NEW REPORTER RESIDUES OF TRIMER FORMATION BY ESCHERICHIA COLI MCPs

MASSAZZA, D. A.; PARKINSON, J.S.; STUDDERT, C.A.

Cuernavaca, México

Congreso; Xth BLAST (Bacterial Locomotion and Signal Transduction) Meeting; 2009

It is currently accepted that E. coli chemoreceptors are organized in vivo in trimer-of-dimer arrangements.  Using receptors bearing a cysteine reporter residue near the trimer axis, we previously demonstrated efficient formation of 2- and 3-subunit crosslinking products upon treatment of intact cells with the trifunctional maleimide reagent TMEA.  In this work, we assessed the in vivo crosslinking behavior of receptors bearing cysteine reporters at different positions along their cytoplasmic domains. We found that the formation of 3-subunit crosslinking products declined with increasing distance of the reporter from the cytoplasmic tip. This result suggests that the in vivo arrangement of the trimer of dimers resembles that observed in the crystal structure of the Tsr cytoplasmic domain, in which the dimers contact one another at the tip and splay apart in the regions that are farther from the tip. We also found that stimulation with repellents immediately before the TMEA treatment caused a slight but reproducible increase in crosslinking efficiency. This behavior is consistent with previous observations suggesting that receptor dimers within the trimer move closer together after repellent stimuli. In cells co-expressing different marked receptors, we analyzed the formation of mixed crosslinking products. When the cysteine reporters were located the same distance from the tip, mixed crosslinking products formed with high efficiency. Mixed products did not form between reporters at different distances from the tip. This result suggests that there are no significant vertical displacements within the trimer of dimers, nor sufficient flexibility between the receptors to get crosslinking between reporters at diferent levels in the dimers.