QM/MM MD simulations on the enzymatic pathway of the human flap endonuclease (hfen1) elucidate common cleavage pathways to RNase H enzymes.

Transcription

1 SUPPORTING INFORMATION QM/MM MD simulations on the enzymatic pathway of the human flap endonuclease (hfen1) elucidate common cleavage pathways to RNase H enzymes. Jacopo Sgrignani a and Alessandra Magistrato b a Institute of research in biomedicine (IRB), Via Vincenzo Vela, 6500 Bellinzona, Switzerland. b CNR-IOM-Democritos National Simulation Center c/o International School for advanced Studies (SISSA/ISAS) via Bonomea 265, Trieste, Italy Dr. Jacopo Sgrignani a Institute of research in biomedicine (IRB), Via Vincenzo Vela, 6500 Bellinzona, Switzerland. jacopo.sgrignani@irb.usi.ch Dr. Alessandra Magistrato CNR-IOM- Democritos National Simulation Center c/o International Studies for Advanced Studies Via Bonomea Trieste (TS) Italy Tel Fax alessandra.magistrato@sissa.it

2 Computational Details of Umbrella Sampling (US) Calculations Step Ic Deprotonation path C, Activation of water by the flaking nucleobase In this case one collective variable (CV1, Figure S1) was considered as reaction coordinate. In particular the difference between the distance between one of the WatR hydrogen atoms and the nearest oxygen of the T2 phosphate group and the distance between the abstracted hydrogen atom and the WatR oxygen. Moreover in order to assure a proper orientation for WatR the distance between O@WatR and the phosphorus atom was constrained between 2.40 and 2.80 Å. with a force constant of 100 kcal/mol. Harmonic bias potential of 200 kcal/mol were placed at different points along the reaction coordinate (-1,-0.50,-0.25, 0, 0.25, 0.50, 0.75, 1, 1.50, 2, 2.25, 2.50). The simulations were started in sequential manner in order to permit to the system to be equilibrated in the constrained state before moving to the subsequent point. The free energy was estimated considering the last ps of simulation. Step IIc Attack of the hydroxide to the posphate group. In this case only CV5 (Figure S3) was considered (i.e. the distance between the oxygen atom of the previously deprotonated Wat-R, the hydroxide group, and P@T1). An harmonic bias potential of 200 kcal/mol was placed at different points along the reaction coordinate (1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4 Å). Each simulation was run for 5 ps and the free energy was estimated considering the last 4 ps of each simulation. Notably these calculations were performed also considering the phosphate after protonation by Lys93. Step IIa-Proton transfer from Asp86 to the leaving oxygen of the phosphorane intermediate For this step we selected CV6 (Figure S4), namely the distance between the Asp86 carboxylic hydrogen and O3 of the phosphorane group. An harmonic bias potential of different strength was used along the reaction

