Divalent cations promote TALE DNA-binding specificity

Abstract

Recent advances in gene editing have been enabled by programmable nucleases such as transcription activator-like effector nucleases (TALENs) and CRISPR-Cas9. However, several open questions remain regarding the molecular machinery in these systems, including fundamental search and binding behavior as well as role of off-target binding and specificity. In order to achieve efficient and specific cleavage at target sites, a high degree of target site discrimination must be demonstrated for gene editing applications. In this work, we studied the binding affinity and specificity for a series of TALE proteins under a variety of solution conditions using in vitro fluorescence methods and molecular dynamics (MD) simulations. Remarkably, we identified that TALEs demonstrate high sequence specificity only upon addition of small amounts of certain divalent cations (Mg2+, Ca2+). However, under purely monovalent salt conditions (K+, Na+), TALEs bind to specific and non-specific DNA with nearly equal affinity. Divalent cations preferentially bind to DNA over monovalent cations, which attenuates non-specific interactions between TALEs and DNA and further stabilizes specific interactions. Overall, these results uncover new mechanistic insights into the binding action of TALEs and further provide potential avenues for engineering and application of TALE- or TALEN-based systems for genome editing and regulation.

Figure 1.

Crystal structure of TALE protein bound to target DNA and design of fluorescence anisotropy experiments. (A) General schematic of the 21.5 repeat TALE protein construct used in our work and an expanded view of one repeat sequence in the central repeat domain (CRD). The repeat variable diresidues in all repeats are shown in the boxes. The lengths of the N-terminal, the central and the C-terminal domains are specified. (B) TALE PthXo1 bound to a 36 bp target DNA sequence (PDB ID: 3UGM (23)), with the NTR shaded in orange and the CRD shaded in green. (C) DNA templates used in fluorescence anisotropy experiments. The binding site for the 21.5 repeat TALE construct used in this work is shown in red and the non-specific elements are shown in black. The green star represents the fluorescein dye molecule conjugated to the 5’ terminus of DNA.

Figure 2.

Effect of monovalent ions on TALE-DNA binding for 21.5 repeat, 15.5 repeat and 11.5 repeat TALEs. Binding of the 21.5 repeat TALE to (A) target (dots for raw data, solid lines for fitted curves) and (B) random (dots for raw data, dashed lines for fitted curves) DNA sequences measured by fluorescence anisotropy. (C) Equilibrium dissociation constants, K_{d,non-specific} and K_d,specific, determined for the 21.5 repeat TALE to random and target DNA sequences. Binding of the 15.5 repeat TALE to (D) target (dots for raw data, solid lines for fitted curves) and (E) random (dots for raw data, dashed lines for fitted curves) DNA sequences, and (F) K_{d,non-specific} and K_d,specific values. Binding of the 11.5 repeat TALE to (G) target (dots for raw data, solid lines for fitted curves) and (H) random (dots for raw data, dashed lines for fitted curves) DNA sequences, and (I) K_{d,non-specific} and K_d,specific values. TALE-DNA binding was measured in 20 mM Tris–Tris HCl buffer with different amounts of KCl. DNA templates were maintained at a constant concentration of 1 nM and TALE concentrations were adjusted. Duplicate experiments were performed to determine the mean and standard deviation of fluorescence anisotropy value.

Figure 3.

Effects of divalent cations on TALE-DNA binding for the 21.5 repeat TALE. Binding of the 21.5 repeat TALE to target (dots for raw data, solid lines for fitted curves) and random DNA (dots for raw data, dashed lines for fitted curves) measured by fluorescence anisotropy in 20 mM Tris–Tris HCl buffer containing 100 mM KCl and 10 mM (A) MgCl₂, (B) CaCl₂, (C) SrCl₂ or (D) ZnCl₂. (E) Equilibrium dissociation constants K_{d,non-specific} and K_d,specific for TALE binding in the presence of various divalent cations. Fluorescence anisotropy results for binding to (F) target DNA and (G) random DNA in 20 mM Tris–Tris HCl buffer with the total ionic strength of added salts held constant at 120 mM. (H) Equilibrium dissociation constants K_{d,non-specific} and K_d,specific for TALE–DNA binding in the presence of KCl and MgCl₂. Fluorescence anisotropy results for the binding to (I) target DNA and (J) random DNA in 20 mM Tris–Tris HCl buffer with increasing concentrations of MgCl₂ in the absence of any monovalent salt. (K) Equilibrium dissociation constants K_{d,non-specific} and K_d,specific for TALE–DNA binding in the presence of only MgCl₂. DNA concentration was held constant at 1 nM, and TALE concentrations were adjusted as noted. Duplicate experiments were performed to determine the mean and standard deviation of fluorescence anisotropy value.

