Supplementary Information
Atomistic Details of the Molecular Recognition of DNA-RNA Hybrid Duplex by
Ribonuclease H enzyme
GORLE SURESH and U DEVA PRIYAKUMAR*
Center for Computational Natural Sciences and Bioinformatics,
International Institute of Information Technology, Hyderabad 500 032, India
e-mail: deva@iiit.ac.in
1
Table of Contents
Supplementary methods
List of Supplementary Figures
Figure S1. Time series of RMS deviations for (A) RNase H -DNA-RNA hybrid complex, RNase H
enzyme and hybrid in holo state, (B) apo-RNase H enzyme and apo-hybrid, and (C) active site and
non-active site regions of RNase H enzyme in both apo and holo states. Time series of radius of
gyration for (D) RNase H-hybrid complex (E) RNase H enzyme, and (F) DNA-RNA hybrid
duplexes in apo and holo states.
Figure S2. Ramachandran plots for the amino acids present in active site region of apo and holo
RNase H enzyme.
Figure S3. Probability distributions of hydrogen bonds for Watson - Crick base pairs in apo and
holo DNA-RNA hybrid duplexes.
Figure S4. Average values of base pair step parameters for base pair steps present in the DNA-RNA
hybrid duplexes in apo and holo states.
Figure S5. Average values of base pair axis and base pair parameters for all the base pairs in DNARNA hybrid in apo (black) and holo (red) states.
Figure S6. Probability distribution of backbone dihedral angles and glycosidic dihedral angle of
DNA-RNA hybrid duplex in apo and holo states.
Figure S7. Times series of surface area buried in between RNase H enzyme and DNA-RNA hybrid
duplex while binding each other.
Figure S8. Time series of hydrogen bonds present at the interface of RNase H enzyme-DNA-RNA
hybrid complex which are showing longer average life time.
2
List of Supplementary Tables
Table S1. List of amino acids present in various secondary structural elements and respective
average rmsd values for Ribonuclease H enzyme in free and complex states.
Table S2. Probabilities of pseudorotation angles of furanose sugar puckering of each residue
present in DNA-RNA hybrid duplex.
Table S3.Stacking interaction energies (kcalmol-1) for non-terminal nucleotides of DNA-RNA
hybrid in apo and holo states.
Table S4. Hydration numbers and solvent accessible surface area values calculated around the
phosphate oxygen atoms, major and minor groove regions of DNA-RNA hybrid duplex
Table S5. Statistics of the hydrophobic interactions present at enzyme-nucleic acid interface
observed in MD simulations.
Table S6. Statistics of the observed electrostatic interactions between enzyme and nucleic acid
during MD simulations.
Table S7. Statistics for the water mediated hydrogen bonds observed at RNase H enzyme-DNARNA hybrid interface during MD simulations on binary complex.
3
Supplementary Methods
Hydration analysis: The hydration numbers, which represent the number of water molecules
present around a particular site, were calculated around the major and minor grooves of each base
pair basing on the definitions for major groove atoms (O6, N7, O4, H61 and H62) and minor
groove atoms (O2, N3, H21, and H22), around backbone oxygen atoms (O1P, O2P, O2’, O3’) in the
range of distance < 3.0 Å from the solute atoms. The criteria for existence of hydrogen bond
between hydrogen bond donor (D) and acceptor (A) in the form D-H…A are a) a maximum H…A
distance of 2.4 Å b) a minimum DHA angle of 120˚. The occupancies, life times, number of events
corresponding to all normal and water mediated hydrogen bond, electrostatic and hydrophobic
interactions were calculated using the cut off 5 ps for life time and 30% for occupancy. The ratio of
number of frames with hydrogen bond to the number of total analysis frames gives the percentage
of occupancy. The life time of a specific hydrogen bond equals to the time interval when onwards
from its first appearance until it was first broken and the average of all its incarnations gives its
average life time. The water mediated hydrogen bonds were also examined in the similar manner.
Statistical analysis had been performed on the independent data sets obtained from the time
blocks which got by dividing the final 80 ns simulation time as 10 ns blocks. All the averages and
errors reported here were the resultant averages of data sets present in all the blocks.