3 coordinate. The strength of the force constant at each point is reported in parenthesis: 1 (350 kcal/mol), 1.25 (350 kcal/mol), 1.4 (550 kcal/mol), 1.5 (350 kcal/mol), 1.6 (400 kcal/mol), 1.68 (350 kcal/mol), 1.75 (350 kcal/mol), 2 (200 kcal/mol), 2.25 (200 kcal/mol), 2.5 (200 kcal/mol), 2.75 (200 kcal/mol), 3.0 (200 kcal/mol). For each point we performed a simulation of 5 ps and the free energy was estimated considering the last 4 ps of simulation. Steps IId and IIe (Proton transfer from Lys93 to the scissile phosphate) CV7 (Figure S5) was considered for the proton transfer from Lys93 to the apical oxygen of the scissile phosphate (OP1), namely the difference of the distance between of the Lys93@NzH and the nearest oxygen of the phosphate group (OP1@T1) and the distance between the lysine nitrogen atom and the proton, which has to be transferred. Harmonic bias potential of 200 kcal/mol were placed at different points along the reaction coordinate (-0.75,-0.50,-0.25, 0, 0.25, 0.50, 0.75, 1, 1.25, 1.50, 1.75 Å). Each point was sampled for 7 ps and the free energy was estimated considering the last 6 ps of simulation. An identical calculation was done carried out also on the substrate before the nucleophile attack (step Ie). Step IVd-Proton transfer from OP1 to In this case only CV8 (Figure S6), namely the distance between the O3 oxygen of T1 and the hydrogen atom bound to the OP1 atom was considered. Harmonic bias potential of 200 kcal/mol were placed at different points along the reaction coordinate, the strength of the force constant at each point is reported in parenthesis: (2.6 Å (200 kcal/mol), 2.4 Å (200 kcal/mol), 2.2 Å (200 kcal/mol), 2 Å (200 kcal/mol), 1.8 Å (200 kcal/mol), 1.7 Å (400 kcal/mol) (200 kcal/mol), 1.65 Å (400 kcal/mol), 1.55 Å (400 kcal/mol), 1.5 Å (400 kcal/mol), 1.50 Å (200 kcal/mol), 1.45 Å (600 kcal/mol), 1.4 Å (300 kcal/mol), 1.2 Å (200 kcal/mol) Å). Each point was simulated for 4 ps of QM/MM MD and the last 3 ps were used to estimate the free energy profile.

4 Figure S1. Graphical scheme of the collective variables considered in the MTD simulations concerning step Ia of the enzymatic reaction. CV1 was the difference of the distance depicted in blue and that in red, while CV2 was the difference of the green and pink distances. CV1 was used also in step IC, deprotonation path C. Figure S2. Graphical scheme of the collective variables considered in the MTD simulations concerning the reaction mechanism proposed by De Vivo et al. 1 step Ib. CV3 was the difference of the distance depicted in green and that in red, while CV4 was the distance in blue.

5 Figure S3. Graphical scheme of CV5 (depicted in green) considered in step IIc of the enzymatic reaction by US calculations. Figure S4. Graphical scheme of CV6 (depicted in magenta) considered in the third step (IIa) of the enzymatic reaction.

6 face Figure S5. Graphical scheme of CV7 (depicted as the difference of the purple and green distances) considered in step IId of the enzymatic reaction. Figure S6. Graphical scheme of CV8 (depicted in green) considered in step IVd of the enzymatic reaction.

7 Figure S7. Top (A) and side view (B) of the three-dimensional plots of the free energy surfaces resulting from three independent metadynamics (one simulation per row) calculations for step Ia. Free energies are reported in kcal/mol.

8 Figure S8. Top (A) and side view (B) of the three-dimensional plots of the free energy surfaces resulting from three independent metadynamics (one simulation per row) calculations for the step Ib. Free energies are reported in kcal/mol.

9 Figure S9. Free energy profile for Ic (activation of water molecule) by the adjacent phosphate, statistical uncertainty is reported using red bars.

10 Figure S10. Free energy profile for IIc (hydroxide attack), statistical uncertainty is reported using red bars.

11 Figure S11. Reaction free energy profile for Asp86 deprotonation (IIa) statistical uncertainty is reported using red bars.

12 Figure S12. Free energy profile for Lys93 deprotonation (IId) statistical uncertainty is reported using red bars.

13 Figure S13. Free energy profile for initial Lys93 deprotonation (IIe) statistical uncertainty is reported using red bars.

14 Figure S14. Free energy profile for IIIe (hydroxide attack) on a protonated phosphate, statistical uncertainty is reported using red bars.

15 Figure S15. Free energy profile for the final proton transfer from O1P to (step IVe) statistical uncertainty is reported using red bars.

16 Table S1. Average coordination distances (Å) for MgA and MgB obtained from the QM/MM MD. Standard deviations are reported in parenthesis. MgA MgB 2.33(0.08) (0.06) (0.08) (0.07) (0.06) 2.25(0.06) 2.29(0.06) (0.06) (0.06) (0.06) 2.36(0.09) 2.31(0.08) (1) De Vivo, M.; Dal Peraro, M.; Klein, M. L. J. Am. Chem. Soc. 2008, 130,