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Introduction Nitrogen is an essential component of all living organisms. The mechanism by which levels of ammonia, the biologically usable form of nitrogen, are regulated in the cell remains largely unknown. However, the ammonia channel protein Amt1, and the signaling protein GlnK1 are believed to play a role in the process of ammonia uptake. By determining the structure of GlnK1 protein bound to different effectors, Yildiz et all (2007), are able to learn more about the interactions and mechanism for the association of these proteins and how they control ammonia uptake. This finding contributes to understanding the role of GlnK1 in nitrogen uptake and metabolism Nitrogen in Biology Converting N2 into a Biologically Useful Form. Nitrogen is the third most abundant element on earth. This element exists in different forms, and the natural conversion of N2 into its many forms is described by the Nitrogen Cycle (Figure 1). Microscopic viruses, plants and animals all require nitrogen as an essential component of life. Over 70% of the earth’s atmosphere is Nitrogen, O2 and CO2 comprise the rest. Cells and living organisms can use nitrogen, only after it is reduced to its primary biologically usable form, ammonia. Reduction of N2 is carried out by a special group of organisms, living in the soil, called nitrogen fixing bacteria which convert the atmospheric nitrogen into ammonia. These bacteria are essential to the persistence of life on this planet being the only organisms able to convert atmospheric N2 into a biologically usable form. Biological Consumption of Nitrogen Compounds. After nitrogen is reduced to ammonia, it can be used by organisms for conversion to useful biological molecules. Ammonia can be oxidized into nitrite and ultimately nitrate by soil bacteria, which derive their energy from these conversion processes called nitrification. Many plants and bacteria can absorb soil nitrate and convert it back into ammonia using a class of enzymes called reductases. Plants use ammonia as a source of nitrogen to make essential components of life including amino acids and nucleic acids. Animals then ingest plants and use these amino acids as building blocks for protein, and other compounds. Decomposing Nitrogen Compounds back to Atmospheric Nitrogen When an organism dies bacteria and fungi decompose microbial protein, returning ammonia back to the soil. This ammonia can either be oxidized back into nitrate by soil bacteria, or to N2 by other denitrifying bacteria. Thus the Nitrogen Cycle maintains a balance of levels of global nitrogen, nitrate and ammonia. Amt Family of Membrane Proteins. Biosynthesis of essential molecules requires reduced nitrogen. In some eukaryotes this nitrogen is obtained as amino acids or nitrates, however in most prokaryotes the source of biological nitrogen is ammonia or ammonium. The diffusion of ammonia across cellular plasma membranes is unregulated and under acidic conditions tends to be slow, and can prohibit the cell from maintaining homeostasis. Therefore, organisms have developed intricate systems to control the uptake of ammonia using a family of membrane proteins, Amt, found in bacteria, archae, fungi, and plants. High-resolution crystal structures have shown that Amt proteins are trimers with 11 membranespanning helices in each monomer is a subunit that forms a bundle around a hydrophobic channel (Khademi et al, 2004). This structure suggests that Amt proteins act as channels across which uptake of ammonia by a cell (Figure 2). The Amt channel, which does not actively transport ammonium, cooperates with other ammonia transporters to regulate the amount of ammonia in the cell. Each monomer of Amt has an ammonium-binding site on the extracellular surface which deprotonates the ammonium ion to ammonia before it passes through the channel. Once inside the cell the ammonia can be protonated to ammonium. Two important features of Amt channels is that they do not require energy to transport ammonia across the plasma membrane, as many active transport channels do, and this channel can be easily regulated by PII proteins. PII Proteins. Nitrogen metabolism in archae and bacteria is regulated by soluble PII proteins. PII proteins have recently been discovered in eukaryotes, and the structure has been determined in plants, as described in Wendy Ingram’s student project Plant PII Protein Structure: Insights into Eukaryotic Nitrogen Metabolism. These