Folding of a DNA hairpin loop structure in explicit solvent using replica-exchange molecular dynamics simulations

Abstract

Hairpin loop structures are common motifs in folded nucleic acids. The 5'-GCGCAGC sequence in DNA forms a characteristic and stable trinucleotide hairpin loop flanked by a two basepair stem helix. To better understand the structure formation of this hairpin loop motif in atomic detail, we employed replica-exchange molecular dynamics (RexMD) simulations starting from a single-stranded DNA conformation. In two independent 36 ns RexMD simulations, conformations in very close agreement with the experimental hairpin structure were sampled as dominant conformations (lowest free energy state) during the final phase of the RexMDs ( approximately 35% at the lowest temperature replica). Simultaneous compaction and accumulation of folded structures were observed. Comparison of the GCA trinucleotides from early stages of the simulations with the folded topology indicated a variety of central loop conformations, but arrangements close to experiment that are sampled before the fully folded structure also appeared. Most of these intermediates included a stacking of the C(2) and G(3) bases, which was further stabilized by hydrogen bonding to the A(5) base and a strongly bound water molecule bridging the C(2) and A(5) in the DNA minor groove. The simulations suggest a folding mechanism where these intermediates can rapidly proceed toward the fully folded hairpin and emphasize the importance of loop and stem nucleotide interactions for hairpin folding. In one simulation, a loop motif with G(3) in syn conformation (dihedral flip at N-glycosidic bond) accumulated, resulting in a misfolded hairpin. Such conformations may correspond to long-lived trapped states that have been postulated to account for the folding kinetics of nucleic acid hairpins that are slower than expected for a semiflexible polymer of the same size.

FIGURE 1

Heavy atom Rmsd of sampled DNA conformations (5′-GCGCAGC) from (A) folded hairpin structure and (B) single-stranded start structure versus simulation time. Results are shown for two independent 75 ns simulations starting from the same single-stranded DNA with different initial atomic velocity assignments (dotted and continuous lines, respectively).

FIGURE 2

(A) Rmsd (heavy atoms) of the 5′-d(GCGCAGC) conformations (from lowest temperature run of each RexMD simulation) with respect to the folded hairpin reference structure versus simulation time. The panel on the right of each Rmsd plot corresponds to the Rmsd probability distribution during the first (continuous line), second (dashed line), and last (dotted line) 12 ns of each simulation. (B) Single-stranded start structure and fully folded hairpin loop structure (sampled as the dominant state of both simulations after ∼20 ns).

FIGURE 3

Comparison of an ensemble of NMR structures of the (A) GCA trinucleotide loop (four structures of PDB entry: 1ZHU; sequence: 5′-dATGCAAT) and four randomly selected structures obtained during the final stage of the (B) RexMD simulation A with a heavy atom Rmsd of <2 Å from the folded reference hairpin structure. (C) Superposition of “misfolded” DNA hairpin structures with the loop guanine (G₃) in a syn conformation and the loop adenine (A₅) partially stacked in the DNA minor groove.

FIGURE 4

Representative structures (stick representation) of conformational clusters obtained during three different phases of the RexMD simulations. Each structure corresponds to a conformation closest to the average structure of a cluster (cluster centroid) with a cluster population around or above 1% of all recorded structures during the corresponding time interval. Cluster analysis was performed with an Rmsd cutoff of 2 Å and using the kclust program of the MMTSB package (48). The color in the stick representation goes gradually from red (5′-DNA end) to blue (3′-DNA end) to get an impression of the chain orientation. For clarity, hydrogen atoms have been omitted.

FIGURE 5

(A) Rmsd of sampled conformations (during lowest temperature run) with respect to the native trinucleotide loop structure (only of the three central nucleotides, in black) and with respect to the stem structure of the folded hairpin conformation (considering only the two stem basepairs, in red). (B) Superposition of five conformations obtained during the 7–10 ns simulation time interval with near-native trinucleotide loop structure but not correctly formed stem structure. Loop nucleotides C₂ (gray) to A₅ (green) are shown as bond sticks and using color coding according to residue number.

FIGURE 6

Deviation of the central three nucleotides (x axis) and four stem nucleotides (y axis) from the folded reference DNA hairpin structure during four different time intervals of the RexMD simulations. Dark/light regions in the 2D plots indicate a high/low probability, respectively, for a given pair of central loop and stem Rmsds.

FIGURE 7

Specific water binding to the hairpin loop motif in the DNA minor groove. (A) Superposition of four sampled structures with the near-native trinucleotide loop structure and a water molecule bridging the O₂ atom of C₂ (gray) and the N₁ atom of the A₅ (green) nucleobase. A water molecule was found at this position in more than 90% of the recorded conformations where the loop had correctly formed. The view is into the minor groove and using the same color coding as in Fig. 5. (B) Accessible surface area representation of one simulation snapshot (color coding of residue numbers) with a bound water molecule bridging C₂ (gray) and A₅ (bold bond stick model). Two minor water binding sites (thin bond stick water model) bridging phosphate groups and the A₅ base (occupancy ∼40% in recorded conformations with a native-like trinucleotide loop structure) are also indicated.

FIGURE 8

Folding intermediates of the DNA-trinucleotide hairpin loop. Each of the snapshots from various stages of the RexMD simulations contains a frequently found structural motif of the central nucleotides (color coded and using bold sticks). “Correctly folded” loop motifs correspond to a similar helical arrangement of the central loop nucleotides as the native hairpin structure. These intermediates are likely to rapidly progress toward the fully folded conformation. The syn-G₃ loop motif is sterically also compatible with a fully folded hairpin, but it retains the misfolded helical arrangement of the central loop nucleotides. “Misfolded loop” motifs strongly deviate from the native trinucleotide loop structure (only a few examples are shown) and are unlikely to progress rapidly toward a fully folded hairpin structure.

FIGURE 9

Contribution of native-like hairpin loop structures (within an Rmsd of <2.0 Å of the folded reference structure) at various stages of the RexMD simulations (indicated by different plot textures). Contributions are given as percentage of the total ensemble at each replica simulation temperature.