Dissociation of minor groove binders from DNA: insights from metadynamics simulations

Supplementary Information

Dissociation of minor groove binders from DNA: insights from metadynamics simulations

(dCMs, nhb) (dCMs, nhph) (dCMs, nhph, nhb)

IMI●DNA 5.2 3.1 7.5

DST●DNA 2.8 3.2 10.1

DST●DNA’ 3.2 - -

Table S1. Total metadynamics simulation time (ns) needed to the ligand to dissociate from the duplex, in the various systems and with different choices of the active CVs.

IMI●DNA Min 1 TS 1-2 TS 1-3 Min 2 Min 3 TS 3-4 Out Free Shortening (%) 4.0 (1.2) 3.7 (1.2) 1.9 (0.8) 3.9 (1.0) 2.2 (0.7) 2.2 (1.0) 1.7 (0.6) 2.3 (0.9) RMSDB-DNA 3.3 (0.4) 3.2 (0.4) 2.8 (0.5) 3.2 (0.3) 2.8 (0.4) 2.8 (0.1) 3.2 (0.3) 3.6 (0.7) RMSDA-DNA 5.5 (0.5) 5.5 (0.4) 5.5 (0.4) 5.8 (0.4) 5.4 (0.4) 5.4 (0.2) 5.6 (0.5) 5.0 (0.6) RMSDFree 2.3 (0.4) 2.3 (0.3) 2.2 (0.4) 2.2 (0.3) 2.2 (0.3) 2.3 (0.2) 2.0 (0.4) - RMSDMin1 - 1.4 (0.2) 2.1 (0.2) 1.2 (0.3) 2.1 (0.2) 2.0 (0.2) 2.6 (0.3) 2.6 (0.5) Dihe1 27 (6) 27 (7) 30 (7) 27 (7) 31 (7) 29 (8) 31 (7) - Dihe2 12 (7) 13 (7) 15 (8) 13 (7) 16 (9) 13 (7) 18 (11) -

Table S2. Row1: Shortening of the duplex calculated along the LFEP path of Figure 1 for IMI•DNA (last column, Free, refers to the simulation of the duplex in solution). Rows 2-5: average RMSD (Å, standard deviations in brackets) of the duplex from canonical B and A DNA, and from ‘Free’ and ‘Min1’ conformations. Rows 6-7: Dihedral angles between benzene and pyrrol rings (Dihe1) and between the pyrrol and the tail CONH2 (Dihe2) of IMI. As can be seen, the structure of

IMI does not change significantly in the process.

DST●DNA Min 1 TS 1-2 Min 2 TS 2-3 Min 3 TS 3-4 Out Free Shortening (%) 1.6 (1.0) 1.7 (0.3) 1.4 (0.6) 1.6 (0.2) 1.8 (0.4) 1.8 (0.2) 2.0 (0.4) 1.3 (0.6) RMSDB-DNA 1.6 (0.4) 1.8 (0.2) 2.1 (0.4) 1.8 (0.2) 1.9 (0.3) 2.3 (0.2) 2.2 (0.3) 2.4 (0.3) RMSDA-DNA 5.0 (0.3) 5.1 (0.2) 5.0 (0.3) 4.8 (0.2) 5.1 (0.3) 4.7 (0.2) 5.2 (0.4) 4.3 (0.3) RMSDFree 2.0 (0.4) 0.9 (0.3) 1.4 (0.4) 1.8 (0.3) 1.0 (0.3) 1.6 (0.3) 1.0 (0.4) - RMSDMin1 - 1.7 (0.2) 1.7 (0.2) 1.5 (0.2) 1.6 (0.3) 1.9 (0.2) 2.1 (0.4) 2.1 (0.3) Dihe1 21 (12) 16 (15) 21 (3) 4 (22) -4 (18) -9 (4) -10 (9) - Dihe2 22 (12) -26 (25) 29 (18) 17 (22) -20 (16) -11 (7) -17 (10) - Dihe3 89 (27) 9 (22) -16 (14) 26 (42) 108 (35) -71 (21) 81 (24) -

Table S3. Row1: Shortening of the duplex calculated along the LFEP path in Figure 2 for DST•DNA (last column, Free, refers to the simulation of the duplex in solution). Rows 2-5: average RMSD (Å, standard deviations in brackets) of the duplex from canonical B and A DNA, and from ‘Free’ and ‘Min1’ conformations. Rows 6-8: Dihedral angles between rings A and B (Dihe1), B and

C (Dihe2), and between ring C and the amidinium tail of (Dihe3) of DST. The larger internal flexibility of DST is reflected in the large variation of its internal structure, in particular of Dihe3.

N H N N H N OH NH H3C

Chart S1. Chemical structure of Hoechst 33258.

Figure S1. _{Orientation of the ligand DST in the complexes DST●DNA (left) and DST●DNA’}

Figure S2.

∆

and transition states along the LFEP are reported, along with the values in kcal/mol of the associated free energies.

Figure S3. Close-up view of the difference between conformations Min 1 and Min 2 in IMI•DNA. The two structures are substantially superimposable, although the ribose sugar and the nucleoside

G8 are rotated relatively to each other.

