Download STRUCTURE AND FUNCTION

Complex Formation
Amt1 and GlnK1 Complex
Before analyzing the proteins, their respective structures and the interactions that occur between
them it is necessary to first isolate and purify the proteins. By cloning Amt1 and GlnK1 from
Methanococcus jannaschii, the resulting proteins are more stable than the forms expressed in E.
coli, due to the fact that M. jannaschii contain two sets of genes for Amt and PII like proteins.
The genes which encode for GlnK1 and Amt1 were removed from the M. jannaschii and
transformed into the E. coli plasmid and allowed to replicate and translate within the host. The
similarity of amino acid identity of the proteins expressed in each organism is comparable
enough that the use of the M. jannaschii gene is justifiable and feasible with 53% of the sequence
being identical (Yildiz).
Following the expression and purification of the target proteins the products were analyzed using
size-exclusion chromatography. When analyzed separately the two proteins each expressed a
single distinct peak, however when the two were allowed to incubate together for a period time
before application to the gel a new peak appeared, along with a decrease in size of the individual
peaks. This phenomenon is explained by the formation of an Amt1/GlnK1 complex, with the
new peak representing the complex, and the decrease in size of the individual peaks supporting
this claim. Further analysis of the new peak’s fraction using SDS-PAGE, and electron
microscopy, verifies that GlnK1 and Amt1 elute at the same time confirming the existence of an
Amt1/GlnK1 complex, as expected in general with Amt and PII proteins (Yildiz).
Preventing Complex Formation
To further understand the interactions between GlnK1 and Amt1 it is necessary to examine
different factors that effect complex formation. Two known effectors hypothesized to inhibit
complex formation are adenosine triphosphate(ATP) and magnesium(Mg), so to further study
this interaction the group incubated the complexed proteins with magnesium and ATP and the
products were run out again using size-exclusion chromatography, and SDS-Page. It was found
that the addition of these two possible effectors caused a 95% decrease in the Amt1/GlnK1
complex.
To test the hypothesis that ATP is necessary for this process to occur and not just any nucleotide,
further tests were done in which the GlnK1 was immobilized and incubated with Amt1 and a
range of different nucleotides including ADP, AMP, and GTP as well as ATP. It was found that
the AMP and GTP had no effect on the binding of the two proteins, whereas ADP had only a
slight effect. The only nucleotide that had a sufficient effect on the dissociation of the complex
was ATP and therefore it was concluded that ATP and not just any nucleotide is important to the
inhibition of complex formation.
It has been previously noted that the addition of 2-ketoglutarate(2-KG) is important in the
binding of the signaling pathways of PII proteins when involved with ATP, this was also found to
be true in the case of GlnK1 (Kamberov et al, 1995). This was tested by using the same methods
of incubating and analyzing the protein complex using size exclusion chromatography. It was
found that the addition of 2-KG alone had no effect on binding however, the addition of 2-KG
with ATP and Mg caused complex formation to cease completely. So to further study these
interactions and to determine what exactly causes them to occur visualizing the structure is
required.
EXPERIMENTS
Size-exclusion chromatography(SEC) – is a chromatographic method in which particles of
different size are separated from a solution. A solution is placed on a gel in a column and
allowed to move down the column. The gel is composed of small polymeric beads with pores of
varying size, so as the solution flows down the gel smaller particles tend to get stuck within the
beads whereas the larger particles can not get into the beads and thus flow faster. This method is
used to separate particles exclusively by size.
SDS-PAGE – Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis is a method used to
separate particles depending on their size. Generally in this process proteins are coated with
SDS at an even charge to mass ratio, and are placed on a polyacrylamide gel. The gel is then
placed in a buffer and an electric current is run through the get causing the negative charges to
migrate down the gel. The larger the particle, the more slowly it will move down the gel and
visa versa. The advantage of this method is the ability to differentiate particles strictly on mass.
Electron microscopy- Involves the use of an electron microscope, which uses electrons to create
an image of the target with magnification of approximately two million times greater than an
ordinary light microscope.
X-ray Crystallography – Is an analytical technique which uses diffraction to determine the
crystal structure of a given protein. The protein is crystallized and then bombarded with x-rays
which produce a diffraction pattern which can be translated into an electron density map and
eventually into an atomic model of the protein.
Structure
To completely understand the mechanisms and interactions which regulate the binding of GlnK1
to Amt, it is necessary to visualize the actual protein. GlnK1 was crystallized without any
effector molecules present, in complex with Mg-ATP, and with both Mg-ATP and 2-KG. The
interactions and structural changes seen in the protein allowed for the hypotheses on the
regulatory mechanism of Glnk1.