Figure 4.

TALE NTR binding to DNA in a variety of solution conditions. Binding of the NTR-only TALE construct (without a CRD or CTR) to random DNA template was measured via fluorescence anisotropy in 20 mM Tris–Tris HCl buffer with increasing concentrations of (A) KCl only, (B) MgCl₂ only or (C) a combination of KCl and MgCl₂ with total ionic strength of either 15 or 25 mM (dots for raw data, dashed lines for fitted curves). Equilibrium dissociation constants for TALE NTR binding to DNA in the presence of (D) KCl, (E) MgCl₂ and (F) KCl and MgCl₂. Binding is rapidly diminished in the presence of MgCl₂, even when accounting for total ionic strength. DNA concentration was held constant at 1 nM. Duplicate experiments were performed to determine the mean and standard deviation of fluorescence anisotropy value.

Figure 5.

Variation in preferential interaction coefficients as a function of distance from the solute surface. For simulations involving DNA, TALE NTR and NTR-DNA complex in (A) 100 mM KCl and (B) 25 mM MgCl₂, and simulations involving DNA, TALE CRD and CRD–DNA complex in (C) 100 mM KCl and (D) 25 mM MgCl₂, the preferential interaction coefficients approach constant values beyond 6 Å from the molecule surface.

Figure 6.

Preferential interactions of the NTR with ions. (A) Change in the solvent-accessible area ΔSAA of the residues in the TALE NTR when the NTR is in the dissociated state compared to the associated state. Ten residues with the largest ΔSAA values are labeled, with seven positively charged residues (red) and three hydrophobic residues (blue). (B) Contact coefficients for the residues in the NTR with the different ionic species. Mg²⁺ shows stronger interactions with parts of the NTR compared to K⁺ and Cl⁻. However, (C) the residues in the NTR that interact with DNA have little interactions with Mg²⁺. The NTR surface is colored based on the contact coefficients of the NTR residues with Mg²⁺ at the dissociated state. Binding of the NTR to DNA requires the exclusion of cations from DNA. In the presence of Mg²⁺, the TALE NTR binding is attenuated due to the strong associations between DNA and Mg²⁺.

Figure 7.

Preferential interactions of the CRD with ions. (A) The average change in the solvent-accessible area ΔSAA of the residues at each location within a repeat when the CRD is in the dissociated state compared to the associated state. (B) The average contact coefficients for (B) the residues at each position of the repeat and (C) different type of residues in the CRD at the dissociated state. Divalent cations show stronger interaction with the CRD residues compared to monovalent cations, which is mostly driven by the negatively charged aspartic acid residue at position 4 of each repeat or position 13 of the repeats with RVD of HD. Snapshots of the simulated CRD–DNA complex structures in the presence of (D) 200 mM KCl and (E) 50 mM MgCl₂. K⁺ and Mg²⁺ preferentially bind to DNA that the CRD only partially contacts with DNA backbone. The 1–21 repeats in the CRD are shown in tube (cyan), and the lysine (pink) and glutamine (blue) residues are shown in stick. K⁺ (green) and Mg²⁺ (magenta) are shown in spheres. (F) Mg²⁺ binds to the major groove of DNA, and interacts with DNA bases and the aspartic acid residue (the second residue in RVD of HD) in repeat 9, which may stabilize the specific interaction.