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.

Graphical Abstract

AT-hook peptides 1, 2 and 3 from HMGA 2 bind to the major and minor groove of AT-rich DNA duplexes to form a 1:1 and 2:1 ‘key-locked’ conformational ensemble.

Scheme 1.

Preparation of PDB template for modeling of DNA.

Figure 1.

Typical mobility profiles, MS distributions (inset) and candidate structures (right) are shown for the [M + 4H]⁺⁴ and [M + 5H]⁺⁵ charge states of ATHP 1, 2, or 3 in complex with the DNA hairpin (FL876) with a ATHP: DNA 1:1 stoichiometry.

Figure 2.

Typical mobility profiles, MS distributions (inset) and candidate structures (right) are shown for the [M + 4H]⁺¹ and [M + 5H]⁺⁵ charge states of ATHP 1, 2, or 3 in complex with the DNA hairpin (FL876) and the minor groove binding (MGB) Hoechst 33258 with a ATHP: DNA : MGB 1:1:1 stoichiometry

Figure 3.

Typical mobility profiles, MS distributions (inset) and candidate structures (right) are shown for the [M + 4H]⁺¹ and [M + 5H]⁺⁵ charge states of ATHP 1, 2, or 3 in complex with the DNA hairpin (FL876) with a ATHP: DNA 2:1 stoichiometry.

Scheme 2.

Mechanism of ATHP attachment to DNA showing the preformed hairpin prior to peptide unfolding upon binding.

Figure 4.

DNA UV melting curves for DNA hairpin (FL876) and DNA hairpin (FL876) complexed with ATHPs 1, 2 or 3 (A). ITC results determining the binding stoichiometry and affinity of ATHP 1, 2 and 3 to both the major and minor groove of FL876 DNA (B) and Collision induced dissociation for FL876 complexed with ATHP1. ATHP2 or ATHP 3 (charge state and degrees of freedom were considered) with the curve derivative shown as an inset (C).