highly conserved proteins are among the most ancient, and versatile signaling proteins. PII proteins are small trimeric signal transduction proteins that regulate gene transcription, modulate activity of regulatory proteins, and control the catalytic activity of nitrogen metabolism. Two PII proteins in E.coli are GlnB and GlnK and they can bind ATP, 2-ketoglutarate, and magnesium (Van Heeswijk et al, 1996). A great deal is still not known about these specific proteins but they form 1:1 complexes with Amt channel proteins. This Amt/GlnK complex tends to dissociate in the presence of ATP, magnesium chloride, and 2ketoglutarate. By studying the structure of GlnK1, Yildiz et al (2007) gain an understanding of the Amt/GlnK complex and its function. Discovery of an Amt/GlnK Complex Formation of the GlnK1/Amt1 Complex. Purified recombinant Amt1 and GlnK1, cloned in E.Coli, were seperated by size-exclusion chromatography (Figure 3). Each protein eluted as a single distinct peak. However when both GnlK1 and Amt1 were mixed and incubated together before chromatography, a new chromatographic peak appeared that represents Amt1/GlnK1 complex, along with a decrease in size of the individual Amt and GlnK1 protein peaks. Further analysis by SDS-PAGE, and electron microscopy, verified that GlnK1 and Amt1 coelute, confirming the existence of an Amt1/GlnK1 complex (Yildiz 2007). Dissociation of the GlnK1/Amt1 Complex. To understand the molecular interactions between GlnK1 and Amt1 it is necessary to examine different factors that affect complex formation. Two compounds hypothesized to inhibit complex formation are adenosine triphosphate (ATP) and magnesium (Mg). The complexed GlnK1 and Amt proteins were incubated with magnesium and ATP and the proteins were analyzed again using size-exclusion chromatography and SDSPAGE. The addition of these two possible effectors caused a 95% decrease in the formation of Amt1/GlnK1 complex. ATP is Required For Dissociation. To test the hypothesis that ATP is necessary for the dissociation of the Amt1/GlnK1 complex GlnK1 was immobilized and incubated with Amt1 and a range of different nucleotides including ADP, AMP, and GTP as well as ATP. AMP and GTP had no affect on the association of the two proteins, whereas ADP had only a slight affect on the Amt/GlnK1 complex. Since only ATP caused dissociation of the complex it is clear that ATP specifically, and not just any nucleotide, is necessary to inhibit complex formation. 2-Ketoglutarate also Affects Dissociation. 2-ketoglutarate (2-KG) is important in the signaling pathways regulated by PII proteins. This is also true for GlnK1 (Kamberov et al, 1995). GlnK1 was again immobilized and incubated, this time in the presence of 2-KG. The protein complex were analyzed by using size exclusion chromatography (Figure 4). 2-KG alone had no effect on the association of the two proteins. However, 2-KG, ATP and Mg together completely prevented complex formation. Visualizing the structure of GlnK1 would be helpful in further understanding and studying the interactions between the two proteins and these ligands. GlnK Structure To understand the mechanisms and interactions which govern the binding of GlnK1 to Amt, it is necessary to visualize the protein structure. GlnK1 was crystallized under several conditions: GlnK1 with No Effectors GlnK1 with Mg-ATP GlnK1 with Mg-ATP and 2-KG Analysis of GlnK1 and Amt structures under these different conditions suggested how regulation of ammonia uptake by GlnK1 occurs. GlnK1 with No Effectors. Four trimers of the GlnK1 protein formed a cluster when crystallized with no effectors bound (Figure 5). Each trimer contained three T-Loops which differed in conformation. Six out the 12 T-loops in the four trimers were disordered, the remaining six were well-defined and in the extended conformation. The overall structures of the T-loops varied greatly indicating a high degree of flexibility of the loop. Although no nucleotides were presented during crystallization, the AMP and ADP present in the binding sites had been purified with the ptoreins isolated from the cells. An important conclusion from the crystal structure of GlnK1 relates to the relative charge of the protein at neutral pH. An overall positive charge found on the interface was proposed to interact with Amt1. Clusters of several positively charged arginines were found on the tips of the extended T-loops. The conformational flexibility of the T-loop and its role in binding to Amt1 