IAL   21557
INSTITUTO DE AGROBIOTECNOLOGIA DEL LITORAL
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
The glycine hinge of bacterial chemoreceptors: functional analysis of its signaling role in Tsr
Autor/es:
MASSAZZA, DIEGO A.; PEDETTA, ANDREA; STUDDERT, CLAUDIA A.; HERRERA SEITZ, MARÍA K.
Lugar:
New Orleans, USA
Reunión:
Congreso; XIVth BLAST (Bacterial Locomotion and Signal Transduction) Meeting; 2017
Institución organizadora:
BLAST Meeting Board
Resumen:
Background: Chemotaxis requires the transmission of information from the environment to the flagellar motors. Chemoreceptors are dimeric transmembrane proteins with a periplasmic domain for ligand binding and a conserved cytoplasmic domain consisting in a long hairpin that forms a four-helix coiled coil bundle. The activity of the CheA kinase, attached to the tip of the cytoplasmic domain, is modulated in response to external signals. The mode of signal propagation along the long chemoreceptor rod is still under study. In this work we focus on the role of three conserved glycine residues, two in the N-terminal and one in the C-terminal helix of the hairpin, that are thought to conform a hinge with signaling function.Methods: We carried out random-codon mutagenesis at the three glycine hinge positions in Tsr and obtained several non-functional variants. We then characterized the mutants with respect to subcellular localization by a fluorescence reporter protein, trimer-of-dimer formation by crosslinking competition assays, influence on Tar function, control of kinase activity by flagellar rotation assays, and methylation pattern by Western blotting with an anti-Tsr antibody. Furthermore, we generated second-site mutations that restore the chemotaxis proficiency of some of the hinge mutant proteins, and characterized them.Results: We obtained 14 non-functional replacements, 13 of which retained native receptor interactions and subcellular localization, but were defective in kinase control. All the mutants in G439 (eight) were unable to activate the kinase and showed a hyper-methylated pattern, indicative of an OFF-biased receptor conformation. Only two of them were able to activate the kinase and got demethylated upon stimulus with the repellent glycerol. Second-site mutations lay in the methylation region (increasing hydrophobic interactions) or near the mutated glycine.Conclusion: The glycine hinge seems to be dispensable for trimer-of-dimer formation but to play a role in ON/OFF switching. The results are consistent with the yin-yang model, in which the glycine hinge separates the signaling hairpin subdomains.