Isolated operator binding and ligand response domains of the TyrR protein of Haemophilus influenzae associate to reconstitute functional repressor.

Highly purified preparations of the TyrR protein of Haemophilus influenzae Rd undergo specific and limited proteolytic cleavage during storage at 4 degreesC to generate two fragments of 28 and 8 kDa. Under nondenaturing conditions, the two fragments remain tightly associated. Nicked TyrR is identical to full-length TyrR in its operator binding characteristics. The 8-kDa fragment containing amino acid residues 258-318 was separated from the 28-kDa fragment (residues 1-257) by gel filtration chromatography in the presence of 4 M urea. Upon renaturation, this fragment bound to operator with an affinity similar to that of full-length TyrR but was unresponsive to ligands that normally modulate operator binding (gamma-S-ATP and L-tyrosine). It was not possible to renature the urea-treated 28-kDa fragment. Highly purified soluble preparations of truncated TyrR containing residues 1-257 were obtained after the overexpression of a shortened form of the tyrR gene via a specific plasmid construct. By several criteria, this species had native secondary and tertiary structure. The 28-kDa fragment was unable to bind to operator but could reconstitute nicked TyrR when added to the renatured 8-kDa fragment, as shown by physical properties and responsiveness to cofactors in operator binding. When either the 28- or 8-kDa species was expressed in vivo, there was no detectable operator binding, as evaluated using a lacZ reporter system driven by the repressible aroF promoter. When the two fragments were co-expressed in a common cytoplasm, an operator-binding species was formed, as demonstrated through partial restoration of repression capability.