were further elucidated in the structure of the MgATP complex bound to GlnK1. GlnK1 with Mg-ATP. Crystals of Glnk1 with both ATP and Mg bound revealed some important clues to the function of the T-loops (Figure 6). The structure has a trimer as a unit cell, instead of the complex four unit crystals characterized with substrate-free Glnk1. The Tloops in the trimer occurred in a compact conformation and not in the extended, flexible state seen in the substrate-free Glnk1 structure. All three binding sites within the trimer contained both Mg and ATP. The Mg coordinates with three water molecules which in turn form hydrogen bridges with several residues within the T-loop. Numerous other interactions between the ATPMg complex and the T-loop of the protein, stabilize its compact conformation. These interactions also contribute to the dissipation of positive charge on the tips of the T-loops and also cause a change in the overall charge of GlnK1 from positive to slightly negative. The MgATP complex inhibits binding of GlnK1 to Amt1, however 2-ketoglutarate can play a role in this process. Glnk1 with Mg-ATP and 2-KG. 2-Ketoglutarate (2-KG) had a stabilizing effect on the compact conformation of GlnK1 by forming favorable interactions with the protein and the MgATP complex (Figure 7). When 2-KG was added to the crystallization buffer, the protein was found to contain ATP in all three binding sites but only one of the three sites contained Mg. Only the T-loop with active site bound Mg-ATP complex was in the compact form. The other two loops were similar to the form found on the GlnK1 structure with no ATP bound. For the Tloop that had Mg-ATP bound, one molecule of 2-KG was bound near the compact loop. A network of hydrogen bonds helps to stabilize the binding of 2-KG to the protein, with the only significant difference between the 2-KG bound and unbound form being a slightly more compact conformation of several amino acid residues. The 2-KG also causes an increase in the overall negative charge of GlnK1 and completely makes up for the positive charges on the tip of the Tloop. Techniques Size-exclusion chromatography (SEC). In this method, proteins in solution are separated on the basis of size. A solution is placed on top of a gel column and allowed to move down the column. A typical gel is composed of small polymeric beads with pores of varying size. As the solution flows through the gel column smaller proteins can enter the pores of the beads and are retarded while the larger proteins do not enter the pores and thus are eluted faster. SDS-PAGE. Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis is another method used to separate proteins depending on their size. SDS is a detergent that can coat proteins evenly, resulting in a uniform negative charge to mass ratio. Proteins in an SDS solution are placed on a polyacrylamide gel which is equilibrated with a buffer. A voltage is applied across the gel and the negatively charged SDS-coated proteins migrate through the gel. Larger proteins migrate more slowly in the gel then do smaller proteins. The advantage of this method is its high resolving power to separate proteins that differ only slightly in mass. Electron microscopy. This method uses a beam of electrons that is focused on a target and which produces an image of it that cane be magnified approximately two million times greater than an ordinary light microscope. X-ray Crystallography. An analytical technique that uses x-ray diffraction by a protein crystal to permit deduction of the 3D structure of a protein. A protein is crystal is bombarded with xrays, producing an x-ray diffraction pattern that can be translated into an electron density map and eventually into a model showing the 3-dimentional arrangement of atoms in the protein crystal. Amt/GlnK Formation of the Amt/GlnK Complex. GlnK and PII proteins regulate Amt-dependent ammonia uptake in prokaryotes. From the results of the incubation and size exclusion chromatography, GlnK and Amt bind in a one-to-one ratio. GlnK and Amt form a complex in the absence of Mg-ATP and 2-KG but the complex does not form when these effectors are present. Electron microscope images of the GlnK1 trimer show that it binds to the central region of cytoplasmic side of the membrane spanning ammonia channel. This, result is significant because the channel through which ammonia passes into the cell exists within this region of the Amt1 membrane protein. T-loop Dynamic Conformation. The T-loop was