Role of the interdomain linker in distance determination for remote cleavage by homing endonuclease I-TevI

Abstract

I-TevI is a modular intron-encoded endonuclease, consisting of an N-terminal catalytic domain and a C-terminal DNA-binding domain, joined by a 75 amino acid linker. This linker can be divided into three regions, starting at the N terminus: the deletion-intolerant (DI) region; the deletion-tolerant (DT) region; and a zinc finger, which acts as a distance determinant for cleavage. To further explore linker function, we generated deletion and substitution mutants that were tested for their preference to cleave at a particular distance or at the correct sequence. Our results demonstrate that the I-TevI linker is multi-functional, a property that sets it apart from junction sequences in most other proteins. First, the linker DI region has a role in I-TevI cleavage activity. Second, the DT linker region participates in distance determination, as evident from DT mutants that display a phenotype similar to that of the zinc-finger mutants in their selection of a cleavage site. Finally, NMR analysis of a freestanding 56 residue linker segment showed an unstructured stretch corresponding to the DI region and a portion of the DT region, followed by a beta-strand corresponding to the remainder of the DT region and containing a key distance-determining arginine, R129. Mutation of this arginine to alanine abolished distance determination and disrupted the beta-strand, indicating that the structure of the DT linker region has a role in cleavage at a fixed distance.

Figure 1. Length rather than sequence of the DI region is needed for cleavage

(a) Representation of I-TevI domains. The I-TevI linker between the catalytic and DNA-binding domains is differentiated into three regions: the DI region (deletion intolerant); the DT region (deletion tolerant); and the zinc finger (Zn) . The α and HTH in the cartoon indicate the α-helix and helix-turn-helix motifs of the DNA binding domain. Numbers on the cartoon (except the last) correspond to the beginning of a domain or segment. The mutated residues are enclosed within boxes. (b) Cleavage assays on linearized pTZtdΔI DNA. Cleavage assays were performed with proteins synthesized in vitro with [³⁵S]-Met as described in Materials and Methods and the reaction products were resolved on agarose gels. S, substrate; P, cleavage products. WG = unprogrammed wheat germ extract. The cleavage reaction mixtures were adjusted to equivalent ³⁵S -labeled protein and diluted step-wise 1/3 from 1 (undiluted) to 1:243.

Figure 2. Ala-substitution mutants in the DI region reduced linker flexibility

(a) Cleavage activity on wild-type, +5 and +10 mutant substrates. DI-1AA, DI-3AA and wild-type I-TevI cleavage activity is shown on each of the three DNA substrates. In vitro-synthesized enzymes were incubated with 5′ end-labeled double-strand substrates (S) to yield products (P), and resolved on a 6% sequencing gel. The cleavage reaction mixtures were adjusted to equivalent ³⁵S -labeled protein and diluted step-wise 1/3 from 1 (undiluted) to 1:9. D = cleavage at distance (black triangles), S = cleavage at sequence (gray triangles). (b) WT, +5 and +10 substrate sequences. The inserted sequences are shaded. D and S are as in (a). The intron IS is indicated by a vertical line.

Figure 3. DT linker deletion mutants affect cleavage-site selection

(a) Schematic representation of mutations in the DT region. The deletion mutants were constructed with two-, four- or five-amino acid deletions, represented below the linker sequence by bars numbered 1–12. Mutants are named by indicating the size of the deletion followed by the location of the starting amino acid. For example, Δ2-125 is a two-amino acid deletion starting at residue 125 and Δ5-138 is a five-amino acid deletion starting from residue 138 . Cleavage assay data are represented as % cleavage at distance (black bars) over sequence (gray bars) for each deletion mutant and WT control, and are averaged over three independent experiments. (b) Top strand cleavage-site mapping on +5 and +10 I-TevI homing site (HS). Numbers correspond to constructs in (a). WT, wild-type I-TevI; 1, Δ2-116; 2, Δ2-118; 3, Δ5-120; 4, Δ2-125; 5, Δ4-127; 6, Δ2-131; 7, Δ2-133; 8, Δ2-135; 9, Δ2-137; 10, Δ5-138; 11, Δ5-143; 12, Δ2-148; Z1, ΔZn; Z2, CZnA; WG, wheat germ extract; and SUB, DNA substrate. Substrate sequences and labeling as in Figure 2. Similar ratios of cleavage at sequence versus distance were observed on the bottom strand (data not shown).

