TAL effector specificity for base 0 of the DNA target is altered in a complex, effector- and assay-dependent manner by substitutions for the tryptophan in cryptic repeat -1

Abstract

TAL effectors are re-targetable transcription factors used for tailored gene regulation and, as TAL effector-nuclease fusions (TALENs), for genome engineering. Their hallmark feature is a customizable central string of polymorphic amino acid repeats that interact one-to-one with individual DNA bases to specify the target. Sequences targeted by TAL effector repeats in nature are nearly all directly preceded by a thymine (T) that is required for maximal activity, and target sites for custom TAL effector constructs have typically been selected with this constraint. Multiple crystal structures suggest that this requirement for T at base 0 is encoded by a tryptophan residue (W232) in a cryptic repeat N-terminal to the central repeats that exhibits energetically favorable van der Waals contacts with the T. We generated variants based on TAL effector PthXo1 with all single amino acid substitutions for W232. In a transcriptional activation assay, many substitutions altered or relaxed the specificity for T and a few were as active as wild type. Some showed higher activity. However, when replicated in a different TAL effector, the effects of the substitutions differed. Further, the effects differed when tested in the context of a TALEN in a DNA cleavage assay, and in a TAL effector-DNA binding assay. Substitution of the N-terminal region of the PthXo1 construct with that of one of the TAL effector-like proteins of Ralstonia solanacearum, which have arginine in place of the tryptophan, resulted in specificity for guanine as the 5' base but low activity, and several substitutions for the arginine, including tryptophan, destroyed activity altogether. Thus, the effects on specificity and activity generated by substitutions at the W232 (or equivalent) position are complex and context dependent. Generating TAL effector scaffolds with high activity that robustly accommodate sites without a T at position 0 may require larger scale re-engineering.

Figure 1. Alignment of N-terminal portions of multiple TAL effector crystal structures shows a conserved conformation for W232 consistent with an important interaction with the 0^th position T.

The N terminus of TAL effector PthXo1 bound to its DNA target (PDB structure 3UGM) [4] is shown in blue, the N terminus of unbound artificial TAL effector dTALE2 (PDB structure 4HPZ) [26] in red, and the N terminus of artificial TAL effector dHAX3 bound to a DNA-RNA hybrid (PDB structure 4GG4) [5] in brown. DNA is from structure 3UGM. Side chains are shown for W232 as well as arginines at positions 236 and 266, which make non-specific contacts to the nucleic acid backbone. T₀, the 0^th position thymine. G_-1, a guanine 5’ of T₀.

Figure 2. Activity of TAL effectors with selected single amino acid substitutions for W232 on targets with A, C, G, or T at the 0^th position.

A. Schematic of a TAL effector with the -1^st and 0^th repeats and the central repeat region (CRR) labeled. The amino acid sequence of the -1^st repeat is shown below; W232 is shown in bold. B. Effects of PthXo1 W232 substitution and target combinations (treatment) on activity. Shown at top are the PthXo1 RVD and EBE sequences. X marks the 0^th position. Activity was measured in an Agrobacterium-mediated transient expression assay in Nicotiana benthamiana leaves, using a GUS reporter gene cloned downstream of a minimal promoter (see Materials and Methods) containing a PthXo1 EBE with the 0^th position thymine (EBE_PthXo1-T), or variants with adenine, cytosine, or guanine as base 0 (EBE_PthXo1-A, EBE_PthXo1-C, and EBE_PthXo1-G, and respectively). Treatment effects were estimated using a mixed linear model to account for variation due to experiment and replicate effects (see Materials and Methods). Effects were computed relative to the negative control EBE_PthXo1-T with no TAL effector and normalized to the effect of wild-type PthXo1 (W232) on EBE_PthXo1-T. Bars indicate one standard deviation. C. Effects of TAL868 W232 substitution and target combinations on activity, as in B. Shown at top are the TAL868 RVD and EBE sequences. X marks the 0^th position.

Figure 3. Activity of TALENs with selected single amino acid substitutions for W232 on targets with A, C, G, or T at the 0^th position.

A. RVD sequence of the golden gate-assembled PthXo1 equivalent and nucleotide sequence of the PthXo1 EBE. X marks the 0^th position. B. Activity of TALENs with the PthXo1 equivalent RVD sequence, in a yeast single strand annealing assay [14]. EBE_PthXo1-A, EBE_PthXo1-C, EBE_PthXo1-G, and EBE_PthXo1-T indicate activity on targets with paired PthXo1 EBEs each with A, C, G, or T at the 0^th position, respectively. Data are normalized to PthXo1 on EBE_PthXo1-T. Values are the mean of seven or more replicates. Error bars represent s.d. See Table S8 for p-values.

Figure 4. Relative binding affinities of TAL868 and selected W232 substitution variants for the TAL868 target DNA with T, A, C, or G at the 0^th position.

A. Electrophoretic mobility shift assay of labeled TAL868 target DNA with T, A, C, or G in the 0^th position, or a scrambled target, (as labeled at top) following incubation with increasing amounts (left to right) of TAL868 (W232) or selected W232 substitution variants, as labeled. Bands across the bottom represent unbound DNA. The next bands up represent DNA bound by the TAL effector. The uppermost bands represent higher order complexes. DNA bound at lower protein concentrations indicates higher affinity. Each interaction was assayed at least twice. Representative gel images are shown. B. Results from all experiments, reported as the fraction of DNA bound by the protein, estimated by band densitometric analysis. Error bars represent standard deviation.

Figure 5. Activity of chimeric TAL effector RTL-PthXo1 with single amino acid substitutions for R298 on targets with A, C, G, or T at the 0^th position.

A. Schematic of the chimeric TAL effector RTL-PthXo1 with the -1^st and 0^th repeats and the central repeat region (CRR) labeled, and showing the RSc1815 -1st repeat sequence. RTL-PthXo1 was created by replacing the N terminal region immediately upstream of the CRR of our PthXo1 construct with the N terminal region of RTL RSc1815 (shown in red). The arginine (R) in the RTL that aligns to W232 is shown in large bold font. Numbers indicate positions in the RTL sequence. B. Left, activity of RTL-PthXo1 with the native arginine at position 298 (R298) and of variants with selected amino acid substitutions at that position (as labeled), measured in an Agrobacterium-mediated transient expression assay in Nicotiana benthamiana leaves, using a GUS reporter gene cloned downstream of a minimal promoter (see Materials and Methods) containing the PthXo1 EBE with the 0^th position thymine (EBE_PthXo1-T), or variants with adenine, cytosine, or guanine as base 0 (EBE_PthXo1-A, EBE_PthXo1-C, and EBE_PthXo1-G, and respectively). Right, the same plot at a larger scale. Activity is normalized to the activity of PthXo1 on EBE_PthXo1-T, set to 1.0. Values are the mean of five replicates. Error bars represent s.d. See Table S9 for p-values. Experiments were repeated at least twice with similar results.

Figure 6. Structural models for representative W232 substitutions.

Models were built for all 20 possible amino acids at position 232 with DNA target sites for all 16 nucleotide combinations at positions 0 and -1. Panels A, B, C, and D depict the models for W232, W232R, W232P, and W232Q, respectively. The amino acid at position 232 is labeled using the single letter Code. T₀ and the nucleotide at position -1 are also labeled. Models shown are of the DNA target with the highest predicted binding affinity for the corresponding W232 substitution, from the flexible backbone simulations. For these substitutions, differences between the fixed and flexible backbone models are slight.