Biology subjects

Andreu, J. M.

Publications and source records attributed to Andreu, J. M..

3 recordsLinked to original sources

bioRxiv2021

Mechanism of FtsZ assembly dynamics revealed by filament structures in different nucleotide states

Treadmilling protein filaments perform essential cellular functions by growing from one end while shrinking from the other, driven by nucleotide hydrolysis. Bacterial cell division relies on the primitive tubulin homolog FtsZ, a target for antibiotic discovery that assembles into single treadmilling filaments that hydrolyse GTP at an active site formed upon subunit association. We determined high-resolution filament structures of FtsZ from the pathogen Staphylococcus aureus in complex with different nucleotide analogues and cations, including mimetics of the ground and transition states of catalysis. Together with mutational and biochemical analyses, our structures reveal interactions made by the GTP {gamma}-phosphate and Mg2+ at the subunit interface, a K+ ion stabilizing loop T7 for co-catalysis, new roles of key residues at the active site and a nearby crosstalk area, and rearrangements of a dynamic water shell bridging adjacent subunits upon GTP hydrolysis. We propose a mechanistic model that integrates nucleotide hydrolysis signalling with assembly-associated conformational changes and filament treadmilling. Equivalent assembly mechanisms may apply to more complex tubulin and actin cytomotive filaments that share analogous features with FtsZ.

biophysics↗

bioRxiv2021

Crystal structures of taxane-tubulin complexes: Implications for the mechanism of microtubule stabilization by Taxol

Paclitaxel (Taxol(R)) is a taxane and a first-line chemotherapeutic drug that stabilizes microtubules. While the interaction of paclitaxel with microtubules is well described, the current lack of high-resolution structural information on a tubulin-taxane complex precludes a comprehensive description of the binding determinants that affect the drugs mechanism of action. Here, we solved the crystal structure of the core baccatin III moiety of paclitaxel lacking the C13 side chain in complex with tubulin at 1.9 [A] resolution. Based on this information, we engineered two tailor-made taxanes with modified C13 side chains, solved their crystal structures in complex with tubulin, and analyzed their effects along with those of paclitaxel, docetaxel, and baccatin III on the microtubule lattice by X-ray fiber diffraction. We then compared high-resolution structures of ligand-bound tubulin and microtubule complexes with apo forms and used molecular dynamics simulations to understand the consequences of taxane binding to tubulin as well as to simplified protofilament and microtubule-lattice models. Our combined approach sheds light on three mechanistic questions. Firstly, taxanes bind better to microtubules as compared to unassembled tubulin due to a dual structural mechanism: Tubulin assembly is linked to a conformational reorganization of the {beta}M loop, which otherwise occludes ligand access to the taxane site, while the bulky C13 side chains preferentially recognize the microtubule-assembled over the unassembled conformational state of tubulin. Second, the occupancy of the taxane site by a ligand has no influence on the straightness of tubulin protofilaments. Finally, binding of the taxane core to the taxane site displaces the S9-S10 loop of {beta}-tubulin resulting in microtubule expansion. Our results provide detailed new insights into the mechanism of microtubule-stabilization by taxanes.

biophysics↗

bioRxiv2016

A polymerisation-associated conformational switch in FtsZ that enables treadmilling

Bacterial cell division in many organisms involves a constricting cytokinetic ring that is orchestrated by the tubulin-like protein FtsZ. FtsZ forms dynamic filaments close to the membrane at the site of division that have recently been shown to treadmill around the division ring, guiding septal wall synthesis.\n\nHere, using X-ray crystallography of Staphylococcus aureus SaFtsZ we reveal how an FtsZ can adopt two functionally distinct conformations, open and closed. The open form is found in SaFtsZ filaments formed in crystals and also in soluble filaments of E. coli FtsZ as deduced by cryoEM. The closed form is found within several crystal forms of two non-polymerising SaFtsZ mutants and corresponds to many previous FtsZ structures from other organisms.\n\nWe argue that FtsZ undergoes a polymerisation-associated conformational switch. We show that such a switch provides explanations for both how treadmilling may occur within a single-stranded filament, and why filament assembly is cooperative.

microbiology↗