POSTERS - BLAST X - University of Utah

BLAST X Poster #67
NEW REPORTER RESIDUES OF TRIMER FORMATION BY ESCHERICHIA COLI MCPs
Diego A. Massazza 1 , John S. Parkinson 2 , Claudia A. Studdert 1
1 Instituto de Investigaciones Biológicas, Universidad Nacional de Mar del Plata, Argentina
2 Biology Department, University of Utah
It is currently accepted that E. coli chemoreceptors are organized in vivo in trimer-ofdimer
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.
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