4
Figure S1.Time series of RMS deviations for (A) RNase H -DNA-RNA hybrid complex, RNase H
enzyme and hybrid in holo state, (B) apo-RNase H enzyme and apo-hybrid, and (C) active site and
non-active site regions of RNase H enzyme in both apo and holo states. Time series of radius of
gyration for (D) RNase H-hybrid complex (E) RNase H enzyme, and (F) DNA-RNA hybrid
duplexes in apo and holo states.
5
Figure S2.Ramachandran plots for the amino acids present in active site region of apo and holo
RNase H enzyme.
6
Figure S3.Probability distributions of hydrogen bonds for Watson - Crick base pairs in apo and holo
DNA-RNA hybrid duplexes.
7
Figure S4. Average values of base pair step parameters for base pair steps present in the DNA-RNA
hybrid duplexes in apo and holo states.
8
Figure S5.Average values of base pair axis and base pair parameters for all the base pairs in DNARNA hybrid in apo(black) and holo(red) states.
9
Figure S6.Probability distribution of backbone dihedral angles and glycosidic dihedral angle of
DNA-RNA hybrid duplex in apo and holo states.
10
Figure S7. Times series of surface area buried in between RNase H enzyme and DNA-RNA hybrid
duplex while binding each other.
11
Figure S8.Time series of hydrogen bonds present at the interface of RNase H enzyme-DNA-RNA
hybrid complex which are showing longer average life time.
12
Table S1.List of amino acids present in various secondary structural elements and respective
average RMS deviation values for Ribonuclease H enzyme in free and complex states.
Segment
Residues
Apo
Holo
β1 strand
7-14
1.22 ± 0.09
0.61 ± 0.03
β2 strand
18-26
0.53 ± 0.01
0.45 ± 0.01
β3 strand
32-42
0.56 ± 0.01
0.61 ± 0.00
β4 strand
68-71
0.32 ± 0.01
0.24 ± 0.00
β5 strand
117-120
0.28 ± 0.01
0.23 ± 0.00
Helix A
43-62
0.46 ± 0.01
0.46 ± 0.03
Helix B
72-82
0.39 ± 0.00
0.34 ± 0.00
Helix D
94-109
0.42 ± 0.01
0.38 ± 0.00
Loop1
1-6
0.53 ± 0.02
0.60 ± 0.03
Loop2
15-17
0.45 ± 0.03
0.34 ± 0.01
Loop3
27-31
0.31 ± 0.00
0.35 ± 0.02
Loop4
63-67
0.73 ± 0.02
0.75 ± 0.01
Loop5
83-90
0.88 ± 0.04
0.55 ± 0.01
Loop6
111-116
0.43 ± 0.01
0.46 ± 0.01
Loop7
126-135
3.85 ± 0.08
1.25 ± 0.01
Turn 1
91-93
0.22 ± 0.01
0.20 ± 0.00
Turn 2
121-125
0.34 ± 0.01
0.28 ± 0.00
13
Table S2. Probabilities of pseudorotation angles of furanose sugar puckering of each residue
present in DNA-RNA hybrid duplex.
South
North
South
Apo
Holo
Apo
Holo
dA2
92
87
5
9
dA3
59
64
29
dT4
58
9
dC5
73
dA6
92
North
Apo
Holo
Apo
Holo
rU2
0
0
100
99
31
rG3
0
0
99
100
17
58
rA4
0
0
99
100
98
14
0
rU5
0
0
99
99
87
2
0
rU6
0
0
99
99
14
Table S3.Stacking interaction energies (kcalmol-1) for non-terminal nucleotides of DNA-RNA
hybrid in apo and holo states.
