Evidence for a New Theory of Allostery
Andrew Michaelson
Northeastern University
Arpil 23rd, 2010
Instructor: Professor Mary Jo Ondrechen
Course: Molecular Modeling CHEM 5638
This report shows that a new theory of allostery can be supported when there is no conformational
change in a protein. Results are described for Uracil Phosphoribosyltransferase for both the inactive
form (PDB # 1XTT) and the active form (PDB # 1XTU). This enzyme catalyzes the reaction of 5phosphate-α-1-diphosphate and uracil to uridine 5‘-monophosphate diphosphate [2]. This enzyme is an
important drug target since mammals do not have this enzyme in their pyrimidine salvage pathways
[2]. It is noted that the inactive and active forms have a similar structural alignment in both 1 chain and
4 chain analysis, but they do have different pKa values at important catalytic residues found near the
allosteric site and active site. Analysis of all catalytic residues at important sites within the protein
found the largest pKa changes to occur near the allosteric site as the protein switched from the inactive
to active form. The weight of these implications that these pKa changes imply that a new theory of
allosteric regulation is useful for understanding the behavior of proteins that do not undergo
characteristic conformational change [2, 3]. Results are summarized to show the evidence for the pKa
changes for the inactive and active forms of the protein.
Introduction:
Understanding how allostery works in proteins that do not behave according to the classic theory of
allostery may lead to designing new drug molecules for allosteric sites and the design of new allosteric
sites that bind better with existing drugs for better treatments for human disease. The classic theory of
allostery describes 2 possible scenarios. The first scenario describes a 2 state system in which a
conformational change occurs in the protein as it goes from relaxed binding state to a tense non-binding
state[3]. The second scenario describes incremental changes as more and more small molecules bind to
domains within the protein that describes a gradual conformational change from the active to inactive
state[3]. The problem is that neither of these classic scenarios take into account what occurs when no
conformational change takes place in the protein as it undergoes a change from the inactive (nonbinding) to the active state (binding a ligand, cofactor, or other small molecule at the allosteric site)
leading to a non-conformational change at the active site. The purpose of this research is to create a
new theory of allostery based on electrostatic differences of ionizable residues at the allosteric site and
active site of a protein to see if there is a relationship between the two, to create a new theory of
allostery that takes into account proteins that do not undergo a conformational change as they go from
inactive to active states.
A prerequisite step is determining that the protein in the inactive and active states is so structurally
similar from one state to the next that either a literature review or structural alignment program will
show a negligible change in whatever differences are present so they can be ignored (such as
differences in loops). Briefly this is done by finding key residues located at catalytic sites within the
protein under consideration using either a software program to predict those residues or reviewing
literature relevant to that protein. After that a prediction of pKa values for ionizable residues of those
sites that were found to be catalytically relevant was determined through a software program. Finally
an examination of those residues are done taking into account how far apart they are, pKa, and what
catalytic site they are found at to determine relevance in supporting a new theory of allostery.
Methods
Using THEMATICS [4] and a literature survey [3] catalytic residues were found the protein Uracil
Phosphoribosyltransferase in both inactive (PDB # 1XTT) and active forms (PDB # 1XTU). In the
active form of this enzyme a 4 chain analysis was done with chains A, D, H, and E which were
compared with chains A, B, C, and D from the inactive form of this protein. In addition a 1 chain
analysis was done using chain A of both the inactive and active forms of this enzyme.
PropKa v2.0 [5, 6] was used to determine the pKa values of the catalytic residues. YASARA
[1] was used to find residues close to the predicted catalytic sites, in addition the Motif algorithm which
is part of YASARA was used to show structural similarity between the inactive and active states of
Uracil Phosphoribosyltransferase. Microsoft Excel was used to quantitatively compare the pKa values
of the predicted catalytically important residues.
A retrieval of the inactive and active forms of the Uracil Phosphoribosyltransferase came from the
Protein Data Bank. These 2 forms of the protein were then loaded into YASARA to be cleaned which is
a process that adds Hydrogens and fixes discrepancies in the molecule such as missing atoms. After
that an examination of the 2 forms of the protein in YASARA was done to determine how many chains
should be compared to one another and what ligand was to be used in the comparison between the 2
forms. After that the parameters were approximated for the ligand using CHARMM 19 [see
supplementary] for use in THEMATICS.
