J. Biomedical Science and Engineering, 2011, 4, 591-598 JBiSE
doi:10.4136/jbise.2011.49075 Published Online September 2011 (http://www.SciRP.org/journal/jbise/).
Published Online September 2011 in SciRes. http://www.scirp.org/journal/JBiSE
Mode of interaction of calcium oxalate crystal with human
phosphate cytidylyltransferase 1: a novel inhibitor purified
from human renal stone matrix
Priyadarshini Pathak1, Pradeep Kumar Naik 1, Dipankar Sengupta1, Shrawan Kumar Singh2,
Chanderdeep Tandon1*
1Biotechnology & Bioinformatics, Jaypee University of Information Technology, Waknaghat, H.P., India;
2Department of Urology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India.
Received 17 June 2011; revised 12 July 2011; accepted 10 August 2011.
ABSTRACT
Nephrolithiasis is a common clinical disorder, and
calcium oxalate (CaOx) is the principal crystalline
component in approximately 75% of all renal stones.
It is widely believed that proteins act as inhibitors of
crystal growth and aggregation. Acidic amino acids
present in these proteins play a significant role in the
inhibition process. In this study, interaction of cal-
cium oxalate with human phosphate cytidylyltrans-
ferase 1(CCT), a novel calcium oxalate crystal growth
inhibitor purified from human renal stone matrix has
been elucidated in silico and involvement of acidic
amino acids in the same. As only sequence of CCT is
available, henceforth its 3-D structure was modeled
via Homology modeling using Prime module of
Schrodinger package. Molecular dynamic simulation
of modeled protein with solvation was done by mac-
romodel (Schrodinger). The quality of modeled pro-
tein was validated by JCSG protein structure valida-
tion (PROCHECK & ERRAT) server. To analyze the
interaction of modeled protein CCT with calcium
oxalate along with role played by acidic amino acids,
‘Docking simulation’ was done using MOE–Dock.
Interaction between calcium oxalate and CCT was
also studied by substituting acidic amino acid in the
active sites of the protein with neutral and positively
charged amino acids. The in silico analysis showed
the bond formation between the acidic amino acids
and calcium atom, which was further substantiated
when substitution of these acidic amino acids with
alanine, glycine, lysine, arginine and histidine com-
pletely diminished the interaction with calcium ox-
alate.
Keywords: Calcium Oxalate; Matrix-Assisted Laser
Desorption/ I onization-Time of Flight
(MALDI-TOF-MS); Antilithiatic; Human Phosphate
Cytidylyltransferase 1; MOE–Dock.
1. INTRODUCTION
Calcium oxalate is the most significant component of
urin ary stones [1]. The mechanisms for the formation of
calcium oxalate urinary stones are still not understood,
though it is thought that organic macromolecules, in
particular proteins, play a significant role. It is widely
believed that proteins act as inhibitors of crystal growth
and aggregation. Prothrombin fragment 1 (PFT1) [2],
nephrocalcin [3], bikunin [4,5] and osteopontin [6] have
all been shown to inhibit growth or aggregation or both
in crystallization experiments. Nucleolin-related protein
(NRP) is found on the surface of inner medullary
collecting d uct (IMCD) cel ls in culture (cI MCD) and sele-
ctively adsorbs to calcium oxalate (CaOx). NRP mediates
attachment to the renal tubular epithelium of Ca stone
crystals through an electro static interaction with a highly
acidic region (acidic fragment (AF)) similar to those of
other proteins that have been reported to affect urinary
crystal formation. In vitro studies have shown that OPN
is a potent inhibitor of COM growth, promotes the
formation of calcium oxalate dihydrate (COD) rather
than monohydrate, and inhibits the aggregation of COM
crystals [7].
In vivo, OPN is the major component of the organic
matrix of oxalate-containing kidney stones. OPN con-
tains; 300 amino acids, including a conserved sequence
of contiguous aspartic acid residues [8]. This polypep-
tide chain undergoes extensive posttranslational modifi-
cation, including glycosylation, phos-phorylation, and
sulfation. The exact pattern of modification depends
upon the species and tissue in which the protein is syn-
thesized. The bovine milk isoform of OPN contains 27

P. Pathak et al. / J. Biomedical Science and Engineering 4 (2011) 591-598 592
sites of serine phosphorylation, 1 of threonine phos-
phorylation, and 3 of O-linked glycosylation [9]. Human
milk OPN is phos-phorylated at 34 serine and 2
threonine residues, and O-glycosylated at five sites [10].
