Biology subjects

Luque-Ortega, J. R.

Publications and source records attributed to Luque-Ortega, J. R..

3 recordsLinked to original sources

bioRxiv2023

Crosslinking by ZapD drives the assembly of short, discontinuous FtsZ filaments into ring-like structures in solution

Bacterial cell division relies on the Z ring, a cytoskeletal structure that acts as a scaffold for the assembly of the divisome. To date, the detailed mechanisms underlying the assembly and stabilization of the Z ring remain elusive. This study highlights the role of the FtsZ-associated protein (Zap) ZapD in the assembly and stabilization of Z-ring-like structures via filament crosslinking. Using cryo-electron tomography and biochemical analysis, we show that, at equimolar concentrations of ZapD and FtsZ, ZapD induces the formation of toroidal structures composed of short, curved FtsZ filaments that are crosslinked vertically, but also laterally and diagonally. At higher concentrations of ZapD, regularly spaced ZapD dimers crosslink FtsZ filaments from above, resulting in the formation of straight bundles. Despite the simplicity of this reconstituted system, these findings provide valuable insights into the structural organization and stabilization of the Z ring by Zap proteins in bacterial cells, revealing the key role of optimal crosslinking density and geometry in enabling filament curvature and ring formation.

biochemistry↗

bioRxiv2022

Crk proteins activate the Rap1 guanine nucleotide exchange factor C3G by segregated adaptor-dependent and -independent mechanisms

C3G is a guanine nucleotide exchange factor (GEF) that activates Rap1 to promote cell adhesion. Resting C3G is autoinhibited and the GEF activity is released by stimuli that signal through tyrosine kinases. Tyrosine phosphorylation of C3G and interaction with Crk adaptor proteins, whose expression is increased in multiple human cancers, participate in C3G activation. However, the molecular details of C3G activation and the interplay between C3G phosphorylation and Crk interaction are poorly understood. Here, we combine biochemical, biophysical, and cell biology approaches to elucidate the mechanisms of C3G activation. CrkL interacts through its SH3N domain with the proline-rich motifs P1 and P2 of inactive C3G in vitro and in Jurkat and HEK293T cells, and these sites are necessary to recruit C3G to the plasma membrane. However, direct stimulation of the GEF activity requires binding of Crk proteins to the P3 and P4 sites. P3 is occluded in resting C3G and is essential for activation, while P4 contributes secondarily towards complete stimulation. Tyrosine phosphorylation of C3G alone causes marginal activation. Instead, phosphorylation primes C3G lowering the concentration of Crk proteins required for activation and increasing the maximum activity. Unexpectedly, optimal activation also requires the interaction of CrkL-SH2 domain with phosphorylated C3G. Phosphorylation and Crk-binding form a two-factor mechanism that ensures tight control of C3G activation. The simultaneous SH2 and SH3N interaction of CrkL with C3G, required for the activation, reveals a novel adaptor-independent function of Crk proteins relevant to understanding their role in physiological signaling and their deregulation in diseases.

molecular biology↗

bioRxiv2021

Assisted assembly of bacteriophage T7 core components for genome translocation across the bacterial envelope

In most bacteriophages, the genome transport across bacterial envelopes is carried out by the tail machinery. In Podoviridae viruses, where the tail is not long enough to traverse the bacterial wall, it has been postulated that viral core proteins are translocated and assembled into a tube within the periplasm. T7 bacteriophage, a member from the Podoviridae family, infects E. coli gram-negative bacteria. Despite extensive studies, the precise mechanism by which this virus translocates its genome remains unknown. Using cryo-electron microscopy, we have resolved the structure two different assemblies of the T7 bacteriophage DNA translocation complex, built by core proteins gp15 and gp16. Gp15 alone forms a partially folded hexamer, which is further assembled by interaction with gp16, resulting in a tubular structure with dimensions compatible with traversing the bacterial envelope and a channel that allows DNA passage. The structure of the gp15-gp16 complex also shows the location in gp16 of a canonical transglycosylase motif essential in the bacterial peptidoglycan layer degradation. Altogether these results allow us to propose a model for the assembly of the core translocation complex in the periplasm, which helps in the understanding at the molecular level of the mechanism involved in the T7 viral DNA release in the bacterial cytoplasm. SIGNIFICANCE STATEMENTT7 bacteriophage infects E. coli bacteria. During this process, the DNA transverses the bacterial cell wall, but the precise mechanism used by the virus remains unknown. Previous studies suggested that proteins found inside the viral capsid (core proteins) disassemble and reassemble in the bacterial periplasm to form a DNA translocation channel. In this article we solved by cryo-electron microscopy two different assemblies of the core proteins that reveal the steps followed by them to finally form a tube large enough to traverse the periplasm, as well as the location of the transglycosylase enzyme involved in peptidoglycan degradation. These findings confirm previously postulated hypothesis and make experimentally visible the mechanism of DNA transport trough the bacterial wall.

biophysics↗