Probing interactions of the homotrimeric PII signal transduction protein with its receptors by use of PII heterotrimers formed in vitro from wild-type and mutant subunits.

The homotrimeric PII signal transduction protein of Escherichia coli interacts with two small-molecule effectors, 2-ketoglutarate and ATP, regulates two protein receptors, the kinase/phosphatase nitrogen regulator II (NRII) and the glutamine synthetase (GS) adenylyltransferase (ATase), and is subject to reversible uridylylation, catalyzed by the uridylyltransferase/uridylyl-removing enzyme (UTase/UR). The site of PII uridylylation, Y51, is located at the apex of the solvent-exposed T-loop (E. Cheah, P. D. Carr, P. M. Suffolk, S. G. Vasudevan, N. E. Dixon, and D. L. Ollis, Structure 2:981-990, 1994), and an internally truncated PII lacking residues 47 to 53 formed trimers that bound the small-molecule effectors but were unable to be uridylylated or activate NRII and ATase (P. Jiang, P. Zucker, M. R. Atkinson, E. S. Kamberov, W. Tirasophon, P. Chandran, B. R. Schefke, and A. J. Ninfa, J. Bacteriol. 179:4342-4353, 1997). We investigated the ability of heterotrimers containing delta47-53 and wild-type subunits to become uridylylated and activate NRII and ATase. Heterotrimers were formed by denaturation and renaturation of protein mixtures; when such mixtures contained a fivefold excess of A47-53 subunits, the wild-type subunits were mostly redistributed into trimers containing one wild-type subunit and two mutant subunits. The resulting population of trimers was uridylylated and deuridylylated by UTase/UR, stimulated the phosphatase activity of NRII, and stimulated adenylylation of GS by ATase. In all except the ATase interaction, the activity of the hybrid trimers was greater than expected based on the number of wild-type subunits present. These results indicate that a single T-loop region within a trimer is sufficient for the productive interaction of PII with its protein receptors. We also formed heterotrimers containing wild-type subunits and subunits containing the G89A alteration (P. Jiang, P. Zucker, M. R. Atkinson, E. S. Kamberov, W. Tirasophon, P. Chandran, B. R. Schefke, and A. J. Ninfa, J. Bacteriol. 179: 4342-4353, 1997). The G89A mutant form of PII does not bind the small-molecule effectors, does not interact with UTase or with NRII, and interacts poorly with ATase. Heterotrimers formed with a 10/1 starting ratio of G89A to wild-type subunits interacted with UTase/UR and ATase to a lesser extent than expected based on the number of wild-type subunits present but activated NRII slightly better than expected based on the number of wild-type subunits present. Thus, intersubunit interactions within the PII trimer can adversely affect the activity of wild-type subunits and may affect the interactions with the different receptors in a variable way. Finally, we formed heterotrimers containing delta47-53 and G89A mutant subunits. These heterotrimers were not uridylylated, did not interact with NRII, and interacted with the ATase only to the extent expected based on the number of G89A subunits present. Thus, the G89A subunits, which contain an intact T-loop region, were not "repaired" by inclusion in heterotrimers along with delta47-53 subunits.     

Escherichia coli PII signal transduction protein controlling nitrogen assimilation acts as a sensor of adenylate energy charge in vitro

PII signal transduction proteins are among the most widely distributed signaling proteins in nature, controlling nitrogen assimilation in organisms ranging from bacteria to higher plants. PII proteins integrate signals of cellular metabolic status and interact with and regulate receptors that are signal transduction enzymes or key metabolic enzymes. Prior work with Escherichia coli PII showed that all signal transduction functions of PII required ATP binding to PII and that ATP binding was synergistic with the binding of alpha-ketoglutarate to PII. Furthermore, alpha-ketoglutarate, a cellular signal of nitrogen and carbon status, was observed to strongly regulate PII functions. Here, we show that in reconstituted signal transduction systems, ADP had a dramatic effect on PII regulation of two E. coli PII receptors, ATase, and NRII (NtrB), and on PII uridylylation by the signal transducing UTase/UR. ADP acted antagonistically to alpha-ketoglutarate, that is, low adenylylate energy charge acted to diminish signaling of nitrogen limitation. By individually studying the interactions that occur in the reconstituted signal transduction systems, we observed that essentially all PII and PII-UMP interactions were influenced by ADP. Our experiments also suggest that under certain conditions, the three nucleotide binding sites of the PII trimer may be occupied by combinations of ATP and ADP. In the aggregate, our results show that PII proteins, in addition to serving as sensors of alpha-ketoglutarate, have the capacity to serve as direct sensors of the adenylylate energy charge.     

