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Supplementary Information
TOWARDS UNDERSTANDING THE CATALYTIC MECHANISM OF HUMAN
PARAOXONASE 1: EXPERIMENTAL AND IN SILICO MUTAGENESIS STUDIES
Rajan K. Tripathy, Geetika Aggarwal, Priyanka Bajaj, Deepika Kathuria, Prasad V. Bharatam
and Abhay H. Pande
A
B
H3
I127
H1
W115
T137
H2
K192
C
Supplementary Fig. 1. Homology model of RT-460 generated by using 1V04 as a template.
Panel A shows a ribbon diagram representing the modelled structure of RT-460 enzyme, viewed
along the axis with the catalytic and the structural calcium (yellow spheres). Mutated amino acid
residues (H115W, R192K, A137T, M127I, D263H) are shown in grey colour stick format and the
three α-helices of the protein are depicted as H1, H2, and H3, respectively. Panel B and C shows
the Ramachandran plot and Errat plot, respectively, of the modelled structure of RT-460 enzyme.
Calculation of the backbone dihedral distribution of all amino acid residues of the modelled
structure of RT-460 protein showed no residue in the disallowed region, 87.5% (260) residues in
the most favoured region, 11.1% (33) residues in the allowed region, and 1.3% (4) residues in the
generously allowed region (Panel B). Analyses of Errat plot revealed that the overall quality of
modelled structure of RT-460 enzyme was 71.17%.
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We have verified rh-PON1(wt) as well as all the mutant proteins before using them for molecular
docking study. The Supplementary Figure 1 shows the overall quality of modelled structure is
71.17% so that we can use the model for molecular docking study. The Errat plot identifies that in
the 3D structure which residues are within the acceptable range and which are not. The darker and
higher is the bar associated with a particular residue, the unacceptable is the structure of that
residue. If the residues of importance are in the dark black colour, we need to redo the modelling;
otherwise the structure can be utilized for molecular docking. Analysis of the Errat plots of RT460 (Supplementary Fig. 1) and rh-PON1(wt) (Supplementary Fig. 2) suggest that in both cases, the
values of important amino acid residues are in the acceptable range and the minor differences in
the values do not carry any significant meaning.
Homology modeling of rh-PON1 by using 3SRG as a template
The crystal structure of rePON1 (PDB identifier 3SRG) was used as a template
(http://www.rcsb.org/pdb). ClustalW (http://www.ebi.ac.uk/Tools/msa/ clustalw2) tool was used
to carry out protein multiple sequence alignments. The three dimensional (3D) model of the
enzymes was built using Modeller 9v8 (http://www.salilab.org/ modeller/). Subsequently, model
validation was carried out using PROCHECK (Laskowski et al., 1993). PROCHECK scrutinizes
the stereochemical quality of a protein structure and presents a Ramachandran plot of the query
structure. Steric clashes observed in the crude models were recognized by the Errat plot and the
structural loops showing error greater than 90 % were refined through loop modeling using
Modeller Loop class (Colovos and Yeates, 1993).
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A
B
C
Overall quality factor**: 87.20
Supplementary Fig. 2. Homology model of rh-PON1(wt) generated by using 3SRG as a
template. Panel A shows a ribbon diagram representing the modelled structure of rh-PON1(wt)
enzyme, viewed along the axis with the catalytic and the structural calcium (yellow spheres). Panel
B and C shows the Ramachandran plot and Errat plot, respectively, of the modelled structure of rhPON1(wt) enzyme.
Based on the information obtained from the sequence alignment of target (rh-PON1(wt))
and template (3SRG) protein sequence (Ben-David, et al 2012, 2013), the homology model of rhPON1(wt) was generated by Modeller 9v8. 100 models were generated and the model with lowest
discrete optimized protein energy (DOPE) was selected for further optimization. Steric clashes
were observed in the loop regions of the crude model, therefore, these regions were further
subjected to loop modeling using Modeller Loop class. After a few cycles of loop refinement, the
3D models of rh-PON1(wt) exhibited an overall quality factor of 87.20. RMSD value of 0.10 Å was
observed on superimposition of 3SRG and rh-PON1(wt) (3SRG as template) (data not shown).
RMSD value of 0.28 Å was observed on superimposition of rh-PON1(wt) (1V04 as template) and
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rh-PON1(wt) (3SRG as template) (data not shown). The RMSD values were in acceptable range
i.e., ≤0.5 Å, suggesting the validity of the models constructed.
B
A
Supplementary Fig. 3. Comparison between the binding poses of 2-HQ with the homology models
of rh-PON1(wt) generated by using 1V04 as a template (panel A) and by using 3SRG as a template
(panel B).