A graphical analysis of which residues were found at catalytic sites was done in Microsoft Excel where
a plot showed which residues at catalytic sites had a noticeable difference between the pKa values.
In Figure 1 four residues are found, Arginine 80 colored in purple spheres which plays a key role in
inhibition by CTP the allosteric ligand, Aspartate 209 colored in green spheres which is a catalytic
residue, Leucine 58 colored in orange spheres which is close to the active site, and Glycine 206 colored
in yellow spheres which is near Leucine 58.
Table1 shows the distances between Leucine 58 colored in orange, Glycine 206 colored in yellow,
Aspartate 209 colored in green, and Arginine 80 colored in purple these colors match the colors of
Figure 1. These distances are given to help determine how far an electrostatic effect is acting between
these residues.
Figure 2 represents an overview of the purposed interactions between the 4 primary residues that are
involved at the catalytic sites. Leucine 58 at the active site interacts with Glycine 206, Leucine 58 also
interacts with Aspartate 209, and Aspartate 209 interacts with Arginine 80 at the allosteric site. The
blue colored sticks represent the proposed electrostatic interactions occurring between the active site
and catalytic site that may help explain the new theory of allosteric regulation.
In Figure 3 six residues are depicted from the literature as important: Arginine 80 which forms a
peptide bond with Leucine 79 in a cis-trans conformation, Arginine 80 also plays a key role in
inhibition of the allosteric site by interacting with Cytidine-5'-Triphosphate (CTP) depicted with balls
and sticks, Arginine also shares a Hydrogen bond with Glycine 216, Glutamine 98 is here as well,
Leucine 58 is close to the active site (but this can not be seen well here, so see Figure 1 and Figure 5 to
see the interactions Leucine 58 makes with the other important catalytic residues). Aspartate 209 is
near Arginine 80 at the allosteric site. All important residues are shown on this picture as spheres. This
picture is chain A of the active form of Uracil Phosphoribosyltransferase.
Figure 4 shows the chain A representation of Uracil Phosphoribosyltransferase of both the inactive
form in pink and the active form in blue. This pictures shows that there is almost no structural change
between the two forms of this enzyme. These 2 forms are separated by a distance of 0.679 angstroms
difference. The four residues are depicted in spheres and orange is Leucine 58, purple is Arginine 80,
green is Asparate 209, and yellow is Glycine 206.
In Table 2 the residues in black capital letters are the residues predicted by THEMATICS as being
catalytically important, in lowercase black letters are the residues that are catalytically important as
described by the literature. In red is the residue predicted by both THEMATICS and the literature as
being catalytically important, and in blue are the residues that have the largest pKa change for the
protein Uracil Phosphoribosyltransferase.
Plot 1 shows the residues predicted by THEMATICS in uppercase black letters and the letters in lower
case are the residues that are described in the literature as important. The 3 circled residues in red are
the places where the pKa values of the inactive versus the active states seem to significantly differ for
Uracil Phophoribosyltransferase. Table 2 corresponds to this picture as well.
In Table 3 there are 3 residues that propKa could predict the pKa for. Leucine 58 is not shown since
propKa could not predict a pKa for it; instead Glycine 206 is shown since it close to the position of
Leucine 58. The 4 chains A, D, H, and E of Uracil Phosphoribosyltransferase are represented by the 4
values for each residue.
Figure 5 shows the four chain representation of Uracil Phosphoribosyltransferase of both the inactive
and active forms. This pictures shows that there is almost no structural change between the two forms
of this enzyme colored in red and light blue chains. These 2 forms are separated by a distance of 1.743
angstroms difference. The four residues are depicted in spheres and orange is Leucine 58, purple is
Arginine 80, green is Asparate 209, and yellow is Glycine 206.
In Plot 2 Arginine 80 which is at the allosteric site and Aspartate 209 which interacts with Arginine 80
both clearly show very large differences in pKa values. However, Glycine 206 which is near the active
site and close to Leucine 58 shows only a slight pKa difference which can assumed to be negligible.
Discussion and Conclusion
Residues Tyrosine 4, Tyrosine 165, Tyrosine 187, Aspartate 69, Tyrosine 174, Lysine 178, and Tyrosine
183 were all found to be clustered near each other. Arginine 105 and Arginine 107 were also near one
another. Aspartate 209, Aspartate 140, and Arginine 80 were not found to be that close together.
