The novel protein phosphatase PphA from Synechocystis PCC 6803 controls dephosphorylation of the signalling protein P II

In Escherichia coli, the ammonia channel AmtB and the P II signal transduction protein GlnK constitute an ammonium sensory system that effectively couples the intracellular nitrogen regulation system to external changes in ammonium availability. Binding of GlnK to AmtB apparently inactivates the channel, thereby controlling ammonium influx in response to the intracellular nitrogen status. We designed an N-terminally histidinetagged version of AmtB with a native C-terminal region in order to purify the AmtB-GlnK complex. Purification revealed a stable and direct interaction between AmtB and GlnK, thereby showing for the first time that stability of the complex does not require other proteins. The stoichiometry of the complex was determined by two independent approaches, both of which indicated a 1:1 ratio of AmtB to GlnK. We also showed by mass spectrometry that only the fully deuridylylated form of GlnK co-purifies with AmtB. The purified complex allowed in vitro studies of dissociation and association of AmtB and GlnK. The interaction of GlnK with AmtB is dependent on ATP and is also sensitive to 2-oxoglutarate. Our in vitro data suggest that in vivo association and dissociation of the complex might not only be dependent on the uridylylation status of GlnK but may also be influenced by intracellular pools of ATP and 2-oxoglutarate.Under nitrogen-limiting conditions, the uptake of ammonium into the cells of many organisms is mediated by a class of ubiquitous membrane proteins, designated Amt (ammonium transporter) proteins (1). They are present in bacteria, archaea, fungi, and plants, whereas in animals, from nematodes to humans, they are represented by the closely related Rh (Rhesus) proteins. The most studied Amt protein is Escherichia coli AmtB, which is a stable homotrimer in the cytoplasmic membrane and retains this structure when purified and reconstituted in two-dimensional crystals (2, 3). The first 22 residues of E. coli AmtB encode a signal peptide (1, 4) that is cleaved from the preprotein upon membrane insertion so that the mature protein has an N out and C in topology (1,5,6). The x-ray crystal structures of E. coli AmtB and Archaeoglobus fulgidus Amt-1 have been solved and are very similar (5-7). They are both trimers in which each subunit is formed by 11 transmembrane helices (TMH).2 The structure has a pseudo-2-fold symmetry with the 2-fold axis in the middle plane of the membrane, relating helices TMH1 to TMH5 and TMH6 to TMH10, with TMH11 across the lipid-accessible face of each monomer. In the E. coli AmtB structures, the last 20 amino acids, which are predicted to constitute a cytoplasmic region, are disordered. However, the equivalent region of A. fulgidus Amt-1 was resolved, and it folds into two short ␣-helices (7). The Amt structures revealed that transport occurs through a narrow mainly hydrophobic pore located at the center of each monomer. Computer simulations and structural and energetic data, together with in vivo and in vitro assays, indicate that ammonium is the substrate recognize...

Section: Discussionsupporting

confidence: 57%

In Vitro Analysis of the Escherichia coli AmtB-GlnK Complex Reveals a Stoichiometric Interaction and Sensitivity to ATP and 2-Oxoglutarate

Durand

Merrick

2006

“…Wild-type tPphA turns over the pT peptide (7.73 Ϯ 1.10 nmol/min/g) more rapidly than any of the other phosphoseryl-containing peptides. The preference for pT over the pS peptides is frequently found in PP2C-like phosphatases (19,23,24). Activity correlates with the length of the peptides as follows: toward longer peptides the activity was higher than toward smaller substrates; the three-residue peptide (G(pS)E) is apparently too short to be recognized as a substrate (Table 4).…”

Section: Resultsmentioning

confidence: 96%

“…After incubation at 30°C, the reactions were stopped by adding 2.5 l of 100 mM EDTA on ice. Subsequently, the phosphorylation state of P II was determined by nondenaturing PAGE and immunoblot analysis as described previously (19).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

A Third Metal Is Required for Catalytic Activity of the Signal-transducing Protein Phosphatase M tPphA

