Molecular basis for the distinct divalent cation requirement in the uridylylation of the signal transduction proteins GlnJ and GlnB from Rhodospirillum rubrum
© Teixeira et al.; licensee BioMed Central Ltd. 2012
Received: 7 February 2012
Accepted: 20 June 2012
Published: 8 July 2012
PII proteins have a fundamental role in the control of nitrogen metabolism in bacteria, through interactions with different PII targets, controlled by metabolite binding and post-translational modification, uridylylation in most organisms. In the photosynthetic bacterium Rhodospirillum rubrum, the PII proteins GlnB and GlnJ were shown, in spite of their high degree of similarity, to have different requirements for post-translational uridylylation, with respect to the divalent cations, Mg2+ and Mn2+.
Given the importance of uridylylation in the functional interactions of PII proteins, we have hypothesized that the difference in the divalent cation requirement for the uridylylation is related to efficient binding of Mg/Mn-ATP to the PII proteins. We concluded that the amino acids at positions 42 and 85 in GlnJ and GlnB (in the vicinity of the ATP binding site) influence the divalent cation requirement for uridylylation catalyzed by GlnD.
Efficient binding of Mg/Mn-ATP to the PII proteins is required for uridylylation by GlnD. Our results show that by simply exchanging two amino acid residues, we could modulate the divalent cation requirement in the uridylylation of GlnJ and GlnB.
Considering that post-translational uridylylation of PII proteins modulates their signaling properties, a different requirement for divalent cations in the modification of GlnB and GlnJ adds an extra regulatory layer to the already intricate control of PII function.
KeywordsPII proteins Post-translational modification Uridylyltransferase
Members of the PII family of signal transduction proteins are fundamental molecular messengers involved in the regulation of nitrogen metabolism in bacteria, archaea and eukarya (plants) [1, 2]. These proteins exert their role at different levels: they regulate gene expression by modulating the activity of several transcription factors , they control the flux through an ammonium transport protein  and influence the activity of key metabolic enzymes, e.g. glutamine synthetase (GS) and nitrogenase [5, 6].
PII proteins are trimers of about 37 kDa, with each monomer containing a double βαβ ferredoxin fold. It has been previously shown that each trimer can bind up to three molecules of 2-oxoglutarate (2-OG) and ATP/ADP allowing the sensing of the carbon/nitrogen and energy status in the cell [7, 8]. In the different structures of PII proteins solved so far, one of the most striking characteristics is the existence of three surface exposed loops per monomer, the B, C and T-loops . The three nucleotide-binding sites (where ATP and ADP bind) are located in the inter-subunit clefts formed by the interaction of the B and C loops. The binding of ATP displays negative cooperativity (as does 2-OG binding), with ADP competing for the same binding site, as was shown for GlnB from Escherichia coli. Recent structures of Synechococcos elongatus GlnB and Azospirillum brasilense GlnZ have convincingly elucidated the 2-OG binding sites within PII proteins and established that this binding influences protein conformation, particularly of the T-loop region [9, 10]. Moreover, the structure of S. elongatus GlnB also provided an explanation for the negative cooperativity observed in the binding of 2-OG, considering that binding of the first 2-OG molecule generates unequal binding sites in the other two subunits .
In most proteobacteria, including the photosynthetic nitrogen-fixing bacterium Rhodospirillum rubrum, PII proteins are covalently modified by reversible uridylylation at tyrosine 51 in the T-loop, yielding 0–3 subunits modified with UMP per trimer. The uridylyltransferase and uridylylremoving activities are catalyzed by the bifunctional enzyme uridylyltransferase GlnD, with the reactions being regulated by the concentration of 2-oxoglutarate, through binding to the PII proteins . The two activities of R. rubrum GlnD occur at distinct active sites, with the N-terminal nucleotidyltransferase domain involved in PII uridylylation and the central HD domain responsible for PII-UMP deuridylylation .
In R. rubrum, three PII proteins have been identified and named GlnB, GlnJ and GlnK . However, only GlnB and GlnJ have been extensively studied and found to have both unique and overlapping functions in the regulation of gene transcription (two-component system NtrBC), ammonium transport (AmtB) and activity of metabolic enzymes GS and nitrogenase (by regulating the DRAT/DRAG system). While both proteins can regulate the activity of the adenylyltransferase GlnE (and thereby controling GS activity), GlnB specifically regulates NtrB and DRAT and GlnJ has a preferential role in the regulation of AmtB and possibly DRAG [5, 6, 13–15].
