Functional and structural analysis of homologous conserved residues CheW-R62 and CheA-R555 within the chemotaxis ternary complex

ANDREA PEDETTA; JOHN S. PARKINSON; CLAUDIA STUDDERT

Tucson, Arizona

Congreso; BLAST XII Meeting; 2013

Bacterial Locomotion and Signal Transcution, Inc

The small protein CheW, structurally homologous to the P5 regulatory domain of CheA, couples the receptors with the kinase, playing an essential role in CheA control. Various studies in recent years indicate that the role of CheW may be more complex than that of a simple bridge between the two proteins. We focused our study on the conserved residues R62 in CheW and R555 in CheA. These arginine residues lie in equivalent positions in CheW and the homologous P5 domain in CheA, and replacements in either of them cause severe impairment of chemotactic behavior. However, the mutant proteins mediate the formation of ternary complexes with normal kinase activity when studied in vitro, using chemoreceptor-containing membranes. To assess the effect of these replacements on the signaling ability of the complex in living cells we performed a FRET-based assay. The assay, based on CheY-CheZ interaction as a read-out of kinase activity, allowed us to compare the sensitivity and cooperativity of the response in cells expressing the wild-type versions of CheA and CheW with those of cells expressing the mutant variants. Whereas in CheW-R62 mutants the sensitivity and cooperativity were slightly reduced, in CheA-R555 mutants both parameters were more significantly affected. In order to contribute to the understanding of the arrangement of CheW within the signaling complex, CheW R62C was co-expressed with Tsr variants carrying single cysteine replacements, and cells were subjected to oxidizing conditions through diamide treatment. The co-expression of CheW R62C with Tsr V398C (but not Tsr carrying other cysteine replacements) at physiological levels led to formation of a disulfide-bonded product between the two proteins, indicating that the crosslinking reflects a specific interaction. Moreover, the presence of Tsr V398C corrects the chemotaxis defect of cells expressing CheW R62C, indicating that this protein pair is part of a fully functional complex. The extent of crosslinking is somehow increased in cells that lack the CheA kinase. This fact could be explained if there exists some competition between CheW and CheA (P5 domain) for interaction with Tsr. However, we could not detect any disulfide bond formation between CheA R555C and Tsr V398C.  Our results suggest that small changes in the conformation of the ternary complexes may lead to reduced sensitivity and/or cooperativity of the system, with consequent alterations in signal amplification and in the chemotactic response.