STRUCTURE FUNCTION ANALYSIS OF THE PII SIGNAL-TRANSDUCTION PROTEIN OFESCHERICHIA-COLI - GENETIC SEPARATION OF INTERACTIONS WITH PROTEIN RECEPTORS/

The PII protein, encoded by glnB, is known to interact with three bifu nctional signal transducing enzymes (uridylyltransferase/uridylyl-remo ving enzyme, adenylyltransferase, and the kinase/phosphatase nitrogen regulator II [NRII or NtrB]) and three small-molecule effectors, gluta mate, 2-ketoglutarate, and ATP. We constructed 15 conservative alterat ions of PII by site-specific mutagenesis of glnB and also isolated thr ee random glnB mutants affecting nitrogen regulation. The abilities of the 18 altered PII proteins to interact with the PII receptors and th e small-molecule effecters 2-ketoglutarate and ATP were examined by us ing purified components, Results with certain mutants suggested that t he specificity for the various protein receptors was altered; other mu tations affected the interaction with all three receptors and the smal l-molecule effectors to various extents, The apex of the large solvent -exposed T loop of the PII protein (P. D. Carr, E. Cheah, P. M. Suffol k, S. G. Vasudevan, N. E. Dixon, and D. L. Ollis, Acta Crytallogr. Sec t. D 52:93-104, 1996), which includes the site of PII modification, wa s not required for the binding of small-molecule effecters but was nec essary for the interaction with all three receptors, Mutations alterin g residues of this loop or affecting the nearby B loop of PII, which l ine a cleft between monomers in the trimeric PII, affected the interac tions with protein receptors and the binding of small-molecule ligands , Thus, our results support the predictions made from structural studi es that the exposed loops of PII and cleft formed at their interface a re the sites of regulatory interactions.