Crystal structures of the apo and ATP bound Mycobacterium tuberculosis nitrogen regulatory PII protein

PII constitutes a family of signal transduction proteins that act as nitrogen sensors in microorganisms and plants. Mycobacterium tuberculosis (Mtb) has a single homologue of PII whose precise role has as yet not been explored. We have solved the crystal structures of the Mtb PII protein in its apo and ATP bound forms to 1.4 and 2.4 Å resolutions, respectively. The protein forms a trimeric assembly in the crystal lattice and folds similarly to the other PII family proteins. The Mtb PII:ATP binary complex structure reveals three ATP molecules per trimer, each bound between the base of the T‐loop of one subunit and the C‐loop of the neighboring subunit. In contrast to the apo structure, at least one subunit of the binary complex structure contains a completely ordered T‐loop indicating that ATP binding plays a role in orienting this loop region towards target proteins like the ammonium transporter, AmtB. Arg38 of the T‐loop makes direct contact with the γ‐phosphate of the ATP molecule replacing the Mg²⁺ position seen in the Methanococcus jannaschii GlnK1 structure. The C‐loop of a neighboring subunit encloses the other side of the ATP molecule, placing the GlnK specific C‐terminal 3₁₀ helix in the vicinity. Homology modeling studies with the E. coli GlnK:AmtB complex reveal that Mtb PII could form a complex similar to the complex in E. coli. The structural conservation and operon organization suggests that the Mtb PII gene encodes for a GlnK protein and might play a key role in the nitrogen regulatory pathway.     

Coexistence of two structurally similar but functionally different PII proteins in Azospirillum brasilense.

The coexistence of two different PII, proteins in Azospirillum brasilense was established by comparing proteins synthesized by the wild-type strain and two null mutants of the characterized glnB gene (encoding PII) adjacent to glnA. Strains were grown under conditions of nitrogen limitation or nitrogen excess. The proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or isoelectric focusing gel electrophoresis and revealed either by [32P]phosphate or [3H]uracil labeling or by cross-reaction with an anti-A. brasilense PII-antiserum. After SDS-PAGE, a single band of 12.5 kDa revealed by the antiserum in all conditions tested was resolved by isoelectric focusing electrophoresis into two bands in the wild-type strain, one of which was absent in the glnB null mutant strains. The second PII protein, named Pz, was uridylylated under conditions of nitrogen limitation. The amino acid sequence deduced from the nucleotide sequence of the corresponding structural gene, called glnZ, is very similar to that of PII. Null mutants in glnB were impaired in regulation of nitrogen fixation and in their swarming properties but not in glutamine synthetase adenylylation. No glnZ mutant is yet available, but it is clear that PII and Pz are not functionally equivalent, since glnB null mutant strains exhibit phenotypic characters. The two proteins are probably involved in different regulatory steps of the nitrogen metabolism in A. brasilense.     

Uridylylation of the PII protein from Herbaspirillum seropedicae

The PII protein is apparently involved in the control of NifA activity in Herbaspirillum seropedicae. To evaluate the probable role of PII in signal transduction, uridylylation assays were conducted with purified H. seropedicae PII and Escherichia coli GlnD, or a cell-free extract of H. seropedicae as sources of uridylylating activity. The results showed that alpha-ketoglutarate and ATP stimulate uridylylation whereas glutamine inhibits uridylylation. Deuridylylation of PII-UMP was dependent on glutamine and inhibited by ATP and alpha-ketoglutarate. PII uridylylation and (or) deuridylylation in response to these effectors suggests that PII is a nitrogen level signal transducer in H. seropedicae.     