Even though GlnB and GlnJ share 68% sequence identity, the conditions for in vitro uridylylation by GlnD are different . In the uridylylation assays with purified R. rubrum GlnD and PII proteins, efficient uridylylation requires the presence of ATP, 2-OG and a divalent cation. However, the uridylylation of GlnJ only occurred when Mn2+ was present, while the uridylylation of GlnB occurred with either Mg2+ or Mn2+. Although the structure of neither of the R. rubrum PII proteins has been determined, it is possible that their T-loop assumes different conformations, by analogy with the recent structural data from PII proteins from A. brasilense and S. elongatus[9, 10]. Considering these probably different conformations, it can be hypothesized that the correct conformation of the T-loop in GlnJ required for the interaction with GlnD is only achieved in the presence of Mn2+ (or Mn-ATP).
Considering that these differences in the divalent cation required to promote uridylylation of the PII proteins might be of functional significance, we have aimed at elucidating the molecular basis for this difference.
Results and discussion
It was previously shown that the in vitro uridylylation of GlnJ catalyzed by purified GlnD requires the presence of Mn2+ ions, in addition to ATP and 2-OG . Moreover, this functional connection between GlnJ and Mn2+ is supported by additional studies. We have shown that dissociation of the complex formed between GlnJ and the membrane embedded ammonium transport protein AmtB1 is favored by 2-OG and ATP but only in the presence of Mn2+. Also, in the same study it was observed that the uridylylation of endogenous R. rubrum GlnJ recovered from the membrane fraction was only possible in the presence of Mn2+. In contrast to GlnJ, GlnB was efficiently uridylylated in the presence of either Mg2+ or Mn2+.
Although GlnD itself is known to bind both Mg2+ and Mn2+, the fact that uridylylation of GlnB occurs with either of the divalent cations present lead us to hypothesize that the reason for the different response to divalent cations in the uridylylation of GlnB and GlnJ is related to the PII protein and not to GlnD itself. Based on this premise we assumed that exchanging specific amino acid residues in GlnJ to the ones in GlnB might enable GlnJ to also be modified in the presence of Mg2+ as the only cation present.
The deuridylylation of both GlnB-UMP and GlnJ-UMP (also catalyzed by GlnD) was shown previously to require Mn2+, but Mg2+ did not support this activity in the R. rubrum enzyme , in contrast to E. coli GlnD .
Although not all the residues selected are located in regions of the PII protein that have previously been shown to be involved in metabolite binding, we decided to analyze amino acids occurring in areas of high conservation as, due to the considerable flexibility of the PII structure, they may also play a role in this response to divalent cations. An example of this high flexibility comes from the recent structure of S. elongatus GlnB, where the very C-terminal portion of the protein displays a large conformational change upon binding of the ligands to the T-loop region .
Uridylylation of GlnJ variants in the presence of Mn2+ and Mg2+
Using purified GlnD and GlnJ variants we analysed the uridylylation profile in the conditions that were previously determined  and described in the Materials and methods, with either Mg2+ or Mn2+ present in the assays.
GlnB variants H42Q and R85K show reduced uridylylation in the presence of Mg2+
Considering the influence of the residues at positions 42 and 85 we hypothesized that exchanging these residues in GlnB for the corresponding residues in GlnJ could affect Mg2+-dependent uridylylation. That was indeed the case, as shown in Figure 2B. The GlnBH42Q, GlnBR85K and GlnBH42QR85K variants show normal uridylylation in the presence of Mn2+, but that it is clearly reduced in the presence of Mg2+, when compared to wt GlnB.
MnATP (but not MgATP) induces a conformational change in GlnJ
The GlnJ and GlnB variants retain functionality
To analyze the functionality of all variants constructed, we tested the ability to activate GS adenylylation by GlnE, resulting in reduced GS activity. As shown in Figure 6B, all variants tested were able to activate the adenylylation activity of GlnE.
The two PII proteins GlnJ and GlnB from R. rubrum show different requirements in terms of divalent cations (Mg2+/Mn2+) for efficient uridylylation by GlnD. Specifically, the uridylylation of GlnJ requires the presence of Mn2+, with Mg2+ not being able to support this modification. Most likely this is due to the fact that only Mn2+ (or MnATP) is able to bind and induce a conformational change in GlnJ, as demonstrated here with CD spectroscopy.
We have shown that it is possible to influence the divalent cation response in the uridylylation of the PII proteins, catalyzed by GlnD, by simply exchanging two amino acid residues in the PII proteins (at positions 42 and 85, located in the T and B loops respectively). Although the substitutions constructed (Q to H and K to R) do not represent dramatic changes in the amino acid properties, these changes have a clear effect on the role of Mg2+ (the Mn2+ dependent uridylylation is retained in all variants studied). Moreover, we have also confirmed that these variants retain functionality in the GlnE-activation assay, suggesting that these substitutions do not greatly perturb the overall structure.