Structure of Glnk1
The GlnK1 crystals were characterized as trimers, as predicted by electron microscopy. The
trimers formed into a cluster of four with each trimer differing from the others only in the T-loop
conformation and the content of the nucleotide binding site. Six out the 12 T-loops in the four
trimers were disordered, and the remaining six were well-defined, and in the extended
conformation. The crystallized structures of the six well-defined T-loops differed significantly
indicating a high degree of flexibility of the loop. Despite the fact that no nucleotides were
added to the buffer, four of the 12 nucleotide binding sites contained an AMP or ADP as a result
of cellular translation. An important aspect determined from the crystallized structure of the
GlnK1 was the relative charge of the molecule at neutral pH. An overall positive charge was
found on the interface proposed to interact with Amt 1 and also clusters of positive charge were
found on the tips of the extended T-loops at several arginine residues. The dynamics of the Tloop and its importance to binding were further elucidated in the structure of Glnk1 complexed
with Mg-ATP.
Glnk1 with Mg-ATP
Glnk1 bound to both ATP and Mg was also crystallized and some important aspects of its
function were elucidated from the structure. The structure was again found as a trimer, however
this time they were able to crystallize individual molecules of the protein instead of the four unit
crystals seen with the substrate-free Glnk1. Also the T-loops in the trimer appeared in a compact
conformation as opposed to the extended, flexible state in the substrate-free Glnk1. All three
binding sites within the trimer contained a Mg and an ATP molecule. The Mg coordinates tree
water molecules in the binding site which in turn form hydrogen bridges with the carbonyl
groups of several residues within the T-loop. Numerous other interactions take place between
the ATP-Mg complex and the T-loop of the protein which help to stabilize the compact
conformation of the loop. These interactions contribute to the dissipation of positive charge on
the tips of the T-loops and also cause a change in the overall charge of the protein from positive
to slightly negative. The Mg-ATP complex is able to inhibit the binding of GlnK1 to Amt1
however, it seems as if 2-ketoglutarate aids in this process.
Glnk1 with Mg-ATP and 2-KG
2-KG was found to have a stabilizing effect on the compact conformation by forming favorable
interactions with the protein and the Mg-ATP complex. After an addition of 2-KG to the
crystallization buffer it was found that the protein contained ATP in all three binding sites but
only one of the three complexed Mg. In this case it was seen that only the T-loop with the MgATP complex bound within the active site of its subunit was in the compact form. The other two
loops were in the free form, as was found in the ATP free form. This suggests that although
ATP is required for conformational change it is not sufficient to bring about a change
independently. For the T-loop which was interacting with Mg-ATP, one molecule of 2-KG was
bound near the compact loop. This means that for 2-KG to bind GlnK1 the protein must first be
complexed to Mg-ATP which puts the T-loop in the compact conformation. A network of
hydrogen bonds help to stabilize the binding of 2-KG to the protein, with the only significant
difference between the 2-KG bound form and the Mg-ATP bound form being a slightly more
compact formation 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 T-loop.
Note: Will also include detailed structural analysis in a pop-up window, slide form with series of
pictures and explanations.
Amt/GlnK
Formation of the Amt/GlnK Complex
It has been found that in general that GlnK and PII proteins regulate Amt-dependent ammonia
uptake in prokaryotes. From the results it has also been seen that the GlnK and Amt bind in a
one-to-one ratio of two trimers. It was also found that GlnK and Amt bound in the absence of
Mg-ATP and 2-KG but the two proteins do not form when in the presence of these effectors.
Essentially the binding of the effectors to GlnK inhibits or causes the dissociation of the
Amt/GlnK complex. From density measurements of the electron microscopy experiments the
GlnK1 trimer was found to bind to the middle of the cytoplasmic side of the membrane spanning
Amt1 trimer. This is significant because the channel through which the ammonia passes into the
cell exists within that same region of the Amt1 membrane protein.
T-loop Conformations
In the experiments the T-loop was found in two different forms, the compact rigid formation, and
the extended flexible formation. When no Mg-ATP is bound the T-loop is found in the extended
shape regardless of what other nucleotides are present in the binding site. Although the T-loop
for this conformation was found to be relatively disordered, it was found to have a tendency
towards a certain orientation which is predicted to maximize the binding ability of the protein to
other proteins, such as Amt.
