Conformational distortions induced by periodically recurring A…A in d(CAG).d(CAG) provide stereochemical rationale for the trapping of MSH2.MSH3 in polyQ disorders

Abstract

CAG repeat instability causes a number of neurodegenerative disorders. The unusual hairpin stem structure formed by the CAG repeats in DNA traps the human mismatch repair MSH2.MSH3 (Mutsβ) complex. To understand the mechanism behind the abnormal binding of Mutsβ with the imperfect hairpin stem structure formed by CAG repeats, molecular dynamics simulations have been carried out for Mutsβ-d(CAG)₂(CAG)(CAG)₂.d(CTG)₂(CAG)(CTG)₂ (1 A…A mismatch) and Mutsβ-d(CAG)₅.d(CAG)₅ (5 mismatches, wherein, A…A occurs periodically) complexes. The interaction of MSH3 residue Tyr₂₄₅ at the minor groove side of A…A, an essential interaction responsible for the recognition by Mutsβ, are retained in both the cases. Nevertheless, the periodic unwinding caused by the nonisostericity of A…A with the flanking canonical base pairs in d(CAG)₅.d(CAG)₅ distorts the regular B-form geometry. Such an unwinding exposes one of the A…A mismatches (that interacts with Tyr₂₄₅) at the major groove side and also facilitates the on and off hydrogen bonding interaction with Lys₅₄₆ sidechain (MSH2-domain-IV). In contrast, kinking of the DNA towards the major groove in Mutsβ-d(CAG)₂(CAG)(CAG)₂.d(CTG)₂(CAG)(CTG)₂ doesn't facilitate such an exposure of the bases at the major groove. Further, the unwinding of the helix in d(CAG)₅.d(CAG)₅ enhances the tighter binding between MSH2-domain-I and d(CAG)₅.d(CAG)₅ at the major groove side as well as between MSH3-domain-I and MSH3-domain-IV. Markedly, such enhanced interactions are absent in Mutsβ-d(CAG)₂(CAG)(CAG)₂.d(CTG)₂(CAG)(CTG)₂ that has a single A…A mismatch. Thus, the above-mentioned enhancement in intra- and inter- molecular interactions in Mutsβ-d(CAG)₅.d(CAG)₅ provide the stereochemical rationale for the trapping of Mutsβ in CAG repeat expansion disorders.

Keywords: A…A mismatch; CAG repeat expansion; MSH2.MSH3; Molecular dynamics simulations; Polyglutamine diseases.

Fig. 1

Cartoon representation of the crystal structure of MSH2.MSH3 and a DNA (colored in cyan) having a bulge (PDB ID: 3THX). Note that the different domains of MSH2 and MSH3 are colored differently.

Fig. 2

MutSβ interaction with the CAG-1AA at the mismatch site. (A) Time vs hydrogen bond distance plot showing the complete loss of hydrogen bond between the mismatched A₈ and A₂₃ in the CAG-1AA substrate of MutSβ-CAG-1AA complex. (B) Time vs hydrogen bond distance plot showing the formation of A₂₃(N7)…Tyr₂₄₅(O), A₂₃(N6)…Tyr₂₄₅(O), A₈(O4′)…Tyr₂₄₅(N), A₂₃(N7)…Lys₂₄₆(NZ) and A₈(N3)…Lys₂₄₆(NZ) hydrogen bonds. (C, D) Snapshot showing (C) the interaction of Tyr₂₄₅ with A₈ and A₂₃ and, (D) the kink at the mismatch site of the DNA substrate at 500 ns. (E) Snapshot illustrating the interaction of Arg₃₁₃(MSH3) to a base of the substrate (500 ns).

Fig. 3

MutSβ interaction with the CAG-5AA substrate. A) Time vs hydrogen bond distance plots corresponding to (A) A₂₃(N7)…A₈(N6), (B) Lys₅₄₆(NZ)… A₈(N7) and Tyr₂₄₅(O)…A₈(N3). Note the on and off interaction of Lys₅₄₆ and Tyr₂₄₅ with A₈ can occur either simultaneously or individually. C) Snapshots showing the simultaneous Tyr₂₄₅(O)…A8(N3) (minor groove) and Lys₅₄₆(NZ)…A₈(N7) (major groove) hydrogen bond formation at 215 ns. (D, E) Hydrogen bond distance plot corresponding to D) A₅…A₂₆ and E) A₁₁…A₂₀. Note the short residence time of hydrogen bonds in (E).

Fig. 4

Na⁺ ion coordination network that tightens the interaction between A₅…A₂₆ and MSH2-domain-I in MutSβ-CAG-5AA. (A) Snapshots showing the Na⁺ mediated network involving A₂₆, G₂₇, C₂₈, G₃, C₄ and Asp-41 residues. (B–D) Distance plots describing the coordination of Na⁺ with DNA/protein residues: Na⁺…C₂₈ (N4) (B), Na⁺…A₂₆(N6) (B), Na⁺…G₂₇(O6) (B), Na⁺…G₃(O6) (C), Na⁺…C₄(N4) (C), Na+…Asp₄₁(OD2) (D) and Asp₄₁(OD1)…C₄(N4) (D). Note that the coordination distances given in (B-D) represent the interactions (indicated as a, b, c, d, e, f and g) shown in (A).

Fig. 5

Cartoon diagram illustrating the nearness (CAG-5AA) or farness (CAG-1AA) of domain-I (colored blue) and domain-IV (colored red) of MSH3. (A-C) MutSβ-DNA substrate complex corresponding to (A) MutSβ-CAG-5AA (500 ns) and (B) MutSβ-CAG-1AA (500 ns) and (C) the crystal structure (PDB ID: 3THX). Note that the arrows indicate (zoomed view) the notable differences seen in the domain movements of the (A-C) three complexes. The proximity of the domain-I and IV can be seen in (A) MutSβ-CAG-5AA which is absent in (B) MutSβ-CAG-1AA as indicated by the arrows. Note that the DNA substrate is shown in cyan color. See also Supplementary Movies S1 and S2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Exposure of the bases towards the major groove in MutSβ-CAG-5AA and its absence in MutSβ-CAG-1AA illustrated by considering 500 ns structure as the representative structure. (A) Extension and (B) compression of the DNA substrate (Left, cyan surface) and the consequent exposure of the bases towards the major groove in (A) MutSβ-CAG-5AA and its absence in (B) MutSβ-CAG-1AA can be seen at the mismatch site. The double headed arrows indicate the extension and compression of the substrates. Note the kink in the DNA towards the major groove in MutSβ-CAG-1AA doesn’t expose the bases to MutSβ (B, Top-Right, Bottom-Right), whereas the exposure of the bases toward the major groove in MutSβ-CAG-5AA facilitates its interaction with MutSβ (A, Top-Left, Bottom-Left, indicated by single headed arrows). Note that the terminal 2 base pairs on both the sides of the DNA substrates are not shown due to the end fraying effect. (C) The crystal structure of 16-mer DNA substrate (Left, cyan surface) (with an A…A mismatch) and its complex with E. coli (PDB ID: 1OH6) homologue of human MutSβ mismatch repair complex is shown for comparison. A compression at the A…A mismatch site as seen in (B) and the consequent inaccessibility of the bases to the protein can readily be seen. Note that the human MutSβ-DNA complex (PDB ID:3THX) is available only with a DNA loop region (viz., not with an A…A or any other mismatches) and thus, is not shown here. The A…A mismatch is indicated in the golden color and the protein is shown in the blue color cartoon. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)