It is presently unclear from the structural point of view, which conformations of either GlnJ or GlnB (particularly of the T-loop) are interacting with GlnD and how these conformations are affected by the binding of different divalent cations (Mg2+ and Mn2+). Additionally, a direct translation of the present results obtained with purified proteins to an in vivo physiological situation is not linear as there is presently no information concerning the concentrations of either Mg2+ or Mn2+ in R. rubrum, and if these concentrations vary in response to the nitrogen status (transitions that require changes in the uridylylation of the PII proteins). Nevertheless, it is certainly possible that Mn2+ has an important role, as we found this divalent cation to be always required in all reactions involving GlnJ. In addition to the Mn2+ requirement for in vitro uridylylation of GlnJ by GlnD, we have also demonstrated that the dissociation of the GlnJ-AmtB1 complex only occurs with Mn2+, ATP and 2-oxoglutarate, and that Mg2+ can not substitute for Mn2+[11, 13]. In addition, Mn2+ ions are essential for the activity of DRAG (the activating enzyme for nitrogenase) [14, 17], a protein that has been suggested to interact with GlnJ [14, 15]. Considering that GlnJ is only expressed under nitrogen fixing conditions [6, 15], all factors that affect uridylylation of GlnJ can be of importance in the regulation of the DRAT/DRAG system and ultimately of nitrogenase.
In summary, considering that GlnJ and GlnB are remarkably similar yet retaining functional specificity, it is possible that differences in divalent cation binding and consequently in the uridylylation status of the proteins can result in different target interaction and ultimately in different physiological roles. This study adds on to the understanding of the complexity of the PII signaling system in bacteria.
Bacterial strains and plasmids
Bacterial strains and plasmids used in the present study
Strain or plasmid
Reference or source
BL21 (DE3) pLysS
Host for expression of PII proteins, Cmr
BL21 Star (DE3)
Host for expression of GlnE
ΔglnD; host for expression of GlnD, Tcr
pET101 derivative containing glnE, Apr
pGEX6P-3 derivative containing glnD, Apr
pET15b derivative containing glnB, Apr
pET15b derivative containing glnJ, Apr
pETGlnJ derivative encoding GlnJR17K, Apr
pETGlnJ derivative encoding GlnJQ42H, Apr
pETGlnJ derivative encoding GlnJN54D, Apr
pETGlnJ derivative encoding GlnJK85R, Apr
pETGlnJ derivative encoding GlnJV100I, Apr
pETGlnJ derivative encoding GlnJE109G, Apr
pETGlnJ derivative encoding GlnJQ42HK85R, Apr
pMJET derivative encoding GlnBH42Q, Apr
pMJET derivative encoding GlnBR85K, Apr
pMJET derivative encoding GlnBH42QR85K, Apr
All GlnJ and GlnB variants were generated by standard PCR-mediated site-directed mutagenesis using the QuikChange kit (Stratagene) and according to the manufacturer’s instruction. The templates used were pETGlnJ  and pMJET .
Purification of R. rubrum PII proteins
All constructs used to express PII proteins were pET15b derivatives, generating proteins with an N-terminal poly-histidine tag. All PII proteins were purified using HiTrap 1 ml columns (GE Healthcare) according to .
Purification of R. rubrum glutamine synthetase, GlnE and GlnD proteins
GlnD was purified as a GST fusion-protein according to . Glutamine synthetase was purified from wild type R. rubrum and GlnE was purified with a C-terminal poly-histidine tag as previously described .
Each reaction (final volume 50 μl) contained 50 mM Tris–HCl pH 7.6, 3.5 μM PII protein (GlnJ, GlnB or a variant), 0.2 μM GlnD, 100 mM KCl, 1 mM ATP, 1 mM dithiothreitol, 0.5 mM UTP and either 3 mM MnCl2 and 60 μM 2-OG or 25 mM MgCl2 and 250 μM 2-OG (in the control reactions the divalent cations were omitted and 2-OG was at 250 μM). After 30 min (or as indicated) the reaction was stopped by the addition of 5X native loading buffer (125 mM Tris–HCl pH 6.8, 50 mM EDTA, 50% glycerol, 5% sorbitol) and a 20 μl sample was loaded onto a 12.5% native PAGE prepared according to . After electrophoresis the gels were stained with Coomassie brilliant blue R250.
Circular dichroism spectroscopy
Far-UV CD measurements were performed on an Applied photophysics chirascan CD spectropolarimeter using a 50 μm quartz cuvette. Wavelengths in the range 190–250 nm were scanned using 0.5 nm step resolution and 100 nm/min scan speed. The spectra recorded were collected and averaged over 1–6 scans. Measurements were recorded with the temperature kept constant at 24°C using a quantum northwest TC125 temperature controller.
This study was supported by grants from the Swedish Research Council to SN. The authors are also indebted to Dr. Jesper Lind and Dr. Lena Mäler (Stockholm University) for their help with CD measurements, Dr. Tiago Selão (presently Nanyang Technological University, Singapore) for mass spectrometry analysis and Dr. Ekaterina Morgunova (Karolinska Institute) for the generation of a structural model of GlnJ.
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