Structure of the Z Ring-associated Protein, ZapD, Bound to the C-terminal Domain of the Tubulin-like Protein, FtsZ, Suggests Mechanism of Z Ring Stabilization through FtsZ Cross-linking

Abstract

Edited by Norma AllewellCell division in most bacteria is mediated by the tubulin-like FtsZ protein, which polymerizes in a GTP-dependent manner to form the cytokinetic Z ring. A diverse repertoire of FtsZ-binding proteins affects FtsZ localization and polymerization to ensure correct Z ring formation. Many of these proteins bind the C-terminal domain (CTD) of FtsZ, which serves as a hub for FtsZ regulation. FtsZ ring-associated proteins, ZapA-D (Zaps), are important FtsZ regulatory proteins that stabilize FtsZ assembly and enhance Z ring formation by increasing lateral assembly of FtsZ protofilaments, which then form the Z ring. There are no structures of a Zap protein bound to FtsZ; therefore, how these proteins affect FtsZ polymerization has been unclear. Recent data showed ZapD binds specifically to the FtsZ CTD. Thus, to obtain insight into the ZapD-CTD interaction and how it may mediate FtsZ protofilament assembly, we determined the Escherichia coli ZapD-FtsZ CTD structure to 2.67 Å resolution. The structure shows that the CTD docks within a hydrophobic cleft in the ZapD helical domain and adopts an unusual structure composed of two turns of helix separated by a proline kink. FtsZ CTD residue Phe-377 inserts into the ZapD pocket, anchoring the CTD in place and permitting hydrophobic contacts between FtsZ residues Ile-374, Pro-375, and Leu-378 with ZapD residues Leu-74, Trp-77, Leu-91, and Leu-174. The structural findings were supported by mutagenesis coupled with biochemical and in vivo studies. The combined data suggest that ZapD acts as a molecular cross-linking reagent between FtsZ protofilaments to enhance FtsZ assembly.FtsZ is a highly conserved prokaryotic tubulin homolog that mediates cell division in most bacteria, chloroplasts, many archaea, as well as the mitochondria of primitive eukaryotes (1-38). It is composed of a short N-terminal region, a globular core that binds GTP, a long and flexible linker of variable sequence and length (ranging from ϳ40 to 257 residues), and a short C-terminal domain (CTD) 3 of ϳ14 residues. The FtsZ CTD region can be further divided into an N-terminal conserved region (CTC) and a short C-terminal variable region (CTV) (19). GTP binding to the globular domains between different FtsZ protomers leads to the formation of linear protofilaments. The FtsZ protofilaments assemble at the cell center to create the Z ring, which then serves as the platform for the assembly of the divisome (9). There is still debate regarding the organization of FtsZ protofilaments within the Z ring. Superresolution analyses on multiple bacteria indicate that the Z ring is composed of dispersed protofilament assemblages (10 -15). However, other data suggest that FtsZ protofilaments form a continuous ring around the cell (18).Although the specific organization of the Z ring is unclear, data show that its assembly is effected by regulatory proteins (19). Indeed, the intracellular levels of FtsZ in Escherichia coli and most bacteria remain largely unchanged throughout the cell cycle and exceed ...

The intrinsically disordered C-terminal linker of FtsZ regulates protofilament dynamics and superstructurein vitro

“…Many proteins that bind the CTC of FtsZ have been shown to affect Z-ring structure in vivo and/or affect FtsZ polymer structure in vitro (24,(37)(38)(39). It is unclear if and how these proteins regulate polymerization of FtsZ without interacting directly with the GTPase domain, since the CTC and the GTPase domain are linked by a long unstructured CTL.…”

A DNA-binding protein tunes septum placement duringBacillus subtilissporulation

“…It has not escaped our attention that many regulators of FtsZ including FtsA, ZapD, and MinD possess glutamate or aspartate residues near the implicated FtsZ C-terminal tail binding site which are proceeded by either a hydrophobic or polar uncharged residue followed by an arginine or lysine. For example, FtsA from Thermotoga maritima possesses LRE 69 , ZapD from E. coli and a variety of Gammaproteobacteria I(R/K)E 70,71 , and MinD a highly conserved ARD 72 . Whether these residues represent a bona fide motif involved in FtsZ regulation remains to be determined, but it is intriguing that two residues identified as critical for RefZ function fall within an IRE sequence.…”

Essential dynamic interdependence of FtsZ and SepF for Z-ring and septum formation inCorynebacterium glutamicum

“…8b). The apparent Kd value for the SepFDML-FtsZCTD interaction, as determined by surface plasmon resonance (SPR), was 8 reported for other FtsZCTD interactors such as ZipA 32 , FtsA 33 or ZapD 34 . The interface was further validated by mutating two FtsZCTD-contact residues in SepF: F131A and K125E.…”

“…Most FtsZ-binding proteins that have been characterized to date recognize the FtsZCTD, which represents a 'landing pad' for FtsZ interactors 35 . Other known structures of regulatory proteins in complex with FtsZCTD include T. maritima FtsA and the E. coli proteins ZipA, SlmA and ZapD [32][33][34]36 . These crystal structures had shown that the FtsZCTD peptide can adopt multiple conformations depending on its binding partner, from full-or partial-helical states as in the FtsA or ZipA complexes to distinct extended conformations as in ZapD or SlmA.…”