found in two different forms. One form was compact and rigid, and the other had an extended flexible formation. In the absence of Mg-ATP bound to GlnK1, the T-loop assumes an extended shape, regardless of the presence of other nucleotide phosphates in the binding site. Although in this conformation the T-loop is relatively disordered, it has a tendency to be oriented in a way that could maximize the binding ability of GlnK1 to other proteins, such as Amt. T-loop Compact Conformation. When Mg-ATP is present in the binding pocket of the GlnK1 protein, the T-loop occurs in a compact form, folded closely to the body of the protein. Many interactions between the binding site, Mg-ATP, and the T-loop help to stabilize the compact form of the protein. Without these bound effectors the T-loop has conformational flexibility in the extended state. 2-KG can only bind to GlnK1 when Mg-ATP is already in the GlnK1 binding site. 2-KG contributes to the stability of the compact T-loop structure through interactions with the protein. Keeping Ammonia Out. From the crystal structures and known features of the Amt and GlnK protein families, a mechanism was developed to explain regulation of ammonia uptake by a prokaryotic cell. One interesting aspect of the interactions between the two proteins is that an overall negative charge is found on the cytoplasmic side of Amt protein concentrated near its ammonia channel, and effector-free GlnK1 has an overall positive charge. Positively charged extended T-loops of GlnK1 could interact with the negatively charged regions, sealing the channels, and effectively inhibiting ammonia uptake. These types of interactions would at low concentrations of Mg-ATP and 2-KG within the cells, indicating ammonia is not needed for biosynthesis. Another reason to exclude ammonia from the cell is when ATP levels are low, and ammonia enters it tends to become protonated, dissipating the proton gradient required for ATP synthesis. This result could be especially harmful to a cell that already has insufficient ATP for its metabolic needs. Letting Ammonia Flow. When ATP levels are high, the Mg-ATP complex can bind to the GlnK1 trimer and the T-loops assume the compact form. When in the compact form GlnK1 is unable to bind Amt1. When 2-KG is bound, a pronounced negative charge occurs at the interacting surface of GlnK1. The combination of these two effects causes the GlnK1 to dissociate from the Amt and electrostatic repulsion occurs between the similarly negative charged proteins. Dissociation of GlnK1 unblocks the Amt ammonia channels and ammonia is free to flow into the cell. When cellular levels of 2-KG and ATP are high, this indicates a sufficient amount of carbon compounds and energy to initiate nitrogen requiring biosynthesis. These conditions induce dissocation of the Amt/Glnk1 complex and allow the necessary ammonia to enter the cell to be incorporated into proteins and nucleic acids. Summary and Future Directions The model for binding of the GlnK1 and Amt proteins presented and supported by the experimental data of Yildiz et al (2007) suggests that GlnK1 is an important regulator of nitrogen uptake by prokaryotic cells. GlnK1 is regulated by biological conditions within the cell which causes it to bind or dissociate from the Amt transmembrane ammonia channel. This model leads to the idea that PII proteins such as GlnK1 are more widely involved in other signaling pathways in nitrogen metabolism and regulation. References Yildiz, O., Kalthoff, C., Raunser, S., Kuhlbrandt, W., (2007) Structure of GlnK1 with bound effectors indicates regulatory mechanism for ammonia uptake. The EMBO Journal. 26, 589-599. [Abstract] Saparov, S.M., Liu, K., Agre, P., Pohl, P., (2007) Fast and Selective Ammonia Transport by Aquaporin-8*. The Journal of Biological Chemistry. Vol. 282, NO. 8, pp.5296-5301. [Abstract] Khademi, S., O’Connell, III. J., Robles-Colmenares, Y., Miercke, L.J., Stroud, R.M., (2004) Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 A. Science. 305: 1587-1594. [Abstract] Kamberov, E.S., Atkinson, M.R., Ninfa, A.J., (1995) The Escherichia coli PII signal transuction protein is activated upon binding 2-ketoglutarate and ATP. The Journal of Biological Chemistry. 270: 17797-17807. [Abstract] Van Heeswijk, W.C., Hoving, S., Molenaar, D., Stegeman, B., Kahn, D., Westerhoff, H.V., (1996) An alternative PII protein in the regulation of glutamine synthetase in Escherichia coli. Molecular Microbiology 21: 133-146. [Abstract]