The structure of the P_II–ATP complex

Abstract

P II is a signal transduction protein that is part of the cellular machinery used by many bacteria to regulate the activity of glutamine synthetase and the transcription of its gene. The structure of P II was solved using a hexagonal crystal form (form I). The more physiologically relevant form of P II is a complex with small molecule effectors. We describe the structure of P II with ATP obtained by analysis of two different crystal forms (forms II and III) that were obtained by co-crystallization of P II with ATP. Both structures have a disordered recognition (T) loop and show differences at their C termini. Comparison of these structures with the form I protein reveals changes that occur on binding ATP. Surprisingly, the structure of the P II /ATP complex differs with that of GlnK, a functional homologue. The two proteins bind the base and sugar of ATP in a similar manner but show differences in the way that they interact with the phosphates. The differences in structure could account for the differences in their activities, and these have been attributed to a difference in sequence at position 82. It has been demonstrated recently that P II and GlnK form functional heterotrimers in vivo. We construct models of the heterotrimers and examine the junction between the subunits.Keywords: ATP binding protein; GlnK; P II ; signal transduction; X-ray structure.P II is a signal transduction protein found in a variety of organisms. It is best characterized in Escherichia coli in which it indicates the nitrogen status of the cell. In many bacteria the level of available nitrogen is closely coupled to the level of glutamine synthetase (GS) activity. P II is involved in the regulation of GS and the transcription of its gene [1±4]. In brief, the nitrogen status of the cell is monitored by the uridylyl transferase/uridylyl removing enzyme (UT/URase). It uridylylates P II at Tyr51 to form P II ±UMP [5±7] under conditions of limiting ammonia and catalyses the removal of UMP under conditions of excess ammonia. The uridylylation status of P II reflects the nitrogen status of the cell. P II dictates the direction of the enzymatic regulation of GS through its interaction with adenylyl transferase (ATase). The transcriptional regulation of GS is mediated by P II and the nitrogen regulatory proteins I and II (NRI and NRII) also known as NtrC and NtrB, respectively. Although the regulation of GS has been studied for many years, much remains to be learnt about this system in the light of some recent discoveries. For example, it is now apparent that many organisms possess two P II -like proteins. The second protein in E. coli is known as GlnK [8,9]. The physical properties of GlnK are very similar to those of P II and under some circumstances GlnK can substitute for P II . When the cell is grown in nitrogen-poor conditions, P II and GlnK form heterotrimers [10] to fine-tune the regulation of GS [11]. The fine regulation of the nitrogen signal cascade by functional heterotrimers of P II and GlnK introduces a new level of complexity to ...

The crystal structure of the Escherichia coli AmtB–GlnK complex reveals how GlnK regulates the ammonia channel

“…An important difference between the ADP complex observed here and the E. coli GlnK-ATP (21) and E. coli GlnB-ATP (26) complexes is that in the latter cases the T-loop is disordered beyond residue G37. However, in our structure the T-loop is fully ordered, and we observe the two hydrogen bonds of the main-chain NH groups of residues R38 and Q39 with ␣-phosphate oxygens (Fig.…”

“…Thus, we observe an ADP-bound state of E. coli P II , with a stoichiometry of 3ADP:GlnK 3 . Previous studies have shown ATP can bind to this site in both E. coli GlnK (21) and GlnB (26). However, given the close contacts with neighboring amino acid main-chain and side-chain atoms all around the diphosphate moiety as verified through omit maps, we rule out that we are observing ATP with a disordered ␥-phosphate.…”

“…Bound ADP has been reported on two previous occasions: in Thermus thermophilus GlnK where ADP was added during crystallization (33), and in Thermotoga maritima where ADP was found in the crystal structure of a P II protein purified from cells where the growth conditions were not defined (34), the latter case being the only one to date where nucleotide was found to copurify with P II . Consequently for E. coli GlnB and GlnK, the only comparisons that can be made are with bound ATP that was added during crystallization (21,26). An important difference between the ADP complex observed here and those GlnK-ATP and GlnB-ATP complexes is that in the latter cases, the T-loops are disordered, whereas in our structure they are fully ordered (21,26).…”

“…Consequently for E. coli GlnB and GlnK, the only comparisons that can be made are with bound ATP that was added during crystallization (21,26). An important difference between the ADP complex observed here and those GlnK-ATP and GlnB-ATP complexes is that in the latter cases, the T-loops are disordered, whereas in our structure they are fully ordered (21,26).…”

“…P II proteins synergistically bind ATP and 2-oxoglutarate (2-OG), a small-molecule signal of cellular nitrogen status (14,15,22) and a metabolite of the Krebs cycle. The crystal structures of E. coli GlnB-ATP and GlnK-ATP co-complexes revealed ATP bound in the lateral clefts between the subunits (21,26).…”