The specific mechanism by which these proteins
might interact with calcium oxalate surfaces is still in its
infancy. Researchers working in other areas of biomin-
eralization [11] have suggested that acidic amino acid
residues such as Asp and Glu, that are expected to be
deprotonated and negatively charged at urinary pH, are
attracted to the po sitively charged calcium ions. It would
therefore be expected that proteins rich in γ-carboxyglu-
tamic acid, Gla, such as PFT1, with two deprotonated
carboxylate groups would be even better at electrostati-
cally binding to calcium sites [12,13].
Very recently, we have isolated and characterized a
novel protein, human phosphate cytidylyltransferase 1,
choline,beta (CCT) which is anionic in nature (MW 42
kDa) from organic matrix of calcium oxalate renal
stones, which inhibits the growth of calcium oxalate [14].
It was identified by MALDI-TOF-MS followed by da-
tabase search on MASCOT server as human phos-phate
cytidylyltransferase 1, beta. Molecular weight of this
novel CaOx crystal growth inhibitor from human renal
stone matrix is also same as that of human phosphate
cytidylyltransferase 1, choline, beta. It is involved in the
biosynthesis of phosphatidylcholine which happens to be
an important constituent of human renal stones and is
also reported to have an antilithiatic effect. Amino acid
sequence of the identified protein revealed, presence of
acidic amino acid. The aim of the present work is to
study the interaction of this novel CaOx crystal growth
inhibitor (CCT) from human renal stone matrix with
calcium oxalate at molecular level in silico.
2. METHODS
2.1. Sequence Alignments, Secondary Structure
Prediction and Protein Fold Recognition
Blast search of the human CCT sequence obtained from
MALDI-TOF-MS and MASCOT was done using non-
redundant Protein Data Bank for sequence alignment
and further selection of appropriate template for secon-
dary structure prediction and fold recognition .
2.2. Homology Modeling of Human CCT 1
Choline Beta
Homology modeling of the identified human phosphate
cytidylyltransferase 1, choline, bet a protei n s tructure wa s
done by using Prime module of Schrodinger (Prime ver-
sion 1.5, Macromodel version 9.1, Schrodinger, LLC,
New York, NY, 2005) software. Considering the high
degree of similarity of crystal structure of mammalian
CTP: phosphocholine cytidylyltransferase (3HL4_A) of
Rattus novergicus with CCT, it was taken as a model
cytidylyltransferase (template) for studying structure-
function relationships [15,16].
The structure of protein was modeled on the basis of
its structural similarity with the chain A, crystal structure
of a mammalian CTP: phosphocholine cytidylyltrans-
ferase (Protein Data Bank ID: 3HL4) of Rattus nover-
gicus as a template. The degree of identity between the
template and the human CCT sequence was 53%, which
enabled a preliminary model to be generated by Schrö-
dinger. The sequence alignment was then improved
manually and comparative homology method was used
to build the structure of CCT. Macromodel program of
Schrödinger software was used for molecular dynamic
simulation. To check the quality of the modeled protein,
JCSG protein structure validation serve r was u sed wh ere
PROCHEK & ERRAT vali dation were done.
2.3. Docking of Homology Model of Human
CCT1 Choline Beta with Calcium Oxalate
Calcium oxalate structure was drawn with the help of
molecular builder of Molecular Operating Environment
(MOE) package developed by the Chemical Computing
Group Inc. Montreal, Canada. Active site of the modeled
protein (CCT) was predicted by using active site finder
tool of MOE software. Then, the docking of modeled
protein with calcium oxalate was done using MOE-
Dock. MOE-dock utilizes a Monte Carlo Simulated An-
nealing (SA) method in docking calculations to search
for favorable binding configurations of a small, flexible
ligand and a rigid macromolecule in a pre-set box. The
docking energy calculation was carried out within a user-
specified three-dimensional docking box (3D docking
box) using the simulated annealing method under the
OPLS-AA force field. The energy grids for docking were
generated as grid-based potential fields by the MOE-
Dock program, to reduce the calculation time. Each
docking energy value was calculated as the sum value of
the electrostatic, Van der Waals, and flexibility energies.