In order to see the effect of molecular docking studies on two different homology models
of rh-PON1(wt) prepared from 1V04 and 3SRG templates, molecular docking studies of 2-HQ were
performed on both the models and it was found that 2-HQ is showing similar interactions with
both the homology models (Supplementary Fig 3 and Table 1). So, the prediction of the binding
mode of all the substrate will be same with any of the two models of rh-PON1(wt). So, the
interactions of all the ligands were analyzed in the rh-PON1(wt) model obtained from 1V04.
Supplementary Table 1
Table showing the distances of 2-HQ with the amino acids of catalytic site of 3SRG (rePON1) and
the comparative analysis of molecular docking study of 2-HQ with rh-PON1(wt) homology models
prepared from 1V04 and 3SRG as template.
Distances of 2HQ with amino
acids
3SRG
(Crystal
structure of
rePON1)
rh-PON1(wt) homology
model
(1V04 as template)
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rh-PON1(wt) homology
model
(3SRG as template)
(Ca….O)
(E53)
(H115)
(N168)
(N224)
(D269)
2.4
2.8
2.8
3.0
3.6
2.7
2.3
3.4
3.3
2.8
3.0
3.1
2.3
3.3
3.3
3.0
3.0
3.5
Effect of mutations on the structure of rh-PON1 enzymes
Supplementary Fig. 4. Panels A and B show superimposition of amino acid residue at position
115 (Panel A) and position 192 (Panel B) on the model structures of rh-PON1(wt). Amino acid
residues are shown in stick format. Note that in rh-PON1(wt) His and Arg occupies positions 115
and 192, respectively, while in all the mutants Trp and Lys are present at positions 115 and 192,
respectively.
Initially, the effect of mutations on the overall structure of rh-PON1 enzymes and the
orientation of proposed catalytically important amino acid residues in the active site of enzymes
were analysed. H115 is in the catalytic domain of the enzyme and H115W mutation is one of the
most important mutation which is common in all mutants. Supplementary Fig 4A shows an overlay
of 3D structures of rh-PON1(wt) and RT-180, highlighting the H115W mutation. It was observed
that there is a significant difference in the steric factors at this position as Trp residue is a much
bulkier in comparison to His. H115W mutation may cause a partial blockage of the catalytic
domain and does not allow a complete access of the electronegative centres of the ligands to the
catalytic Ca2+ centre. Moreover, His residue exists as its protonated form at physiological pH, it
can cause electrostatic interactions with the phosphate centre (or other electronegative centres) of
the substrate, but Trp does not show the same stabilizing interactions.
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R192K mutation is the next important and common mutation found in all the mutant
enzymes under consideration. Amino acid at position 192 is present at the entrance of the active
site of enzyme through which the substrate molecules enter into the active site cavity of the
enzyme. Supplementary Fig 4B shows the superimposition of the 3D structures of enzymes,
highlighting the differences in the orientation of the Arg/ Lys residues at position 192 in the rhPON1(wt) and RT-180 proteins, respectively. Since the position of these residues is far from the
catalytic domain and also they do not block the pathway of the substrate entry into the active site
cavity of the enzyme, sterical factors are minimal. However, electrostatically there are a few
differences; at physiological pH, positive charge in the Arg residue, due to its guanidine group, is
highly dispersed whereas the charge is localized over Lys residue. Therefore, due to localized
positive charge over Lys, it may show stronger electrostatic interactions with the substrate or other
residues in the active site as compared to Arg. This may lead to improved interactions with the
substrates.
Besides H115W and R192K mutations, other mutation has occurred at position D263.
D263 residue is present in the flexible loop region of the enzyme and is present in anionic state at
physiological pH. D263H mutation changes the electrostatic environment (from negative to
positive) at this position. As this mutation has occurred on flexible loop region of the enzyme, this
could facilitate either the easy access of the substrate to the active site due to the electrostatic
interaction between positively charged H263 and negatively charged phosphate group of the
substrate, and thus could accelerate the catalytic activity of the enzyme.
A137T mutation is present far from the catalytic domain on the loop. This mutation
increases the steric effect and cause only a marginal change in the electronic character (data not
shown). Similarly, M127I, L130F, S211T, D94H, S81R and P165A are the mutations, which are
far from the active site of the enzyme and hence, they are not expected to cause any direct
modulation of activity in steric or electrostatic terms.
References:
Laskowski, R.A., MacArthur, M.W., Moss, D.S. and Thornton, J.M. (1993) J. Appl. Crystallogr,
26, 283-291.
Colovos, C and Yeates T.O. (1993) Protein Sci 9, 1511-1519.
Ben-David,M., Elias,M., Filippi,J.J., Dunach,E., Silman,I., Sussman,J.L. and Tawfik,D.S. (2012)
J Mol Biol, 418, 181-196.
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Ben-David,M., Wieczorek,G., Elias,M., Silman,I., Sussman,J.L. and Tawfik,D.S. (2013) J Mol
Biol, 425, 1028-1038.
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