The C Terminus of FtsZ Regulates FtsZ Assembly Dynamics and Is Required for Bacillus subtilis Cell Division.

摘要

Bacterial cell division is initiated by the assembly of the tubulin homolog FtsZ into a ring (Z ring) at the nascent division site. Once formed, the Z ring serves as a scaffold for recruitment of the division machinery and helps provide some of the constrictive force for cytokinesis. In vitro FtsZ undergoes GTP-dependent assembly where individual subunits form single-stranded protofilaments and laterally-associated filament bundles. How the filamentous FtsZ structures observed in vitro translate into the behavior of the Z ring in vivo remains a fundamental question. In this dissertation I establish important roles for the previously uncharacterized FtsZ C-terminal domains during both FtsZ assembly and Z ring formation. My work provides significant insight into how FtsZ behavior at the protein level impacts its cellular function.;Structurally, the FtsZ monomer is divided into 5 domains: an unstructured N-terminal peptide, a highly conserved N-terminal globular core, an unstructured C-terminal linker (CTL), a conserved set of ~11 residues referred to here as the C-terminal constant region (CTC), and a small, highly variable group of residues at the extreme C-terminus of FtsZ termed the C-terminal variable region (CTV). For simplicity, the N-terminal peptide and core are treated here as a single unit. The core shows a high degree of sequence conservation amongst bacterial species and contains residues required for GTP binding and hydrolysis as well as forming the contacts necessary to make filaments. The entire FtsZ C terminus consists of the CTL, CTC, and CTV. The CTL displays very little conservation between species both in primary sequence and length, is irresolvable by X-ray crystallography, and is presumed to be intrinsically disordered. The CTC and CTV are implicated in interactions between FtsZ and modulatory proteins. To reflect this function the combined domains have been termed the grappling hook peptide (GHP). Prior to this work, the roles that the C-terminal domains had in FtsZ assembly were unknown.;In this dissertation, I demonstrate that these domains do have distinct functions. First I show the CTV is important for regulating lateral interactions between FtsZ protofilaments. B. subtilis FtsZ readily forms bundled structures in vitro. In contrast, I show E. coli FtsZ typically assembles into single-stranded protofilaments. Through deletion analysis and domain swapping, I determine these phenotypes to derive from differences in the CTVs of each species. I also establish that electrostatic interactions are a driving force behind FtsZ bundling. Alterations to the CTV sequence also greatly affect cell division in B. subtilis cells, suggesting filament bundling is important for a stable Z ring in vivo..;Finally, I demonstrate the FtsZ CTL is essential for FtsZ protofilament assembly and cell division. I determine that a functional CTL must behave as an intrinsically disordered peptide with little primary sequence requirement but must be between 25 and 100 residues in length. These findings lead to a model for FtsZ in which the CTL behaves as a flexible tether anchoring FtsZ filaments to the membrane through interactions between the GHP and FtsZ modulatory proteins like FtsA. The linker can undertake different conformations and allow FtsZ filaments bundle through positioning the CTV near adjacent filaments and to respond to the curvature of the membrane, having implications for how the constrictive force for cytokinesis is generated.