Exploring the Conformational and Binding Dynamics of HMGA2·DNA Complexes Using Trapped Ion Mobility Spectrometry-Mass Spectrometry

Abstract

The mammalian high mobility group protein AT-hook 2 (HMGA2) is an intrinsically disordered DNA-binding protein expressed during embryogenesis. In the present work, the conformational and binding dynamics of HMGA2 and HMGA2 in complex with a 22-nt (DNA₂₂) and a 50-nt (DNA₅₀) AT-rich DNA hairpin were investigated using trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) under native starting solvent conditions (e.g., 100 mM aqueous NH₄Ac) and collision-induced unfolding/dissociation (CIU/CID) as well as solution fluorescence anisotropy to assess the role of the DNA ligand when binding to the HMGA2 protein. CIU-TIMS-CID-MS/MS experiments showed a significant reduction of the conformational space and charge-state distribution accompanied by an energy stability increase of the native HMGA2 upon DNA binding. Fluorescence anisotropy experiments and CIU-TIMS-CID-MS/MS demonstrated for the first time that HMGA2 binds with high affinity to the minor groove of AT-rich DNA oligomers and with lower affinity to the major groove of AT-rich DNA oligomers (minor groove occupied by a minor groove binder Hoechst 33258). The HMGA2·DNA22 complex (18.2 kDa) 1:1 and 1:2 stoichiometry suggests that two of the AT-hook sites are accessible for DNA binding, while the other AT-hook site is probably coordinated by the C-terminal motif peptide (CTMP). The HMGA2 transition from disordered to ordered upon DNA binding is driven by the interaction of the three basic AT-hook residues with the minor and/or major grooves of AT-rich DNA oligomers.

Figure 1.

Sequences of (a) HMGA2 and (b) DNA hairpins, and (c) chemical structure of Hoechst 33258 minor groove binder. The three AT-hook DNA binding regions (red) and the negatively charged CTMP (blue) are highlighted. The underlined residues represent the conserved core of each AT-hook. The AT-rich domains of the DNA hairpins are outlined in red.

Figure 2.

HMGA2 (11.6 kDa) native nESI generated mass spectrum (a), native ion mobility distributions (b), representative intramolecular interactions, for which AT-Hook segments and CTMP are colored in gray and blue, respectively (c), and CIU fingerprints (d). Voltage difference (ΔV) of the CIU is denoted in white and eV z/DoF (in meV) values in pink for each CIU profile. Note that individual TIMS spectra for each CIU conditions are available in Figure S2.

Figure 3.

HMGA2·DNA₂₂ (green) and HMGA2·DNA₂₂·HOE (purple) native nESI-generated mass spectra (a), native ion mobility distributions (b), representative intra-and intermolecular interactions, for which DNA, AT-Hook segments, and CTMP are colored in orange, gray, and blue, respectively (c), CIU fingerprints (d), and collision-induced dissociations curves (e). Voltage difference (ΔV) of the CIU is denoted in white and the eV z/DoF (in meV) values are denoted in pink for each CIU profile. Note that individual TIMS spectra for each CIU conditions are shown in Figure S4. The CID data represent the mean value ± standard deviation (SD) of three independent experiments.

Figure 4.

HMGA2·DNA₅₀ (blue) and HMGA2·DNA₅₀·HOE (magenta) native nESI generated mass spectra (a), native ion mobility distributions (b), representative intra- and intermolecular interactions, for which DNA, AT-Hook segments, and CTMP are colored in orange, gray, and blue, respectively (c), CIU fingerprints (d), collision-induced dissociations curves (e), and solution fluorescence anisotropy (f). Ion mobility distributions of HMGA2·DNA₂₂ 1:1 (green squares) and 1:2 (green triangles) are shown for direct comparison with the HMGA2·DNA₅₀ 1:1 complex. The voltage difference (ΔV) of the CIU is denoted in white, and the eV z/DoF (in meV) values ae denoted in pink for each CIU profile. Note that individual TIMS spectra for each CIU condition are shown in Figure S5. The CID and anisotropy data represent the mean value ± standard deviation (SD) of three independent experiments.