Mapping Flexibility and the Assembly Switch of Cell Division Protein FtsZ by Computational and Mutational Approaches

Antonio J Martín-Galiano,Rubén M Buey,Marta Cabezas,José M Andreu

doi:10.1074/jbc.m110.117127

Abstract

The molecular switch for nucleotide-regulated assembly and disassembly of the main prokaryotic cell division protein FtsZ is unknown despite the numerous crystal structures that are available. We have characterized the functional motions in FtsZ with a computational consensus of essential dynamics, structural comparisons, sequence conservation, and networks of co-evolving residues. Employing this information, we have constructed 17 mutants, which alter the FtsZ functional cycle at different stages, to modify FtsZ flexibility. The mutant phenotypes ranged from benign to total inactivation and included increased GTPase, reduced assembly, and stabilized assembly. Six mutations clustering at the long cleft between the C-terminal beta-sheet and core helix H7 deviated FtsZ assembly into curved filaments with inhibited GTPase, which still polymerize cooperatively. These mutations may perturb the predicted closure of the C-terminal domain onto H7 required for switching between curved and straight association modes and for GTPase activation. By mapping the FtsZ assembly switch, this work also gives insight into FtsZ druggability because the curved mutations delineate the putative binding site of the promising antibacterial FtsZ inhibitor PC190723.

Highlights

Cell division protein FtsZ and its eukaryotic structural homolog tubulin are cytoskeletal GTPases, which assemble into different polymers that play crucial roles in living cells
Conservation pressure in T1–T7 could be ascribed to the necessity of maintaining the interaction with the nucleotide, in helices and in particular in sheets, it may concern folding stability or structural changes involved in the activation switch
FtsZ Monomer Flexibility and Predicted Subdomain Movements Modify the Association Interfaces in the FtsZ Filament— The ability to assemble cooperatively is built into the FtsZ structure by a sophisticated system of motion subdomains, allosteric networks, and interacting surfaces

Summary

Introduction

Cell division protein FtsZ and its eukaryotic structural homolog tubulin are cytoskeletal GTPases, which assemble into different polymers that play crucial roles in living cells. GTP binding displaces the balance to the straight filaments and sheet and bundle condensates, whereas GDP-FtsZ filaments tend to be curved and eventually depolymerize. Upon filament formation, the C-domain from the upper subunit contacts the N-domain and the top of H7 of the lower subunit, whereas the T7 loop from the upper subunit acts synergistically by providing the co-catalytic aspartate residues that complete the GTPase site [19, 22,23,24] This engages cooperative polymerization and GTP binding/hydrolysis, which requires the fine coupling of both domains, probably by the evolutionary development of a distinctive combination of elements including hinges, moving parts, and energetically coupled residue networks providing signal transmission across monomers. Insight into the structural switch of tubulin was provided by the comparison of the curved and straight structures [27, 28] and by VOLUME 285 NUMBER 29 JULY 16, 2010

Methods

Results

Conclusion