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. 2012 Feb 10;335(6069):716-9.
doi: 10.1126/science.1216211. Epub 2012 Jan 5.

The crystal structure of TAL effector PthXo1 bound to its DNA target

Amanda Nga-Sze Mak  1 , Philip Bradley, Raul A Cernadas, Adam J Bogdanove, Barry L Stoddard
Affiliations

The crystal structure of TAL effector PthXo1 bound to its DNA target

Amanda Nga-Sze Mak et al. Science. .

Abstract

DNA recognition by TAL effectors is mediated by tandem repeats, each 33 to 35 residues in length, that specify nucleotides via unique repeat-variable diresidues (RVDs). The crystal structure of PthXo1 bound to its DNA target was determined by high-throughput computational structure prediction and validated by heavy-atom derivatization. Each repeat forms a left-handed, two-helix bundle that presents an RVD-containing loop to the DNA. The repeats self-associate to form a right-handed superhelix wrapped around the DNA major groove. The first RVD residue forms a stabilizing contact with the protein backbone, while the second makes a base-specific contact to the DNA sense strand. Two degenerate amino-terminal repeats also interact with the DNA. Containing several RVDs and noncanonical associations, the structure illustrates the basis of TAL effector-DNA recognition.

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Figures

Figure 1
Figure 1. Domain organization of PthXo1 and structure of a single TAL effector repeat
TAL effectors contain N-terminal signals for bacterial type III secretion, tandem repeats that specify the target nucleotide sequence, nuclear localization signals, and a C-terminal region that is required for transcriptional activation. PthXo1 contains 23.5 canonical repeats (color coded to match Figure 2) that contact the DNA target found in the promoter of the rice Os8N3 gene (17). Blue bases correspond to positions in the target where the match between protein and DNA differs from the optimal match specified by the recognition code (3,4). Arrows indicate the start and end of the crystallized protein construct. In the structure, repeats 22 to 23.5 are poorly ordered, as are the C-termini of the two N-terminal cryptic repeats. The sequence and structure of a representative repeat (#14) is shown; RVD residues (HD) that recognize cytosine are red.
Figure 2
Figure 2
Structure of the PthXo1 DNA binding region in complex with its target site. The coloring of individual repeats matches the schematic in Figure 1.
Figure 3
Figure 3. Topology and contacts between TAL effector repeats and DNA bases
Panel a: 8 distinct combinations of RVDs and DNA bases are observed in the structure. HD forms a steric and electrostatic contact with cytosine; HG and NG both form nonpolar interactions between the glycine α-carbon and the thymine methyl group. A “mismatch’ between NG and a cytosine results in a longer distance from the RVD to the base. NN associates with either guanine (repeat 16) or with adenine (which would interact with the same N7 nitrogen of the purine base). NI forms a desolvating interface with either adenine (repeat 3) or cytosine (repeat19). The reduction in loop length by one residue in the ‘N*’ RVD (repeat 7) results in an increased distance to the base. Panel b: Two adjacent repeats form a tightly packed left-handed bundle of helices that position the second amino acid of each RVD in proximity to corresponding consecutive bases in an unperturbed B-form DNA duplex. The first residue of each RVD (position 12, either His or Asn) forms H-bonds to the backbone carbonyl oxygen of amino acid position 8 of the same repeat.
Figure 4
Figure 4. N-terminal cryptic repeats and contacts with 5′ thymine
a: 2Fo-Fc electron density maps contoured around thymine at position ‘0’ and tryptophan 232 in the ‘−1’ repeat. b: Residues 221 to 239 and residues 256 to 273 each form a helix and an adjoining loop that resembles helix 1 and the RVD loop in the canonical repeats; the remaining residues in each region are poorly ordered. W232 forms a non polar van der Waals contact with the methyl carbon of the thymine base at position 0.
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References

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