Figure 4. Defining R129 as a key residue in distance determination

(a) Sequence of DT region. Ala-substitution mutations are indicated by boxes and brackets. Cleavage assay data for +5 and +10 substrates are represented as % cleavage at distance (black bars) over sequence (gray bars) for each substitution mutant and WT control, and are an average over three independent experiments. (b) Representative cleavage reactions on +5 and +10 substrates. Cleavage reactions and gels are as in Figure 2(a). Cleavage on WT substrate is shown in Supplementary Fig. 2. The square bracket represents spurious cleavage on the +10 substrate, as previously reported .

Figure 5. R129A mutation changes the secondary structure of I-TevI_93-148

(a) NMR ¹H{¹⁵N} HSQC spectrum of I-TevI_93-148. Chemical shift assignments of I-TevI_93-148 are indicated. A peak marked by an asterisk is from the uncut thrombin restriction site. (b). Overlay of the ¹H{¹⁵N}-HSQC spectra of I-TevI_93-148 (black) and R129A-I-TevI_93-148 (red). (c). Chemical shift changes, ΔΩ, due to the R129A mutation. Chemical shift changes were calculated by using $\mathrm{\Delta }\mathrm{\Omega }=\sqrt{\mathrm{\Delta }{\mathrm{\Omega }}_H^2+0.25\mathrm{\Delta }{\mathrm{\Omega }}_N^2}$, where ΔΩ_H and ΔΩ_N are changes in amide proton and nitrogen chemical shifts expressed in ppm, respectively. (d) Differences in ¹³C_α chemical shifts of I-TevI_93-148 from the random coil values, $\mathrm{\Delta }{\mathrm{\Omega }}_{C_{\alpha }}^{WT}$. Consistent negative values of $\mathrm{\Delta }{\mathrm{\Omega }}_{C_{\alpha }}^{WT}$ indicate the preference of the protein 123–148 region to form a β-strand.

Figure 6. I-TevI linker sequence alignment

Sequences A-D were recovered from a BLAST search of the environmental subdivision database using the I-TevI linker as query sequence (amino acids 93-167). Residue numbering of I-TevI is below the sequence. Sequence alignments are shown as blocks that correspond to the I-TevI catalytic domain, the linker, the DNA-binding domain, and residues C-terminal to the DNA binding domain. Black shading represents residues that are absolutely conserved among the five sequences and gray shading represents conserved residues with conservative changes. Residues that correspond to the GIY-YIG signature sequences are boxed. GenBank IDs to the hypothetical proteins (Marine Metagenome ID) are: A, 140852551 (Gos_3956749); B, 136178932 (Gos_8413892); C, 136165525 (Gos_8427889); D, 140538853 (Gos_4034147).

Figure 7. Summary of function and structure of the I-TevI linker

(a) I-TevI linker between the catalytic and DNA-binding domains. The linker is cartooned as a loop beside the zinc finger (Zn), the structure of which was solved along with the DNA binding domain . The catalytic domain structure is from ref . Where the protein sits on the DNA is marked by the intron insertion site (IS) and I-TevI cleavage site (CS), which are a fixed distance apart. The asterisk corresponds to R129, which disrupts distance determination function and NMR structure when mutated. The rightward arrows correspond to the beginning of the β-strand (residue 123) and the distance determination transition (residue 134) of the DT region. (b) Summary of activity of deletion mutants ; . (c) Summary of activity of mutant types with respect to linker segments: Δ = deletion; A = Ala-substitution. HS = homing site DNA substrate (WT DNA or +5, +10 insertions). In colored boxes (green = WT phenotype, red = mutant phenotype): + = active; - = inactive; S = cleavage at sequence; S > D = preferred cleavage at sequence over distance, D ≫ S = greatly preferred cleavage at distance over sequence. Green box indicates mutant’s general preference for cleavage at distance, like WT. Red box indicates mutant’s general preference for cleavage at sequence. (d) Requirements for function corresponding to structure.