A key residue for function of CheW, the coupling protein in bacterial chemotaxis

ANDREA PEDETTA; RODRIGO ASILI; CLAUDIA STUDDERT

Puerto Madryn

Congreso; XLVI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2010

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

The coupling protein CheW is essential to transmit the signals sensed by chemoreceptors to the histidine kinase CheA. The precise role of CheW during signaling and its arrangement within the signaling complex have not been elucidated.The conserved residue R62 in CheW has been proposed to function as a “light switch” by displaying important interactions with both CheA and chemoreceptors.Cells expressing the point mutant CheW R62H do not mediate chemotaxis in soft agar plates. However, this mutant protein is able to mediate kinase activation in vitro. In order to identify its functional defect, we performed several in vivo assays aimed to characterize the ability of the mutant protein to modulate kinase activity, adapt to stimuli and mediate proper clustering of receptors at the poles of the cell. We found a slight defect in the clusteringability of the mutant protein, whereas no defects are apparent in the other signaling abilities. We also characterized previously isolated suppressors of R62H in both CheA and chemoreceptors.In order to define the structural arrangement of CheW within the signaling complex, we introduced cysteine residues in the surface of both CheW and receptors and assessed their ability to form disulfides in vivo. We found a specific pair of cysteine replacements that get crosslinked. Our results support the relevance of residue R62 in chemotaxis signaling.