NEW CRYSTAL FORMS OF ESCHERICHIA COLI P_II COMPLEXED WITH VARIOUS LIGANDS AND STRUCTURE SOLUTION OF P_II/ATP/2-KETOGLUTARATE. Karen Edwards, Peter Suffolk, Paul Carr, Eong Cheah and David Ollis. Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia

P_II is a signal transduction protein involved in bacterial nitrogen regulation and plays a key role in regulating both the activity and level of expression of the enzyme glutamine synthetase (GS). Regulation of GS is achieved via a number of protein-protein interactions involving P_II, adenylyl transferase, nitrogen regulatory proteins I and II and uridylyl transferase (UTase/UR). The function of the regulatory enzymes is reversed upon uridylation of residue Tyr51 in P_II which occurs in response to a drop in cellular nitrogen levels.

The structure of unliganded E. coli P_II has been solved and refined to 1.9 Å resolution (Carr et al., 1996). Recent biochemical evidence suggests an important role for ligands in effecting allosteric changes in P_II (Kamberov et al., 1995). ATP, 2-ketoglutarate and glutamate have all been shown to bind to P_II and are necessary for the uridylylation of P_II by UTase/UR.

In order to elucidate the potential structural changes which may occur upon ligand binding, P_II has been crystallised with a number of effector ligands including 2-ketoglutarate (2-KG), glutamate and ATP, and ATP in combination with 2-ketoglutarate and glutamate. We anticipate that these structure determinations will provide us with new insights into the way P_II influences the enzymes involved in nitrogen regulation and how its interaction with various ligands plays a crucial role in signal transduction.

A molecular replacement solution for the P_II/ATP/2-KG crystals has been obtained and refinement of the structure is in progress. Details of the crystallisation, structure solution and current model will be presented.

Carr, P.D., Cheah, E., Suffolk, P.M., Vasudevan, S.G., Dixon, N.E. & Ollis, D.L. (1996). Acta Cryst. D52, 93-104.

Kamberov, E.S., Atkinson, M.R. & Ninfa, A.J. (1995). J. Biol. Chem. 270, 1-11.