IFI16 Preferentially Binds to DNA with Quadruplex Structure and Enhances DNA Quadruplex Formation

Abstract

Interferon-inducible protein 16 (IFI16) is a member of the HIN-200 protein family, containing two HIN domains and one PYRIN domain. IFI16 acts as a sensor of viral and bacterial DNA and is important for innate immune responses. IFI16 binds DNA and binding has been described to be DNA length-dependent, but a preference for supercoiled DNA has also been demonstrated. Here we report a specific preference of IFI16 for binding to quadruplex DNA compared to other DNA structures. IFI16 binds to quadruplex DNA with significantly higher affinity than to the same sequence in double stranded DNA. By circular dichroism (CD) spectroscopy we also demonstrated the ability of IFI16 to stabilize quadruplex structures with quadruplex-forming oligonucleotides derived from human telomere (HTEL) sequences and the MYC promotor. A novel H/D exchange mass spectrometry approach was developed to assess protein interactions with quadruplex DNA. Quadruplex DNA changed the IFI16 deuteration profile in parts of the PYRIN domain (aa 0-80) and in structurally identical parts of both HIN domains (aa 271-302 and aa 586-617) compared to single stranded or double stranded DNAs, supporting the preferential affinity of IFI16 for structured DNA. Our results reveal the importance of quadruplex DNA structure in IFI16 binding and improve our understanding of how IFI16 senses DNA. IFI16 selectivity for quadruplex structure provides a mechanistic framework for IFI16 in immunity and cellular processes including DNA damage responses and cell proliferation.

Fig 1. Binding of IFI16 protein to supercoiled DNAs.

(A) 100 ng sc pBluescript (lane 1–6) and sc pCMYC (lane 7–12) were incubated with increasing concentrations of IFI16 (molar ratio DNA:protein 1:0 / 1:1.25 / 1:2.5 / 1:5/ 1:10 / 1:20) in binding buffer (5 mM Tris-HCl, pH 7.0; 1 mM EDTA, 50 mM KCl and 0.01% Triton X-100) on ice for 15 min. The electrophoresis ran for 3 h at 100 V at 4°C. (B) 100 ng linear pBluescript (lane 1–6) and linear pCMYC (lane 7–12) were incubated with increasing concentrations of IFI16 (molar ratio DNA: protein 1:0 / 1:1.25 / 1:2.5 / 1:5/ 1:10 / 1:20) in binding buffer (5 mM Tris-HCl, pH 7.0; 1 mM EDTA, 50 mM KCl and 0.01% Triton X-100) on ice for 15 min. The electrophoresis ran for 3 h at 100 V at 4°C.

Fig 2. CD spectroscopy of quadruplexes and their stabilization by IFI16.

(A) CD spectra of oligonucleotide HTEL in TE buffer after denaturation (blue line), in TE buffer + 50 mM NaCl (red line) and in TE buffer + 50 mM KCl (green line). (B) CD spectra of oligonucleotide NHEIII in TE buffer after denaturation (blue line), in TE buffer + 50 mM NaCl (red line) and in TE buffer + 50 mM KCl (green line). The schematic drawings represent quadruplex structures of HTEL and NHEIII sequences. (C) The effect of recombinant IFI16 on HTEL quadruplex formation in potassium ions. CD spectra description: HTEL oligonucleotide in TE buffer (blue line), HTEL in TE buffer with 50 mM KCl (red line), HTEL in TE buffer + protein buffer with final concentration 3.4 mM KCl (green line), HTEL in TE buffer + IFI16 in protein buffer at molar ratio 1:1 and final concentration 3.4 mM KCl (violet line), IFI16 protein in protein buffer with final concentration 3.4 mM KCl in TE buffer (black line). (D) The effect of recombinant IFI16 on NHEIII quadruplex formation in potassium ions. The same description of curves as in C (NHEIII instead of HTEL). (E) The effect of recombinant IFI16 on HTEL quadruplex formation in sodium ions. CD spectra description: HTEL oligonucleotide in TE buffer (blue line), HTEL in TE buffer with 50 mM NaCl (red line), HTEL in TE buffer + protein buffer with final concentration 3.2 mM NaCl (green line), HTEL in TE buffer + IFI16 in protein buffer at molar ratio 1:2 and final concentration 3.2 mM NaCl (violet line), IFI16 protein in protein buffer with final concentration 3.2 mM NaCl in TE buffer (black line). (F) The effect of recombinant IFI16 on NHEIII quadruplex formation in sodium ions. The same description of curves as in E (NHEIII instead of HTEL).

Fig 3. H/D exchange of IFI16 in response to DNA interaction.

(A) H/D exchange of IFI16 in response to DNA interaction was analyzed in four reactions: IFI16 protein without DNA as a control, IFI16 with single stranded DNA oligonucleotide (SS DNA), IFI16 with double stranded DNA oligonucleotide (DS NHE III) and IFI16 with DNA forming quadruplex structure (Q NHEIII). H/D exchange was quenched at 900 s after addition of deuterium. The graph shows percentage deuteration of individual amino acids of IFI16 calculated as weighted average of corresponding peptides [53]. Shaded area of the graph shows the areas not covered by peptides. The deuteration spectrum is aligned with the domain structure of IFI16 and with prediction of disordered regions (FoldIndex [54]). (B) Structure of the first HIN-A domain (PDB 2OQ0) corresponding to amino acids 198–389 of IFI16 [14]. (C) Complex of the second HIN-B domain with DNA (PDB 3RNU) corresponding to amino acids 516–710 of IFI16 [23]. In (B) and (C) the helical linker peptide exhibiting the most significant changes in percentage of deuteration in the presence of quadruplex DNA is highlighted in red.

Fig 4. Comparison of IFI16 DNA binding to structurally different DNA targets.

EMSA was performed with 5 pmol of labeled oligonucleotides forming DS from human telomere sequence–DS HTEL (A) and G-quadruplex from one strand of the same sequence–Q HTEL (B), DS from NHE III region from MYC promoter–DS NHEIII (C) and G-quadruplex from one strand of the same sequence–Q NHEIII (D), SS (E) and cruciform (F) and increasing IFI16 concentrations (0 / 1.25 / 2.5 / 5 / 10 / 20 / 40 pmol), incubated in binding buffer (5 mM Tris-HCl, pH 7.0, 1 mM EDTA, 50 mM KCl and 0.01% Triton X-100) at 4°C for 15 min. Samples were electrophoresed on 4% non-denaturing polyacrylamide gel at 50V and 4°C for 3h. (G) Graphical representation of results obtained from densitometry analysis of free DNA bands from gels of IFI16 binding with SS A50, DS NHEIII, Q NHEIII and cruciform DNA targets from three independent experiments with SD. Schemes of DNA structures in A-F are not to scale.