Atomistic Details of the Molecular Recognition of DNA-RNA Hybrid Duplex by Ribonuclease

SupplementaryInformation

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:

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) activesite and non-activesite 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 activesite region ofapo and holoRNase H enzyme.

Figure S3.Probability distributions of hydrogen bonds for Watson - Crick base pairs in apo and holoDNA-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 holostates.

Figure S5.Average values of base pair axis and base pair parameters for all the base pairs in DNA-RNA 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.

List of Supplementary Tables

Table S1.List of amino acids present in various secondary structural elements and respective averagermsd 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-DNA-RNA hybrid interface during MD simulations on binary complex.

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 cutoff 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.

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) activesite and non-activesite 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 activesite region ofapo and holoRNase H enzyme.

Figure S3.Probability distributions of hydrogen bonds for Watson - Crick base pairs in apo and holoDNA-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 holostates.

Figure S5.Average values of base pair axis and base pair parameters for all the base pairs in DNA-RNA 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.

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

Table S2. Probabilities of pseudorotation angles of furanose sugar puckering of each residue present in DNA-RNA hybrid duplex.

South / North / South / North
Apo / Holo / Apo / Holo / Apo / Holo / Apo / Holo
dA2 / 92 / 87 / 5 / 9 / rU2 / 0 / 0 / 100 / 99
dA3 / 59 / 64 / 29 / 31 / rG3 / 0 / 0 / 99 / 100
dT4 / 58 / 9 / 17 / 58 / rA4 / 0 / 0 / 99 / 100
dC5 / 73 / 98 / 14 / 0 / rU5 / 0 / 0 / 99 / 99
dA6 / 92 / 87 / 2 / 0 / rU6 / 0 / 0 / 99 / 99

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

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
Hydration No.
Major Groove / 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
Overall / 12.7 ± 0.04 / 13.0 ± 0.0 / 35.8 ± 0.03 / 27.8 ± 0.1
Minor Groove / 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
Overall / 13.1 ± 0.02 / 10.6 ± 0.10 / 32.5 ± 0.04 / 25.8 ± 0.1
SASA
Major Groove / 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
Minor Groove / 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

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 (ps) / Number of 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
Ser13:C--rU6:C5' / 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

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

Table S7.Statistics for the water mediated hydrogen bonds observed at RNase H enzyme-DNA-RNA 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
Lys85 O...w...dG7 O2P / 52 / 24.3 / 1715
Thr87 OG1...w...dC5 O1P / 53.5 / 11 / 3899

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