Simulations predict preferred Mg2+ coordination in a nonenzymatic primer-extension reaction center

The mechanism by which genetic information was copied prior to the evolution of ribozymes is of great interest because of its importance to the origin of life. The most effective known process for the nonenzymatic copying of an RNA template is primer extension by a two-step pathway in which 2-aminoimidazole-activated nucleotides first react with each other to form an imidazolium-bridged intermediate that subsequently reacts with the primer. Reaction kinetics, structure-activity relationships, and X-ray crystallography have provided insight into the overall reaction mechanism, but many puzzles remain. In particular, high concentrations of Mg2+ are required for efficient primer extension, but the mechanism by which Mg2+ accelerates primer extension remains unknown. By analogy with the mechanism of DNA and RNA polymerases, a role for Mg2+ in facilitating the deprotonation of the primer 3′-hydroxyl is often assumed, but no catalytic metal ion is seen in crystal structures of the primer-extension complex. To explore the potential effects of Mg2+ binding in the reaction center, we performed atomistic molecular dynamics simulations of a series of modeled complexes in which a Mg2+ ion was placed in the reaction center with inner-sphere coordination with different sets of functional groups. Our simulations suggest that coordination of a Mg2+ ion with both O3′ of the terminal primer nucleotide and the pro-Sp nonbridging oxygen of the reactive phosphate of an imidazolium-bridged dinucleotide would help to pre-organize the structure of the primer/template substrate complex to favor the primer-extension reaction. Our results suggest that the catalytic metal ion may play an important role in overcoming electrostatic repulsion between a deprotonated O3′ and the reactive phosphate of the bridged dinucleotide and lead to testable predictions of the mode of Mg2+ binding that is most relevant to catalysis of primer extension.

Figure S1.Distances observed in the crystal structure PDB 6C8E used for constraints during equilibration.(A) Structure of PDB 6C8E with RNA duplex shown in cartoon representation, and the 2AI-bridged dimer in licorice representation.(B) Zoomed in view from (A) showing O3'-P and O2'-P distances when 2AI is facing the major groove.(C) Zoomed in view from (A) showing O3'-P and O2'-P distances when 2AI is facing the minor groove.

Figure S2 .
Figure S2.RMSD of the duplex, bridged dinucleotide, and reaction center suggest increased dynamics with a deprotonated 3′-OH.(A) Overlay of simulation snapshots over a 500 ns simulation trajectory of the complex with primer 3′-OH w/o Mg 2+ .The first frame of the simulation can be seen partly as a dark gray ribbon representation.The G*G bridged dinucleotide is highlighted in an orange box.(B) Time series of root mean-squared deviation (RMSD) of the entire duplex for five simulation replicates.Comparing RMSD probability distribution for the (C) nucleic acid region and (D) G*G bridged dinucleotide (2AI-bridged dinucleotide only) for the simulation systems, 3′-OH w/o Mg 2+ (Sim1; dark blue) and 3′O -w/o Mg 2+ (Sim2; light blue).(E) Time series of the G*G dinucleotide RMSD of the 3′-OH w/o Mg 2+ (Sim1a-e; left) and 3′O -w/o Mg 2+ (Sim2a-e; right) simulation systems for all simulation replicates showing the 1 ns running averages.(F) Time series of the nucleic acid region RMSD of the 3′O -w/o Mg 2+ (Sim2a-e) simulation system for all simulation replicates showing the 1 ns running averages.(G) Time series of the reaction center RMSD, comprised of the G*G bridged dinucleotide and 3′terminal primer guanosine, of the 3′OH w/o Mg 2+ (Sim1; left), and the 3′O -w/o Mg 2+ (Sim2; right).