苏

Schlicker

et al. 2011

Self Cite

Serine/threonine phosphatases of the PPM 2 family are widely present in eukaryotes and prokaryotes and regulate key signaling pathways involved in cell proliferation, stress responses, or metabolic control (1). PPM phosphatases are metalloenzymes requiring Mg 2ϩ or Mn 2ϩ ions, which are coordinated by a universally conserved core of aspartate residues.The human PPM member PP2C␣ has been the defining representative of this family. As shown by structural analysis and site-directed mutagenesis studies, a binuclear metal center (with metals M1 and M2) activates a catalytic water molecule for nucleophilic attack of the phosphate group (2, 3). The importance of the M1-M2 core was also demonstrated for other PPM members, such as BA-Stp1, a bacterial PPM member from Bacillus anthracis (4). Recently, the structure of several bacterial members (tPphA from Thermosynechococcus elongatus (5), MtPstP from Mycobacterium tuberculosis (6), MspP from Mycobacterium smegmatis (7), STP from Streptococcus agalactiae (8), as well as the structure of Hab1 from Arabidopsis thaliana in co-crystal with its inhibitor, abscisic acid receptor Pyl2 (9)) has been solved. A third metal ion (M3) in proximity of the M1-M2 core could be revealed in several crystal forms of these proteins; however, the function of the third metal was controversially discussed. It was proposed to directly take part in catalysis in the case of SaSTP (8) or to have a regulatory role in the conformation of the flexible FLAP subdomain, which may be important for substrate recognition (10).The PPM tPphA from the thermophilic cyanobacterium T. elongatus is ideally suited to elucidate the role of M3. Because of its thermophilic origin, it has a robust structure, and the M3 coordination was almost invariant in various crystal forms ( Fig. 1) (5), whereas M3 coordination was variable in other PPM members (7,8). Furthermore, tPphA reacts readily with the artificial substrate pNPP, and moreover, its natural substrate, the phosphorylated P II signal transduction protein (P II -P), is available (11). In this study, mutant tPphA variants affected in M3 coordination were generated and analyzed with respect to enzymatic properties, metal binding, and three-dimensional structures, demonstrating a catalytic role of M3 in the dephosphorylation reaction. EXPERIMENTAL PROCEDURESDetailed protocols are available in the supplemental material.Cloning, Overexpression, and Purification of tPphA and Its Variants-The tPphA gene was cloned into the His tag vector pET15b (Novagen) according to standard procedures. Site-directed mutagenesis of tPphA was carried out with the QuickChange XL site-directed mutagenesis kit (Stratagene).Artificial genes for human PP2C fragment (residues 1-297) and the corresponding PP2C-variant D146A were synthesized and cloned into His tag pET15b vector by GENEART (Regensburg, Germany). Overexpression and purification of Histagged proteins were performed as described in the supplemental material.His tag-free tPphA variants D119A and D193A for crystallization were cons...

“…The ATP-binding sites were resolved by crystallographic analysis and were shown to be located in the lateral clefts between the subunits (6). Binding of ATP and 2-oxoglutarate to P II proteins mutually depends on each other (7)(8)(9), and the ␥-phosphate of ATP was shown to be crucial for this interaction (10,11). In addition to binding ligands, P II -like proteins may be subjected to covalent modification in response to changes in the nitrogen availability.…”

mentioning

confidence: 99%

Interaction of the Membrane-bound GlnK-AmtB Complex with the Master Regulator of Nitrogen Metabolism TnrA in Bacillus subtilis

Heinrich

Woyda

Brauburger

et al. 2006

Self Cite

P II proteins are widespread and highly conserved signal transduction proteins occurring in bacteria, Archaea, and plants and play pivotal roles in controlling nitrogen assimilatory metabolism. This study reports on biochemical properties of the P IIhomologue GlnK (originally termed NrgB) in Bacillus subtilis (BsGlnK). Like other P II proteins, the native BsGlnK protein has a trimeric structure and readily binds ATP in the absence of divalent cations, whereas 2-oxoglutarate is only weakly bound. In contrast to other P II -like proteins, Mg 2؉ severely affects its ATP-binding properties. BsGlnK forms a tight complex with the membrane-bound ammonium transporter AmtB (NrgA), from which it can be relieved by millimolar concentrations of ATP. Immunoprecipitation and co-localization experiments identified a novel interaction between the BsGlnK-AmtB complex and the major transcription factor of nitrogen metabolism, TnrA. In vitro in the absence of ATP, TnrA is completely tethered to membrane (AmtB)-bound GlnK, whereas in extracts from BsGlnK-or AmtB-deficient cells, TnrA is entirely soluble. The presence of 4 mM ATP leads to concomitant solubilization of BsGlnK and TnrA. This ATP-dependent membrane re-localization of TnrA by BsGlnK/AmtB may present a novel mechanism to control the global nitrogen-responsive transcription regulator TnrA in B. subtilis under certain physiological conditions.