proteolysis of FtsZ. To date, FtsZ is the first and only folded bacterial protein that was shown to be degraded by ADEP-activated ClpP (ADEP-ClpP) in vitro as well as in vivo, whereas other tested proteins resist degradation. In this thesis, we unraveled the structural and physicochemical features of FtsZ that make it a preferred and especially vulnerable target for ADEP-ClpP. A closer look at the structure of FtsZ shows that it is a globular protein with a disordered C terminus that protrudes from the core domain and is important for the interaction with other cell division proteins. Therefore, the C terminus of FtsZ would represent a very likely target structure for the attack by ADEP-ClpP. Surprisingly, we showed that the flexible C terminus of FtsZ is not the preferred target site, but revealed that the short N-terminus of FtsZ is preferably targeted by ADEP-ClpP. Furthermore, we showed that N terminal attack of FtsZ by ADEP-ClpP leads to N-terminal unfolding of the protein. To date, ClpP has never been shown to degrade a folded protein without the help of a cognate energy-driven ATPase. In this context, it emerged that the hydrophobicity of the N terminus is decisive for the degradation of FtsZ and we suggest that N-terminal unfolding of FtsZ is driven by means of hydrophobic interactions between the hydrophobic N terminus of FtsZ and the hydrophobic rim of the entrance pore of the ClpP barrel. Furthermore, we demonstrated that unfolding and degradation of FtsZ is prevented upon binding of either GTP or GTPꝩS (the latter represents a non-hydrolyzable derivative of GTP that inhibits FtsZ polymerization). These results imply that nucleotide-devoid FtsZ may be characterized by a rather loose protein fold, and that ADEP-ClpP is therefore capable of N terminally unfolding and degrading FtsZ. Further investigations of the degradation process at elevated concentrations of ADEP/ClpP revealed that the C terminus of FtsZ becomes an additional target, suggesting a broadening of the target spectrum of ADEP-ClpP at increased concentrations. Therefore, our results elaborate the molecular basis for the earlier observed distinct phenotypes of Bacillus subtilis cells that were treated with different concentrations of ADEP. In this context, at low inhibitory concentrations of ADEP, cells proceed biomass production and other cellular processes, while cell division is abrogated due to the degradation of FtsZ. This results in a filamentous growth of rod-shaped B. subtilis cells. We suggest that under these conditions, ADEP-ClpP preferably targets and degrades FtsZ due to the here described characteristics of FtsZ that make this protein an especially attractive and vulnerable substrate for ADEP-ClpP. In contrast, when cells are treated with high concentrations of ADEP, biomass production ceases and cells display an uneven cell morphology indicating that severe additional damage has occurred to the cells. Hence, we propose that under these conditions, the target spectrum of ADEP-ClpP is broadened and other putative protein targets are degraded in addition to FtsZ. The different phenotypes described here indicated that at low ADEP concentrations the cytoplasmic FtsZ pool is rapidly depleted. This allowed us to further use ADEP as a tool to study the molecular principles of Z-ring assembly and progression. The Z-ring represents a highly dynamic structure that treadmills around the division plane, thereby guiding peptidoglycan synthesis in order to build the septal cell wall. The dynamic treadmilling motion of FtsZ filaments results from the constant incorporation of FtsZ monomers at one end, while depolymerization occurs from the opposite end. Hence, FtsZ dynamics strictly rely on a cytoplasmic FtsZ pool that ensures the constant supply with new monomers. By employing time-lapse fluorescence microscopy we investigated the fate of different stages of the Z ring upon the depletion of the cytoplasmic FtsZ pool at low inhibitory concentrations of ADEP. We observed that the initiation of Z ring formation was inhibited and that established early-stage Z rings disintegrated under these conditions. However, Z rings that had already entered a later stage finalized septum formation and cell division. Therefore, a two-step model of Z ring assembly and progression is suggested: (i) During the early stages of cell division, the cytoplasmic FtsZ pool is required for Z ring assembly, and Z ring dynamics are important to drive the assembly of the early divisome. However, (ii) at the later stage, Z ring dynamics are less important and the divisome finalizes cell division independent of the depletion of the cytoplasmic FtsZ pool. In summary, this work contributes to a better understanding of the mechanism of action of ADEP and characterizes the structural and physicochemical features of FtsZ that make the central cell division protein a preferred target for the degradation by ADEP-ClpP. Furthermore, this work validates ADEP as a molecular tool to study bacterial cell division, and we provided new insights into the principles of cell division.