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. 2012 Feb 10;335(6069):720-3.
doi: 10.1126/science.1215670. Epub 2012 Jan 5.

Structural basis for sequence-specific recognition of DNA by TAL effectors

Dong Deng  1 , Chuangye Yan, Xiaojing Pan, Magdy Mahfouz, Jiawei Wang, Jian-Kang Zhu, Yigong Shi, Nieng Yan
Affiliations

Structural basis for sequence-specific recognition of DNA by TAL effectors

Dong Deng et al. Science. .

Abstract

TAL (transcription activator-like) effectors, secreted by phytopathogenic bacteria, recognize host DNA sequences through a central domain of tandem repeats. Each repeat comprises 33 to 35 conserved amino acids and targets a specific base pair by using two hypervariable residues [known as repeat variable diresidues (RVDs)] at positions 12 and 13. Here, we report the crystal structures of an 11.5-repeat TAL effector in both DNA-free and DNA-bound states. Each TAL repeat comprises two helices connected by a short RVD-containing loop. The 11.5 repeats form a right-handed, superhelical structure that tracks along the sense strand of DNA duplex, with RVDs contacting the major groove. The 12th residue stabilizes the RVD loop, whereas the 13th residue makes a base-specific contact. Understanding DNA recognition by TAL effectors may facilitate rational design of DNA-binding proteins with biotechnological applications.

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Figures

Fig. 1
Fig. 1
Structure of the TAL repeats in DNA-free dHax3. (A) The 11 TAL repeats of dHax3 form a right-handed superhelical assembly. Two perpendicular views are presented with the RVDs highlighted in red in the right image. (B) All TAL repeats exhibit a nearly identical conformation. Each repeat is organized into short (a) and long (b) α helices connected by a short loop where the two (RVDs at positions 12 and 13 are located. All structure figures were prepared with PyMOL (30). Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
Fig. 2
Fig. 2
Overall structure of dHax3 bound to DNA. The superhelical structure of dHax3 (residues 231 to 720) binds to the major groove of DNA. Shown on the right are the DNA sequence of the sense strand and the corresponding RVDs in TAL repeats of dHax3. dHax3 contains 11.5 repeats with flanking N- and C-terminal helices shown in cyan. Two perpendicular views are presented, with the DNA duplex shown in sticks.
Fig. 3
Fig. 3
Structural comparison of DNA-free and DNA-bound TAL repeats in dHax3. (A) DNA-free and DNA-bound dHax3 are shown for residues 323 to 675, which comprise TAL repeats 2 to 11. The two structures are superimposed by using the N-terminal 23 amino acids, which encompass helix a and the first half of helix b of TAL repeat 2. (B) Superimposition of TAL repeat 2 from DNA-free and DNA-bound dHax3. The structures are superimposed by using the first 23 amino acids. Only the main chains are shown. The orange circle highlights where the structures exhibit variations. (C) Interrepeat interactions in the DNA-bound dHax3. TAL repeats 2 and 3 are shown here. The H bonds and van der Waals interactions are shown in the left and right images, respectively. Water molecules are shown as red spheres, and H bonds are represented by red dashed lines. (D) Interrepeat interactions in the DNA-free dHax3.
Fig. 4
Fig. 4
DNA recognition by TAL repeats. (A) The phosphate groups of the DNA sense strand is embraced by the positively charged ridge of the dHax3 TAL repeats. The surface electrostatic potential was calculated with PyMOL (30) (left). The invariant residues Lys16 and Gln17 (yellow sticks), located at the beginning of helix b in each TAL repeat, contribute to the positive electrostatic potential (right). The RVD loops are highlighted in red. (B) The two hypervariable residues in each TAL repeat are placed in the major groove of DNA. The sense and antisense strands of DNA are colored gold and gray, respectively. (C) The hypervariable residues at position 12 do not contact DNA bases. These residues, either His or Asn in dHax3 repeats, form hydrogen bonds with the carbonyl oxygen of Ala8 in the same repeat, which may help stabilize the conformation of the RVD loop. When consecutive repeats containing HD are present, His12 forms a water-mediated H bond with Asp13 from the previous repeat. (D) The hypervariable residues at position 13 are direct determinants of DNA base specificity. Shown here are repeats 7 to 11 and the corresponding nucleotides from the DNA sense strand. (E) Recognition of base T by NG. A close-up view on the RVD loops in TAL repeats 4 to 6 in molecule A is shown. Note that the RVD loop of repeat 6 adopts a conformation different from all other RVD loops. All distances are shown in the unit of Å.
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References

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