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

Kevin Jeanne Dit Fouque, Sarah N Sipe, Alyssa Garabedian, German Mejia, Linjia Su, Md Lokman Hossen, Prem P Chapagain, Fenfei Leng, Jennifer S Brodbelt, Francisco Fernandez-Lima

Journal of the American Society for Mass Spectrometry 2022 Jul 06

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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.

Citation

Kevin Jeanne Dit Fouque, Sarah N Sipe, Alyssa Garabedian, German Mejia, Linjia Su, Md Lokman Hossen, Prem P Chapagain, Fenfei Leng, Jennifer S Brodbelt, Francisco Fernandez-Lima. Exploring the Conformational and Binding Dynamics of HMGA2·DNA Complexes Using Trapped Ion Mobility Spectrometry-Mass Spectrometry. Journal of the American Society for Mass Spectrometry. 2022 Jul 06;33(7):1103-1112