AT-hook peptides bind the major and minor groove of AT-rich DNA duplexes

Abstract

The mammalian high mobility group protein AT-hook 2 (HMGA2) houses three motifs that preferentially bind short stretches of AT-rich DNA regions. These DNA binding motifs, known as ‘AT-hooks’, are traditionally characterized as being unstructured. Upon binding to AT-rich DNA, they form ordered assemblies. It is this disordered-to-ordered transition that has implicated HMGA2 as a protein actively involved in many biological processes, with abnormal HMGA expression linked to a variety of health problems including diabetes, obesity, and oncogenesis. In the current work, the solution binding dynamics of the three ‘AT-hook’ peptides (ATHPs) with AT-rich DNA hairpin substrates were studied using DNA UV melting studies, fluorescence spectroscopy, native ion mobility spectrometry-mass spectrometry (IMS-MS), solution isothermal titration calorimetry (ITC) and molecular modeling. Results showed that the ATHPs bind to the DNA to form a single, 1:1 and 2:1, ‘key-locked’ conformational ensemble. The molecular models showed that 1:1 and 2:1 complex formation is driven by the capacity of the ATHPs to bind to the minor and major grooves of the AT-rich DNA oligomers. Complementary solution ITC results confirmed that the 2:1 stoichiometry of ATHP: DNA is originated under native conditions in solution.

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

“…The high K d of the HMGA2·DNA 50 ·HOE complex stems from the fact that the minor groove of the DNA 50 AT-rich regions was occupied by the minor groove binder HOE. As a result, HMGA2 binds to the major groove, resulting in a much higher K d in agreement with the gas-phase experiments . If this hypothesis is correct, a further increase of HOE concentration in the binding reaction mixture should shift the binding curve to the right.…”

“…Each AT-hook is comprised of a conserved central Arg-Gly-Arg-Pro core sequence (underlined residues in Figure a) that deeply penetrates into the minor groove of DNA and forms a well-defined AT-hook:DNA complex . In addition, a recent study reported that synthesized AT-hooks also tightly bind to the major groove of DNA when the minor groove is occupied by a minor groove binder Hoechst 33258 (HOE) . The particularity of HMGA2 to be able to transition from unstructured to a defined conformation, when bound to DNA, allows the protein to actively participate in diverse nuclear activities, including DNA replication, DNA repair, chromatin remodeling, and gene transcription and regulation. − One main roadblock in the structural elucidation of these conformational changes is the lack of three-dimensional structures owing to the biological heterogeneity of conformations for HMGA2 and its DNA-bound forms.…”

Exploring the Conformations and Binding Location of HMGA2·DNA Complexes Using Ion Mobility Spectrometry and 193 nm Ultraviolet Photodissociation Mass Spectrometry

“…An overall suppression in the abundance of sequence ions is observed for the HMGA2·DNA complexes relative to HMGA2, occurring primarily between residues 21 and 73 (Figure ), and is shown as negative values beginning at the first AT-hook, spanning the second AT-hook, and terminating at the third AT-hook. This is consistent with previous reports that all three AT-hooks are capable of binding to AT-rich DNA. , As illustrated in Figure S17, the overall fragmentation efficiency of the third AT-hook region is similar. The greater suppression of fragmentation of the first and second AT-hooks relative to the third suggests preferential binding of DNA to these regions of the HMGA2·DNA complexes, which is supported by a previous study in which AT-hooks 1 and 2 induced greater thermal stability of DNA 22 (referred to as FL876) than did AT-hook 3 .…”

“…This is consistent with previous reports that all three AT-hooks are capable of binding to AT-rich DNA. , As illustrated in Figure S17, the overall fragmentation efficiency of the third AT-hook region is similar. The greater suppression of fragmentation of the first and second AT-hooks relative to the third suggests preferential binding of DNA to these regions of the HMGA2·DNA complexes, which is supported by a previous study in which AT-hooks 1 and 2 induced greater thermal stability of DNA 22 (referred to as FL876) than did AT-hook 3 . This does not discount any DNA binding to AT-hook 3, and it is likely that all three binding modes for both DNA hairpins exist in equilibrium in solution.…”

“…Each DNA-binding region, or AT-hook, possesses a conserved Arg-Gly-Arg-Pro motif surrounded by additional basic residues. These AT-hooks can bind to the minor groove of AT-rich DNA (or major groove if the minor groove is otherwise occupied) with possible multivalent binding of one protein to multiple AT tracts, , contributing to its functions involving DNA replication and repair as well as gene transcription and regulation. , The absence of HMGA2 has been linked to dwarfism, and overexpression has been attributed to lung cancer, , prostate cancer, , breast cancer, and obesity . The important functions and specificity of HMGA proteins make them key biomarkers and drug targets, yet their intrinsically disordered regions have impeded their biophysical characterization.…”