TubZ filament assembly dynamics requires the flexible C-terminal tail

Maria E Fuentes-Pérez,Fernando Moreno-Herrero,Rafael Núñez-Ramírez,Maria A Oliva,Oscar Llorca,Alejandro Martín-González,David Juan-Rodríguez

doi:10.1038/srep43342

Abstract

Cytomotive filaments are essential for the spatial organization in cells, showing a dynamic behavior based on nucleotide hydrolysis. TubZ is a tubulin-like protein that functions in extrachromosomal DNA movement within bacteria. TubZ filaments grow in a helical fashion following treadmilling or dynamic instability, although the underlying mechanism is unclear. We have unraveled the molecular basis for filament assembly and dynamics combining electron and atomic force microscopy and biochemical analyses. Our findings suggest that GTP caps retain the filament helical structure and hydrolysis triggers filament stiffening upon disassembly. We show that the TubZ C-terminal tail is an unstructured domain that fulfills multiple functions contributing to the filament helical arrangement, the polymer remodeling into tubulin-like rings and the full disassembly process. This C-terminal tail displays the binding site for partner proteins and we report how it modulates the interaction of the regulator protein TubY.

Highlights

Cytomotive filaments are essential for the spatial organization in cells, showing a dynamic behavior based on nucleotide hydrolysis
There are structural differences between unassembled and assembled monomers unrelated to the nucleotide chemical state, known as structural plasticity, that are key in the modulation of the polymer dynamics[2]
We have measured the nucleotide content of BtTubZ filaments assembled with GTP- γ-S and GTP and have found 20% and 80% of GDP-bound molecules, respectively (Methods)

Summary

Introduction

Cytomotive filaments are essential for the spatial organization in cells, showing a dynamic behavior based on nucleotide hydrolysis. We show that the TubZ C-terminal tail is an unstructured domain that fulfills multiple functions contributing to the filament helical arrangement, the polymer remodeling into tubulin-like rings and the full disassembly process This C-terminal tail displays the binding site for partner proteins and we report how it modulates the interaction of the regulator protein TubY. The understanding of plasmid segregation is key in order to design new strategies to prevent the spread of virulence genes in growing bacterial populations Both TubZ and the regulator protein TubY may be ideal targets to block DNA movement within growing bacteria. Recent cryo-EM structures of 2- and 4-stranded TubZ filaments showed an opening in the longitudinal interface due to GTPase activity[13] These structures displayed contacts between the C-terminal tail and upper TubZ monomers within the protofilament, a lack in continuous electron density made it impossible to describe the interaction mechanism[13]. The flexible C-tail participates as a multifunctional domain on filament assembly and disassembly

Methods

Results

Conclusion