Intra strand
Inter strand
Total
Residue
Apo
Holo
Apo
Holo
Apo
Holo
dA2
-17.76 ± 0.6
-15.94 ±0.6
-0.59 ± 0.0
-1.18 ±0.1
-18.36 ± 0.6
-17.12 ±0.6
dA3
-12.37 ± 0.4
-11.42 ±0.4
-3.96 ± 0.1
-3.64 ±0.1
-16.34 ± 0.6
-15.06 ±0.6
dT4
-9.05 ± 0.3
-8.95 ±0.3
0.19 ± 0.0
0.75 ±0.0
-8.86 ± 0.3
-8.20 ±0.3
dC5
-7.81 ± 0.3
-7.79 +0.3
-2.12 ± 0.1
-2.58 ±0.1
-9.92 ± 0.4
-10.37 ±0.4
dA6
-12.67 ± 0.5
-12.24±0.4
-6.12 ± 0.2
-6.12 ±0.2
-18.79 ± 0.7
-18.35 ±0.7
rU2
-8.68 ± 0.3
-8.46 ±0.3
-2.93 ± 0.1
-2.63 ±0.1
-11.61 ± 0.4
-11.09 ±0.4
rG3
-13.44 ± 0.5
-11.86 ±0.4
-6.54 ± 0.2
-6.56 ±0.2
-19.98 ± 0.7
-18.42 ±0.7
rA4
-14.38 ± 0.5
-13.63 ±0.5
-1.54 ± 0.1
-2.03±0.1
-15.92 ± 0.6
-15.66 ±0.6
rU5
-6.87 ± 0.2
-6.78 ±0.2
-0.74 ± 0.0
-0.07 ±0.0
-7.61 ± 0.3
-6.86 ±0.2
rU6
-3.81 ± 0.1
-2.58 ±0.1
-3.64 ± 0.1
-3.92 ±0.2
-7.45 ± 0.3
-6.51±0.2
15
Table S4.Hydration numbers and solvent accessible surface area values calculated around the
phosphate oxygen atoms, major and minor groove regions of DNA-RNA hybrid duplex.
Base pair
Apo
Holo
Backbone O
Apo
Holo
dA2-rU6
2.5 ± 0.01
2.8 ± 0.04
dA2
5.5 ± 0.01
5.4 ± 0.02
dA3-rU5
2.5 ± 0.04
2.2 ± 0.02
dA3
5.5 ± 0.02
5.6 ± 0.01
dT4-rA4
2.4 ± 0.02
2.7 ± 0.01
dT4
5.5 ± 0.01
5.3 ± 0.02
dC5-rG3
1.3 ± 0.01
1.2 ± 0.01
dC5
5.5 ± 0.01
5.1 ± 0.01
dA6-rU2
2.3 ± 0.01
2.4 ± 0.01
dA6
5.4 ± 0.01
0.2 ± 0.04
12.7 ± 0.04
13.0 ± 0.0
35.8 ± 0.03
27.8 ± 0.1
dA2-rU6
1.3 ± 0.01
1.3 ± 0.01
rU2
5.3 ± 0.01
5.4 ± 0.01
dA3-rU5
1.4 ± 0.01
1.4 ± 0.03
rG3
5.4 ± 0.01
5.2 ± 0.02
dT4-rA4
1.3 ± 0.01
0.0 ± 0.00
rA4
5.4 ± 0.01
3.2 ± 0.05
dC5-rG3
2.4 ± 0.01
1.4 ± 0.02
rU5
5.4 ± 0.01
4.4 ± 0.01
dA6-rU2
1.3 ± 0.01
0.7 ± 0.03
rU6
5.3 ± 0.01
3.4 ± 0.13
13.1 ± 0.02
10.6 ± 0.10
32.5 ± 0.04
25.8 ± 0.1
dA2-rU6
20.9 ± 0.8
28.1±1.2
dA2
78.9 ± 2.8
77.4 ± 2.8
dA3-rU5
19.5 ± 1.0
11.3±0.5
dA3
79.7 ± 2.9
77.5 ± 2.8
dT4-rA4
16.1 ± 0.7
20.7±0.7
dT4
78.3 ± 2.9
75.9 ± 2.9
dC5-rG3
14.9 ± 0.6
10.8±0.4
dC5
79.5 ± 2.8
61.9 ± 2.2
dA6-rU2
19.8 ± 0.7
24.1±0.9
dA6
79.0 ± 2.8
0.00 ± 0.0
dA2-rU6
12.2 ± 0.5
13.2±0.5
rU2
73.0 ± 2.6
72.1 ± 2.6
dA3-rU5
10.2 ± 0.5
8.9±0.3
rG3
75.3 ± 2.7
52.2 ± 2.0
dT4-rA4
10.1 ± 0.5
0.0±0.0
rA4
75.7 ± 2.7
21.7 ± 0.8
dC5-rG3
7.2 ± 0.3
0.0±0.0
rU5
74.1 ± 2.7
14.8 ± 0.5
dA6-rU2
8.2 ± 0.4
1.4±0.2
rU6
67.7 ± 2.4
7.47 ± 0.4
Hydration No.