The interaction energy was calculated using the electro-
static and Van der Waals potential fields sampled on a
grid overlaying the 3D docking box. The 3D docking
box was interpolated at the atom positions by tri-linear
interpolation. The Van der Waals parameters were taken
from the currently active force field. The electrostatic
field was calculated based on forcefield in the Coulom-
bic manner using the constant dielectric of 1.0 for solva-
tion. MOE-Dock performed 25 independent docking
runs, and wrote the resulting conformations and their
energies to a molecular database file. The lowest dock-
ing energy conformation for each active site was chosen
for LIGPLOT.

P. Pathak et al. / J. Biomedical Science and Engineering 4 (2011) 591-598 593
2.4. Point Mutation of Acidic Amino Acid in the
Active Site of 1 and 2 of the Wild Type
Protein WITH Neutral and Positively
Charged Amino Acids
To investigate the significance of acidic amino acids
present in the active sites of the wild type modeled pro-
tein human CCT 1 choline beta, they were substituted
with alanine, glycine, lysine, arginine and histidine in
active sites 1 and 2. After incorporating these mutations,
protein was docked with calcium oxalate using MOE-
Dock with same parameter of docking as was used for
wild type.
3. RESULTS
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3.1. Sequence Alignments, Secondary Structure
Prediction and Protein Fold Recognition
The amino acid sequence of human CCT was 53% simi-
lar (Figure 1(a)) to the chain A Ctp: phosphocholine
cytidylyltransferase (Protein Data Bank ID: 3HL4) of
Rattus novergicus.
3.2. Homology Modeling of Human CCT1
Choline Beta and Docking with Calcium
Oxalate
The protein phosphate cytidylyltransferase 1, choline,
beta (CCT) was homology modeled based on sequence
and structure align ments with the chain A Ctp: phos-
phocholine cytidylyltransferase (Protein Data Bank
3HL4_A) of Rattus novergicus (Figure 1(b)). After mo-
lecular simulation reliable structure was obtained (Fig-
ure 2). Two binding sites were predicted by the MOE
site finder in the wild type CCT and were named site1
and site2. Calcium oxalate was docked within the active
site using the Monte Carlo docking procedure of MOE.
The best-ranking docking modes of the ligands were
identified and energy minimized in the protein, while
allowing full side chain flexibility. Docking of calcium
oxalate with the two binding sites gave best docking
score of –31.07 & –10.68 with site1 & site2 respectively
(Table 1).
LIGPLOT analysis of the binding site reveals in-
volvement of different amino acids of protein with cal-
cium oxalate. In site1 (first binding site) Asp 82, Gly 83,
Ile 84, His 168, Asp 169, Tyr 173 and Tyr 182 were in-
volved in interaction with calcium oxalate (Figure
3(a)).In the site 2, Gln 54 and Tyr 107 were found to be
participating in the interaction with calcium oxalate
(Figure 3(b)). In the first binding site, most of the atoms-
(a)
(b)
Figure 1. (a) Amino acid sequence analysis of human CCT and chain A Ctp: phosphocholine cytidylyltransferase (Protein Data Bank
3HL4_A) of Rattus novergicus. (b) Manual alignment of human CCT and chain A Ctp: phosphocholine cy tidylyltransferase (Protein
Data Bank 3HL4_A) of Rattus novergicus.

P. Pathak et al. / J. Biomedical Science and Engineering 4 (2011) 591-598 594
Table1. Docking score of wild type human CCT model with calcium oxalate.
W i ld Type
Human CCT
Site 1
Y80, D82, G83, I84, F85, D86, H89, G91,
H92, R94, A95, Q98, C113, A149, P150,
W151, T152, L153, H168, D169, Y173,
S175, V181, Y182, T194, Q195, R196, T197,
E198, G199, I200, S201, T202, S203, D204,
R208
Site 2
A48, T51, N52, C53, Q54, A57, P58,
H59, E60, K61, L62, Q66, T71, A73,
D74, R75, P76, R78, Y107, L109, E144,
I146, K160, H161, L162, I163
Docking Score –31.07 –10.68
Figure 2. Homology modeled structure of CCT.
of calcium oxalate are highly exposed. Asp 82 and Tyr
182 are covalently bound to the calcium of the ligand
(calcium oxalate), while Ile 84 is bound to the oxygen,
where it is acting as side chain receptor (Figure 3(a)). In
both of these active sites of the wild type modeled pro-
tein human CCT 1 choline beta, it was observed that
acidic amino acids played a significant role in the inter-
action with calcium oxalate.