Figure S4. View of the widening of the minor groove in configurations TS1-3 (left) and Min 3 (right) of IMI•DNA. This widening allows for the hydration of the region between the drug and strand1, which appears to be an important pre-requisite for the exit of the compound

T ra n si ti o n 3 T ra n si ti o n 2 T ra n si ti o n 1

Twist Roll Tilt

“Transitions” between two minima: Transition 1 (bottom row) corresponds to Min1↔ TS1-2 ↔ Min2, Transition 2 (middle row) to Min1 ↔ TS1-3 ↔ Min3, Transition 3 (upper row) to Min3 ↔ TS3-4 ↔ Out. In this latter transition, instead of the values corresponding to this latter configuration, we have reported those from a 6 ns MD of the solvated DNA duplex (Free). In each graph data corresponding to Min 1 are also reported to give a reference of the changes along the dissociation pathway.

Configurations considered at Transition 1 feature very similar values of all parameters, while some differences are seen between conformations involved in Transition 2, mainly in the region around the drug. A local unwinding at steps d[G5T6]2 and d[T7G8]2 is recognizable in Min3 and TS1-3,

which is correlated to the opening of the minor groove at the binding region (see Figure 3a and in particular the “bump” clearly visible at TS1-3 in Figure 1). Such a distortion is slightly compensated by the winding characterizing the adjacent steps d[C4G5]2 and d[G8G9]2. In addition,

the jumps in the value of the tilt at the tract d[T5…G8]2, also correlated to small and local kinks in

the duplex (see Figure S7) are significantly reduced in TS1-3 and Min3.

Along Transition 3 the conformational changes with respect to Min1, as well as to conformation “Free”, are even more evident. First, the unwinding at step d[G5T6]2 and the compensating effect at

adjacent steps increase in TS3-4 with respect to Min 3. Notice the flatter profile of the twist in “Free” with respect to all configurations along the dissociation pathway. Changes in the roll and tilt profiles are also clearly present.

However, it is important to note that all these forementioned local distortions do not cause an appreciable bending of the DNA duplex (Figure S7, Table S2). This is in agreement with suggestions by Lavery and Sklenar, who pointed out that variations in local parameters like roll and tilt does not necessary induces global distortion in the helix (1).

T ra n si ti o n 3 T ra n si ti o n 2 T ra n si ti o n 1

Twist Roll Tilt

Transition1 is accompanied by an unwinding at the adjacent steps d[T6A7]2 and d[A7G8]2, which is

consistent with the increase of the minor groove width affecting the same tract (see Figure 3b). Additionally, the unwinding is probably correlated with the augmented tilt (opening towards strand #1) observed at the central step d[T5T6]2 adjacent to the unwound tract. The roll does not undergo

major significant changes.

Along Transition 2 the configuration Min3 is characterized by a local twisting at the tract d[A7G8]2

with respect to Min 1 and Min 2, while roll and tilt decrease with respect to Min 2 and a flattening of the profiles in the central region of the duplex is observed.

Transition 3: the profile of the twist at TS3-4 exhibits the most relevant changes at the ends of the duplex with respect to conformation Min3 (and Min1). In particular, the inversion in the profile at d[T6…G8]2 is consistent with the difference in the minor groove width profile in the same tract (Figure 3b). In contrast, the trend is similar in the DST-binding tract d[A4…A7]2. Note that, as in

the case of IMI●DNA, the profile of the twist associated to the free oligonucleotide is quite flat. At opposite to the twist, the roll features a consistent increase in the DST-binding tract; in particular the peaks at d[G3A4]2 and d[T6A7]2 (the former only present in this conformation and in that “Free”)

are responsible for a local bending towards the major groove (see Figure S8 below). Additionally, a slight opening of the duplex towards strand #1 is caused by the increase in tilt at steps T5T6 and A7G8 with respect to Min3 (and Min 1).

As in the case of IMI●DNA, these local distortions do not cause an appreciable bending of the DNA duplex (Figure S8).

Transition 1 Transition 2 Transition 3

Figure S7. DNA axis bending upon dissociation of IMI: configurations involved in the three transitions are reported (Figures generated with VMD (2) from output of Curves (3)). Color legend: red, Min1; dark pink, TS1-2; pink, Min2; silver, TS1-3; white, Min3; iceblue, TS3-4; blue, Free.

Transition 1 Transition 2 Transition 3

Figure S8. DNA axis bending upon dissociation of DST: configurations involved in the three transitions are reported (Figures generated with VMD (2) from output of Curves (3)). Color legend: red, Min1; dark pink, TS1-2; pink, Min2; silver, TS2-3; white, Min3; iceblue, TS3-4; blue, Free.

References

1. Lavery, R. and Sklenar, H. (1988) The definition of generalized helicoidal parameters and of axis curvature for irregular nucleic acids. J. Biomol. Struct. Dyn., 6, 63-91.

2. Humphrey, W., Dalke, A. and Schulten, K. (1996) VMD: visual molecular dynamics. J. Mol. Graph., 14, 33-38.

3. Ravishankar, G., Swaminathan, S., Beveridge, D.L., Lavery, R. and Sklenar, H. (1989) Conformational and helicoidal analysis of 30ps of molecular dynamics on the

d(CGCGAATTCGCG) double helix: "Curves", Dials and Windows. J. Biomol. Struct. Dyn., 6, 669-699.