Primarily the 3 residues that were most important were Leucine 58 at the active site, Arginine 80 at the
allosteric site, and Asparate 209 which was in between those 2 residues. Glycine 206 was used as a
substitute residue for Leucine 58 since it was relatively near Leucine 58 and not so far from the other
important catalytic residues; this was done since propKa could not calculate a pKa change for Leucine
58. A result of the pKa values changing more than 2 can be seen in Plot 2 for these 2 residues Arginine
80 which is found near the allosteric site and Aspartate 209 which is found near Arginine 80 and
Leucine 58.
Table 4 shows Arginine 80 in green which is at the allosteric site. Arginine 80 shows a range of 0.711.18 for the difference in pKa for 4 chains and only 0.39 for 1 chain. Aspartate 209 in red which is
between the active site and allosteric site has a range of 1.88 - 2.71 for the difference in pKa for 4
chains and only 0.27 for 1 chain. Table 4 also shows the pKa changes for the residues that
THEMATICS, the literature, and propKa collectively find [2, 4, 5, & 6].
There is strong evidence that a change in electrostatic differences as can be seen in Table 4 between the
inactive and active states of Uracil Phosphoribosyltransferase supports that this new theory of allostery
is correct since it does not rely on conformational changes to show how this enzyme changes as a result
of changes at the allosteric site. Unfortunately since no direct measurements of how Lecuine 58
changes at the active site could be made this result can not be certain. Also since propKa is an empirical
method for pKa determination a better way to calculate the pKas should be used to ascertain the
certainty of these results such as using Multicoformational Continuum Electrostatics [10, 11]. The next
step for this research would be to look at how other pKa values of other proteins change. If enough
evidence can be gathered then this new theory of how allostery works when there is no conformational
change may actually work .
Acknowledgments:
I would like to thank the Ondrechen Group, Divya Ruth, and Tripti Kulkarni for all their advice with
this project.
References:
1. YASARA Biosciences (2007) YASARA: Yet another scientific artificial reality application. Available:
http://www.YASARA.org/. Accessed 32 April 2010.
2. Arent S, Harris P, Jensen KF & Larsen S (2005) Allosteric regulation and communication between
subunits in uracil phosphoribosyltransferase from Sulfolobus solfataricus. Biochemistry 44, 883–892
3. Freeman and Company L Stryer, WH "Biochemistry Fifth Edition" - New York, 1995
4. Mary Jo Ondrechen, James G. Clifton, Dagmar Ringe, (2001) THEMATICS: A simple computational
predictor of enzyme function from structure, PNAS.
5. Hui Li, Andrew D. Robertson, and Jan H. Jensen "Very Fast Empirical Prediction and Interpretation
of Protein pKa Values" Proteins, 2005, 61, 704-721.
6. Delphine C. Bas, David M. Rogers, and Jan H. Jensen "Very Fast Prediction and Rationalization of
pKa Values for Protein-Ligand Complexes" Proteins, 2008, 73, 765-783.
7. 1XTU. F.C.Bernstein, T.F.Koetzle, G.J.Williams, E.E.Meyer Jr., M.D.Brice, J.R.Rodgers,
O.Kennard, T.Shimanouchi, M.Tasumi, "The Protein Data Bank: A Computer-based Archival File For
Macromolecular Structures," J. of. Mol. Biol., 112 (1977): 535.
8. 1XTT. F.C.Bernstein, T.F.Koetzle, G.J.Williams, E.E.Meyer Jr., M.D.Brice, J.R.Rodgers,
O.Kennard, T.Shimanouchi, M.Tasumi, "The Protein Data Bank: A Computer-based Archival File For
Macromolecular Structures," J. of. Mol. Biol., 112 (1977): 535.
9. Bioinformatics-Biostatistics & Computational Biology Greece. YASARA the absolute structure
viewer. Triantafillos Paparountas. Available: http://bio-informatics.gr/content/view/45/26/ . Accessed
32 April 2010.
10. Georgescu R.E., Alexov E.G., Gunner M.R.(2002). Combining conformational flexibility and
continuum electrostatics for calculating pKa's in proteins. Biophys J. 83, 1731-1748
11. Alexov, E. and Gunner, M.R. (1997) Incorporating protein conformational flexibility into pHtitration calculations: Results on T4 Lysozyme. Biophys. J. 74, 2075-2093
Supplementary
Note: I am still not sure why propKa can determine empirical values for Glycine.