From Ramchandran plot, we found that for the CCT
structure predicted by homology modeling 89.7% of
residues have their torsion angles in the most favored
regions, while remaining 10.3% have in additional al-
lowed regions (Figure 4(a)). Further quality of modeled
protein was checked using Errat program which revealed
an overall quality factor 74.66% (Figure 4(b)) before
and 80.14% (Figure 4(c)) after molecular simulation.
These results indicate that quality of modeled protein is
highly reliable.
3.3. Point Mutation of Acidic Amino Acid of
Active Site and Docking with Calcium
Oxalate
Upon substitution of acidic amino acids present in the
active sites (site 1 and site 2) of the wild type modeled
protein CCT with alanine, glycine, lysine, arginine and
histidine, positive docking scores were obtained indicat-
ing a poor interaction with calcium oxalate. No docking
score was found when acidic amino acid present in
binding site1was mutated to arginine and histidine. In all
of these active sites of the wild type modeled protein
CCT, it was observed that acidic amino acids played a
significant role in the interaction with calcium oxalate.
These studies clearly demonstrate that acidic amino ac-
ids present in the wild type CCT showed a good docking
score, an indicator of interaction with calcium an indi-
cator of interaction with calcium oxalate, while substitu-
tion of these acidic amino acids with alanine, glycine,
lysine, arginine and histidine gave a poor docking score
(Table 2).
4. DISCUSSION AND CONCLUSION
Of all types of renal stones, calcium oxalate (CaOx) is
the most common composition found by chemical
analysis [17]. CaOx crystal growth inhibitors (proteins,
lipids, glycosami-noglycans, and inorganic compounds)
have been proposed to play an important role in renal
stone disease [18,19]. During the last few years more
and more research has been done at the cellular and mo-
lecular levels. In spite of these advances however, the
clinical treatment of urolithiasis remain far from satis-
factory. Stone recurrence in human beings cannot be
predicted an d is beyond the con trol of urologists, mainly
because the mechanism of stone formation at molecular
level is not yet fully understood [20]. Thus, determining
the molecular mechanisms by which urinary con stituents
modulate calcium oxalate crystallization is crucial for
understanding and controlling urolithiassis in humans.
Although a few initial molecular-scale investigations
of the con-trolling mechanisms of kidney stone formation
by these inhibitory molecules have been recently per-
formed [21-24], the majority of previous studies have
been concerned with the overall kinetics of crystallization,
rather than molecular mechanisms which remain poorly
defined. Therefore, in the present work, we have focused
our attention on the interaction of the purified (in our lab)
human phosphate cytidylyltransferase 1 (CCT) [14] with
calcium oxalate using bi oi nf o rmatics tools.
Cytidylyltransferases are critical enzymes involved in
the biosyn-thetic pathways of lipids and complex carbo-
hydrates. These enzymes catalyze a major step of energy
input into biosynthesis by forming the activated inter-
mediates, CDP-alcohols and CMP-sugars [25]. In fact
CCT is involved in the biosynthesis of phosphatid ylcho-
line which happens to be an important constituent of
human renal stones and is also reported to have an anti-

P. Pathak et al. / J. Biomedical Science and Engineering 4 (2011) 591-598 595
lithiatic effect.
Several cytidylyltransferases belong to a single family
of structures, as defined by sequence similarities and sig-
nature sequence that occur in their catalytic domains.
Mammalian CCTα contains several functional regions:
an N-terminal nuclear localization signal, a central cata-
lytic domain, a membrane/lip id activation segment and a
C-terminal phosphorylation region. There is a high de-
gree of sequence similarity within the catalytic domain
of all known forms of CCT, with the yeast catalytic do-
main being 56% identical to that of mammalian CCTα,
and the catalytic domains of CCTα, and CCTβ being
90% identical [16].