Heterotrimerization of PII-like signalling proteins: implications for PII-mediated signal transduction systems

PII-like signalling molecules are trimeric proteins composed of 12-13 kDa polypeptides encoded by the glnB gene family. Heterologous expression of a cyanobacterial glnB gene in Escherichia coli leads to an inactivation of E. coli's own PII signalling system. In the present work, we show that this effect is caused by the formation of functionally inactive heterotrimers between the cyanobacterial glnB gene product and the E. coli PII paralogues GlnB and GlnK. This led to the discovery that GlnK and GlnB of E. coli also form heterotrimers with each other. The influence of the oligomerization partner on the function of the single subunit was studied using heterotrimerization with the Synechococcus PII protein. Uridylylation of GlnB and GlnK was less efficient but still possible within these heterotrimers. In contrast, the ability of GlnB-UMP to stimulate the adenylyl-removing activity of GlnE (glutamine synthetase adenylyltransferase/removase) was almost completely abolished, confirming that rapid deadenylylation of glutamine synthetase upon nitrogen stepdown requires functional homotrimeric GlnB protein. Remarkably, however, rapid adenylylation of glutamine synthetase upon exposing nitrogen-starved cells to ammonium was shown to occur in the absence of a functional GlnB/GlnK signalling system as efficiently as in its presence.     

The PII protein in the cyanobacterium Synechococcus sp. strain PCC 7942 is modified by serine phosphorylation and signals the cellular N-status.

The glnB gene product (PII protein) from Synechococcus sp. has previously been identified among 32P-labeled proteins, and its modification state has been observed to depend on both the nitrogen source and the spectral light quality (N. F. Tsinoremas, A. M. Castets, M. A. Harrison, J. F. Allen, and N. Tandeau de Marsac, Proc. Natl. Acad. Sci. USA 88:4565-4569, 1991). As shown in this study, modification of the PII protein primarily responds to the N-status of the cell, and its light-dependent variations are are mediated through nitrate metabolism. Modification of the PII protein results in the appearance of three isomeric forms with increasing negative charge. Unlike its homolog counterparts characterized so far, PII in Synechococcus sp. is modified by phosphorylation on a serine residue, which represents a unique kind of protein modification in bacterial nitrogen signalling pathways.     

Interactions between the nitrogen signal transduction protein PII and N-acetyl glutamate kinase in organisms that perform oxygenic photosynthesis

PII, one of the most conserved signal transduction proteins, is believed to be a key player in the coordination of nitrogen assimilation and carbon metabolism in bacteria, archaea, and plants. However, the identity of PII receptors remains elusive, particularly in photosynthetic organisms. Here we used yeast two-hybrid approaches to identify new PII receptors and to explore the extent of conservation of PII signaling mechanisms between eubacteria and photosynthetic eukaryotes. Screening of Synechococcus sp. strain PCC 7942 libraries with PII as bait resulted in identification of N-acetyl glutamate kinase (NAGK), a key enzyme in the biosynthesis of arginine. The integrity of Ser49, a residue conserved in PII proteins from organisms that perform oxygenic photosynthesis, appears to be essential for NAGK binding. The effect of glnB mutations on NAGK activity is consistent with positive regulation of NAGK by PII. Phylogenetic and yeast two-hybrid analyses strongly suggest that there was conservation of the NAGK-PII regulatory interaction in the evolution of cyanobacteria and chloroplasts, providing insight into the function of eukaryotic PII-like proteins.     

Cascade control of Escherichia coli glutamine synthetase. Purification and properties of PII protein and nucleotide sequence of its structural gene

A procedure was developed to purify large quantities of PII protein from an Escherichia coli strain which contains a multicopy plasmid harboring the structural gene of PII (the glnB gene). Ultraviolet spectra of uridylylated and unuridylylated PII were obtained using the purified PII and empirical formulas to calculate the concentration of protein and the average number of uridylylated subunits per molecule were derived. A continuous fluorometric assay for the measurement of uridylylated PII (PIID) and adenylyltransferase (ATase) was also established. Rate measurements at various concentrations of PIID and at a fixed concentration of ATase showed that a tetrameric PIID molecule interacts with only one ATase molecule at a time. The complete nucleotide sequence of the glnB gene was determined and parts of the deduced amino acid sequence were confirmed by the results of amino acid sequence analysis of peptides. The PII subunit consists of 103 amino acids (Mr = 11,580). Two tyrosines reside at positions 46 and 51, where Tyr51 is the site of uridylylation. Nucleotide sequence analysis of the upstream region showed no obvious sites for the binding of RNA polymerase, indicating that the glnB gene is a part of an as yet unidentified operon.