Major Groove
Overall
Minor Groove
Overall
SASA
Major Groove
Minor Groove
16
Table S5.Statistics of the hydrophobic interactions present at enzyme-nucleic acid interface
observed in MD simulations.(Resolution 5.0ps, occupancy ≥30%, life time 5.0ps).
Atom pair
Occupancy (%) Average life time Number of
(ps)
events
Distance (Å)
MD
Exp
Val11:CG1--rU5:C5'
44.9
345.6
104
4.6 (0.3)
3.5
Val11:CG2--rU5:C5'
51.6
101.5
407
4.5 (0.3)
5.7
Val11:CG2--rU5:C4'
30.8
12.8
1921
4.7 (0.2)
5.5
34
11.6
2341
4.0 (0.1)
3.7
Asn44:CB--rU5:C4'
72.1
21.1
2732
3.8 (0.1)
3.7
Asn44:CB--rU5:C1'
55.1
13.5
3270
3.9 (0.1)
3.5
Asn71:CB--rA4:C5'
70.6
18.5
3049
3.8 (0.1)
4.3
Asn71:CG--rA4:C5'
77.6
28.1
2210
3.7 (0.1)
3.9
Asn71:C--rA4:C5'
67.4
22.1
2444
3.8 (0.1)
4.6
Thr43:CG2--rC5:C4'
39.2
8.7
3606
3.9 (0.1)
4.1
Thr74:CG2--rA6:C4'
62.2
15.6
3198
3.8 (0.1)
4
Thr74:CG2--rG7:C5'
34.4
8.5
3230
4.0 (0.1)
4.4
Trp78:CZ2--rA6:C5'
60.3
13.3
3630
3.8 (0.1)
4
Trp78CZ2--rA6:C4'
83.1
35.1
1895
3.7 (0.1)
3.7
Ser13:C--rU6:C5'
17
Table S6.Statistics of the observed electrostatic interactions between enzyme and nucleic acid
during MD simulations.(Resolution 5.0ps, occupancy≥30%, life time 5.0ps).
Atom pair
Occupancy (%)
Average life
time (ps)
Number of
events
Distance (Å)
MD
Exp
Glu127:OE2--RU5:P
93.2
74.9
996
3.7 (0.1)
4.5
Arg134:NH1--RU6:P
91.7
90.9
807
3.5 (0.1)
12.5
Arg134:NH1--RC7:P
85.9
51.9
1324
3.7 (0.1)
11.3
Arg134:NH2--RU6:P
94.7
101.2
749
3.6 (0.1)
13
Lys77:NZ--DG7:P
30
16.2
1459
5.6 (0.4)
5.8
Lys119:NZ--RA4:P
15.8
20.2
625
5.6 (0.3)
3.2
18
Table S7.Statistics for the water mediated hydrogen bonds observed at RNase H enzyme-DNARNA hybrid interface during MD simulations on binary complex.
(Resolution 5.0ps, life time cut off 5.0ps, occupancy cut off≥30%)
Atom pair
Occupancy (%)
Average life
time (ps)
Number of events
Val11 O...w...rU6 O2P
71.9
58
991
Ser13 O...w...rC7 O1P
39.8
14.9
2143
Asn45 OD1...w...rA4 O2'
94.8
103.5
733
Ser72 HG1...w...rA4 O2'
94.7
101.7
745
Gln73 HN...w...rG3 N3
44.5
11.1
3198
Thr74 HN...w...rG3 N3
70.5
18.8
2996
Trp120 O...w...rA4 O1P
31.9
15.4
1662
Asp123 OD1...w...rG3 O1P
60.4
19.5
2478
Asp123 OD2...w...rG3 O1P
53.5
18.2
2349
Glu127 OE1...w...rU5 O2P
93.8
42
1785
Thr74 OG1...w...dG7 O4'
54.4
14.2
3072
Gln73 HE22...w...dG7 O4'
30.3
15.3
1589
52
24.3
1715
53.5
11
3899
Lys85 O...w...dG7 O2P
Thr87 OG1...w...dC5 O1P
19