Figure S3 .
Figure S3.QM/MM simulations of the 3′O -w/ Mg 2+ @SP, 2-NH2-Im:RP system (Sim10).(A) Molecular figure of the simulation system in licorice representation showing the portion of the duplex modeled in MM (grey) and the 49 atoms modeled using QM (colored by atom type).(B) The angle of attack during the QM/MM simulation, measured between O3′-P-N atoms, which agrees well with the CHARMM simulations (Figure 5B).(C) Time series of the O3′ (primer)-P (bridged dinucleotide) distance during the QM/MM simulation.The inset shows a probability distribution with the mean value indicated, obtained from a normal fit.This distance agrees well with the average distance from the CHARMM simulations (Figure 4D).(D) Molecular figure showing the O3′-Mg 2+ -O(Sp) angle measured during the QM/MM simulation (E) Time course of the angle measured in (D).The inset shows a probability distribution with the mean value indicated, obtained from a normal fit.This angle agrees well with the average angle from the CHARMM simulations (Figure S16).(F) Circular histogram of pseudorotation angles of the terminal primer nucleotide sugar during the QM/MM simulation.The phase angles are based on the Altona-Sundaralingam 1 definition and are assigned to the puckering modes in multiples of 36°.(G) A molecular figure of the QM system with calculated SCF electron density colored by the electrostatic potential with positive charge in blue and negative charge in red.(H) A zoomed in view of (G), showing the polarization on the H2′ and O3′.(I) Time series of the H2′ and O3′ distance for the MM and QM/MM simulations, showing the tighter interaction in QM/MM due to polarization of the H2′.The time indicated corresponds to the QM/MM simulations, while the trace corresponding to the MM simulation goes to 3 ns.(J) Summary of measured parameters.

Figure S4 .
Figure S4.Terminal primer sugar puckering in the absence of bound Mg 2+ and a major groove-facing 2AI.Circular histogram of pseudorotation angles and amplitudes of the terminal primer nucleotide sugar for the (A) 3′-OH w/o Mg 2+ (Sim1) and (B) 3′O -w/o Mg 2+ (Sim2) simulation systems.The phase angles are based on the Altona-Sundaralingam 1 definition and are assigned to the puckering modes in multiples of 36°.Sugar pucker conformations of the primer nucleotide from the crystal structure, denoted with ⨯ markers in the circular plot show the sugars are in the C3′-endo conformation.Configurations that were more likely to be occupied are colored in blue, while less probable configurations are colored red.

Figure S5 .
Figure S5.Time course of the terminal primer sugar pucker phase angle.(A) Phase angle of the terminal primer sugar for Sim1a.Individual data points shown as black circles, with a 0.15 ns running average.Horizontal lines show the range of angles corresponding to the O4'-endo conformation (72˚-108˚).(B) Phase angle of the terminal primer sugar for Sim2a.Individual data points shown as black circles, with a 0.15 ns running average.Horizontal lines show the range of angles corresponding to the O4'-endo conformation (72˚-108˚).For some transitions from C3'-endo to C2'-endo in both systems, data points between 72˚-108˚ indicate a transition through the O4'-endo conformation.

Figure S6 .
Figure S6.Time series of the distance O3′ (primer)-P (bridged dinucleotide) and primer nucleotide sugar pucker angle for the major groove-facing 3′-OH w/o Mg 2+ simulation system (Sim1) for all five replicates.The horizontal dashed lines indicate the crystal structure value.Intervals with larger distance values and C2′-endo sugar conformations are highlighted in gray.For all time series plots, dark traces show the data averaged over a 1 ns window, while the lighter envelope shows the full range of the data recorded at 10 ps time steps in our simulations.

Figure S7 .
Figure S7.Time series of the distance O3′ (primer)-P (bridged dinucleotide) and primer nucleotide sugar pucker angle for the major groove-facing 3′O -w/o Mg 2+ simulation system (Sim2) for all five replicates.The horizontal dashed lines indicate the crystal structure value.Intervals with larger distance values and C2′-endo sugar conformations are highlighted in gray.For all time series plots, dark traces show the data averaged over a 1 ns window, while the lighter envelope shows the full range of the data recorded at 10 ps time steps in our simulations.

Figure S8 .
Figure S8.Terminal primer sugar puckering in the absence of bound Mg 2+ and a minor groove facing 2AI.Circular histogram of pseudorotation angles of the terminal primer nucleotide sugar for the (A) 3′-OH w/o Mg 2+ (Sim3) and (B) 3′O - w/o Mg 2+ (Sim4) simulation systems where 2-NH2-Im orientation is minor groove-facing.The phase angles are based on the Altona-Sundaralingam 1 definition and are assigned to the puckering modes in multiples of 36°.Configurations that were more likely to be occupied are colored in blue, while less probable configurations are colored red.

