Download STRUCTURE AND FUNCTION

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]