The degree of identity between the template and the
human CCT sequence was 53%, which enabled a pre-
liminary model to be generated by Schrodinger. Aspartic
acid, isoleucine, tyrosine and glycine were found to be
interacting with calcium oxalate at site1. More negative
the docking score, stronger is the binding between ligand
and protein’s active site [26]. The strong interaction be-
tween CCT’s active site and calcium oxalate predicts
inhibition of the same. Highest docking score (–31.079)
(a)

P. Pathak et al. / J. Biomedical Science and Engineering 4 (2011) 591-598 596
(b)
Figure 3. (a) Asp 82, Gly 83, Ile 84, His 168, Asp 169, Tyr 173 and Tyr 182 were involved in
interact i o n with calcium o xalate. ( b ) In the site 2, Gln 54 and T yr 1 07 we re fou nd to be pa rt icipa ting
in the interaction with calcium oxalate.
with site1 indicates good binding of modeled protein
with the ligand calcium oxalate. LIGPLOT of site1
showed involvement of aspartic acid (Asp) at position 82
with calcium while isoleucine (Ile) at position 84, inter-
acts with oxygen of oxalate group. The binding site1 was
found to be better for interaction with calcium oxalate
compared to the other site. Whether a protein or other
macromolecule acts as an inhibitor of growth and ag-
gregation or a promoter of nucleation and aggregation
implies that there must be some mechanism to explain
the interaction with the mineral oxalate surfaces. The
interaction between calcium and acidic Asp, Glu and Gla
is certainly plausible, but it is equally conceivable that
basic residues that are normally protonated at urinary pH
and positively charged might experience an attraction
toward negatively charged oxalate groups [27]. This is
corroborated by the presence of basic amino acids too in
the inhibitory proteins [28]. In either case steric cons-
traints from 3D conformation of the molecule might
limit the number of these simple interactions [27]. Posi-
tive docking score with mutated binding sites confirms
the inhibitory role of acidic amino acids [29-31].

P. Pathak et al. / J. Biomedical Science and Engineering 4 (2011) 591-598 597
(a)
(b)
(c)
Figure 4. (a) In Ramchandran plot 89.7% of residues have
their torsion angles in the most favored regions, while remain-
ing 10.3% have in additional allowed regions. b & c Errat pro-
gram which revealed an overall quality factor 74.66% (b) be-
fore and 80.14% (c) after molecular simulation.
There are reports that members of cytidylyltransferase
family have similar conserved sequence [32]. In our
study, we found 100% sequence in binding site1 and
29% sequence in site2, in the conserved region of CCT.
Interestingly, conserved amino acids are contained
within the catalytic core of the CCTs [32].
We found that the acidic amino acid is interacting with
the calcium of calcium oxalate. This was further sub-
Table 2. Docking score of mutated human CCT modelwith
calcium oxalate.
Amino acids with
which point muta-
tion is done at spe-
cific positions in Site
1 & 2
Site 1
Point mutation of
Aspartic acid at
position 82, 86,
169, 204 & Glu-
tamic acid at posi-
tion 198
Site 2
Point mutation of
Aspartic acid at
position 74 &
Glutamic acid at
position 60 & 144
Alanine (A) 63.29 87.81
Glycine (G) 193.39 159.26
Ly s in e ( K) 98.33 138.05
Arginine (R) 105.27 151.53
Histidine (H) 163.29 153.29
stantiated when substitution of these acidic amino acids
with alanine, glycine, lysine, arginine and histidine
completely diminished the interaction with calcium ox-
alate. These findings are in conformity with the presence
of acidic amino acids in the various inhibitors of calcifi-
cation from human beings [29-31] as well as acidic na-
ture of antilithiatic proteins from plants [33,3 4].
5. FUNDING
We thank Jaypee University of Information Technology,
Solan, HP, India for providing funds to carry out this
research work.
6. ACKNOWLEDGEMENTS
We express our gratitude to the Department of Urology, Post Graduate
Institute of Medical Education and Research (PGIMER) Chandigarh,
India for providing kidney stones. We would also like to thank Mr.
Deeptak Ver ma for helping us with MOE simulation studies at Univer-
sity of North Carolina, Charlotte.
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