Figure S11 .
Figure S11.Comparison of CHARMM (Sim1) and AMBER (Sim9) 3′-OH w/o Mg 2+ simulations where 2-NH2-Im orientation is major groove-facing.Terminal primer sugar puckering for (A) 0-100 ns CHARMM, (B) 75-175 ns CHARMM, and (C) AMBER simulation systems.In (C), data points from the first 100 ns are shown on top, while data points from the second 100 ns are shown underneath in blue.The phase angles are based on the Altona-Sundaralingam 1 definition and are assigned to the puckering modes in multiples of 36°.(D) RMSD values (calculated for nucleic acid and bridged dinucleotide heavy atoms, see methods) of the 0-100 ns CHARMM and two 100 ns AMBER sub-trajectories.The inset shows a probability distribution for each, with mean values indicated in the corresponding color, obtained from a normal fit.Time series of the O3′ (primer)-P (bridged dinucleotide) distance for the 0-100 ns CHARMM and two 100 ns AMBER sub-trajectories (E), and the 75-175 ns CHARMM and two 100 ns AMBER sub-trajectories (F).The insets show a probability distribution for each.(G) The angle of attack during the 0-100 ns CHARMM (top) and two 100 ns AMBER (bottom) sub-trajectories, measured between O3′-P-N atoms.(H) Time series of the distances d(N1,N3) between the G-C base pairs flanking the bridged dinucleotide in the 0-100 ns CHARMM and AMBER simulations.The inset indicates which color traces correspond to the upstream and downstream positions for each simulation.(I) Summary of measured parameters comparing between the AMBER and CHARMM systems.

Figure S14 .
Figure S14.A bridging Mg 2+ in the reaction center stabilizes the primer extension complex.Comparing mean ± standard deviation RMSD of (A) nucleic acid region and (B) bridged dinucleotide heavy atoms among the seven simulation ensembles discussed in this work.Systems highlighted in the main text are outlined.

Figure S17 .
Figure S17.The presence of Mg 2+ reduces electrostatic repulsion in the reaction center complex.A molecular figure of the QM system with calculated SCF electron density colored by the electrostatic potential with positive charge in blue and negative charge in red for the system without (A; Sim10) and with (B; Sim11) Mg 2+ present, visualized at the same isovalue.Total Gibbs free energy (G) and Gibbs free energy minus electronic energy (G-Eel) are shown calculated using an HF-3c method with an implicit solvent model for each system.(C) Zoomed in view of (A), showing the O3'-P distance at the end of the simulation.(D) Zoomed in view of (B), showing the O3'-P distance at the end of the simulation, and a quantitatively reduced potential compared to (C) suggested by the lighter color of the electrostatic map.(E) Coulombic force calculated between the indicated atoms for the system without Mg 2+ (Sim10), with Mg 2+ (Sim11), and the system where Mg 2+ was removed (Sim12).(F) Coulombic force calculated between the indicated atoms for the system with Mg 2+ (Sim11), showing that in addition to the electrostatic influences demonstrated in (E), the presence of Mg 2+ also exerts significant attractive forces between the relevant O atoms in the reactive center.

Figure S18 .Figure S19 .Figure S20 .
Figure S18.Hydrogen bonds h1 and h2 between 2-NH2-Im and non-bridging oxygens on the bridged dinucleotide suggest a preferred preorganization geometry.(A) The major groove-facing and (B) minor groove-facing orientation of the bridged dinucleotide are shown.(C-F) Probability distribution of the hydrogen bond distances h1 and h2 for the various simulation systems.Median distance values from simulation with major and minor groove-facing orientations of bridged dinucleotide are shown in solid and dashed lines, respectively.Lines in black show hydrogen bond distances observed in the crystal structure.3′-OH w/o Mg 2+ , 2-NH 2 -Im:R P 3′O -Mg 2+ @S P , 2-NH 2 -Im:R P 3′O -Mg 2+ @R P , 2-NH 2 -Im:R P