When Mg-ATP was found in the binding pocket of the protein the T-loop was found in the
compact form folded closely to the body of the GlnK1 protein. It was found that this compact
formation would not occur simply in the presence of Mg, ATP or 2-KG alone but when the three
compounds were found in and near the binding site of the protein the T-loop was found in the
compact state. Many interactions occur between the binding site, Mg-ATP and the T-loop that
help to stabilize the compact form of the protein, and without the bound effectors the T-loop is
free to move freely in the extended state.
The 2-KG is only able to bind when Mg-ATP is already in the binding site of the GlnK1 protein,
and it contributes to the stability of the compact T-loop structure by hydrogen bonding with the
protein. The crystallized structures of GlnK1 bound to the different cofactors, and without any
cofactors allows for the creation of a model to explain the binding mechanism and the signaling
process for ammonia uptake regulation.
Proposed Mechanism for Regulation
From the crystallized structures and known features of the Amt and GlnK protein families a
mechanism was developed for the overall regulation of ammonia uptake by the prokaryotic cells.
One interesting aspect of the interactions is that an overall negative charge is found on the
cytoplasmic side of Amt protein concentrated near the ammonia channels. The positively
charged extended T-loops of the GlnK2 could possibly interact with these areas and seal the
channels and effectively inhibit ammonia uptake. These types of interactions would occur in the
event that there are low concentrations of Mg-ATP and 2-KG within the cells, or when the cell
does not need nitrogen for biosynthesis. Another reason why you would want to keep ammonia
out of the cell when ATP levels are low is because once in the cell the ammonia would tend to
protonate and dissipate the proton gradient used for ATP synthesis. This effect could be
especially harmful to a cell that already has low concentrations of ATP to begin with.
When high levels of ATP are found, the Mg-ATP complex can form and bind in the binding
pocket of the GlnK1 trimer. When this occurs the T-loops assume the compact form and
especially with the binding of 2-KG a negative charge occurs at the interacting surface of GlnK1.
The combination of these two effects causes the GlnK1 to dissociate from the Amt and the
electrostatics cause repulsion between the similarly charged proteins. This disassociation of the
two proteins causes the Amt ammonia channels to open once again and ammonia is free to flow
within the cell. When high levels of 2-KG and ATP are found within a cell it represents a
sufficient amount of carbon compounds and energy to initiate biosynthesis, so it allows the
necessary ammonia to enter the cell to be incorporated into proteins and nucleic acids.
The model presented and supported by the experimental data suggests that GlnK1 is an important
molecule in the regulation of nitrogen uptake by prokaryotic cells. This proteins confirmation is
regulated and effected by biological conditions within the cell which causes it to bind or
dissociate from the Amt ammonia channels in the cell. In general this leads to the belief that P II
proteins are involved in more signaling pathways in nitrogen metabolism and regulation.
Importance of Nitrogen
Nitrogen Cycle
Nitrogen is the third most abundant element on earth behind only oxygen and carbon, and an
essential component of all living material. From microscopic bacteria to plants and animals,
nitrogen is a required component of all biological material. Nitrogen makes up over 70% of the
earth’s atmosphere and is found mostly in the elemental N2 gaseous form. For cells and living
organisms to be able to use this important molecule, it must first be converted into its
biologically usable form, ammonia. This process is done by a special group of organisms called
nitrogen fixing bacteria, which convert the atmospheric nitrogen into ammonia through nitrogen
fixation. These nitrogen fixing bacteria are essential to the persistence of life on this planet
because they are one of the only classes of organism which are able to convert nitrogen into its
usable form.
Once nitrogen is fixed into ammonia, usually occurring in the soil, it is taken up by different
organisms and converted into useful molecules for use in the biological process. Once ammonia
is made it is quickly converted into nitrite and ultimately nitrate by bacteria in the earth’s soil,
which derive their energy from this conversion process called nitrification. Although most plants
and animals are able to use ammonia, because of the abundance of these bacteria the majority of
ammonia is converted into nitrate. Once the nitrate is in the soil many plants and bacteria are
able to take up this compound and convert it back into ammonia through a class of enzymes
called reductases. At this point plants are able to use ammonia to make essential components of
life including amino acids and nucleic acids through many complicated mechanisms. These
plants are then ingested by animals which use the amino acids to make proteins.
When the plants and animals die nitrogen is returned back to N2 and the cycle continues. When
an organism dies microbial degradation occurs to the proteins and returns ammonia back to the
soil. This ammonia can either be converted back into nitrate by soil bacteria, or they can be
converted into N2 by other bacteria through nitrification. Through this process a balance of the
levels of nitrogen, nitrate, and ammonia is maintained on earth.
Note: Could possibly go into amino acid/ protein synthesis/ amt and PII proteins in general.