Structure of TALEs

The structure of TALEs are unique and have been selected for and perfected for function throughout evolution. They have a characteristic structure which allows them to bind transcription activation sites in their host cells effectively. 

They are made up of TAL repeats, each repeat is between 33 and 35 amino acids in length. The repeat consists of two alpha-helices joined together by a loop region. This loop region contains the 12^th, 13^th and 14^th amino acid residues and is the region of the repeat which is specific for a particular DNA base. The 12^th and 13^th amino acids are hypervariable and confer the specificity. They are also known as the Repeat Variable Diresidues or RVDs, and give the loop its name, the RVD loop. (Deng et al., 2012)

dHax3 is an artificially engineered TALE which has been produced to study the mechanism of TALE binding to DNA. This TALE has 11.5 TAL repeats. Each individual TAL repeat in this protein consists of 34 amino acid residues. The 3^rd to 11^th amino acids in each repeat make up an α-helix labelled as helix "a", and positions 15 to 33 make up α-helix "b", which is characterised by its bent structure. The RVD loop of dHax3 contains a conserved glycine at position 14. (Deng et al., 2012)

Figure 2. This image shows the structure of a single TAL repeat, the structure of the two helices, a and b, joined by the RVD loop are clearly defined. The bend in helix b is obvious in this image, and the structure helps the repeat to bind the DNA base. You can also see the loops of two other repeats in the image. It has been adapted from the pdb structure of DNA bound dHax3 in pymol. (DOI: 10.1126/science.1215670).

 

The hypervariable residues target specific bases in the DNA. Each repeat has its own specificity, which allows the TALE to recognise a specific sequence of DNA bases in the host. Different TALEs have varying numbers of TAL repeats, from 1.5 to 33.5. The residue at position 12 stabilises the binding of the TAL repeat to the host DNA using hydrogen bonding whereas the amino acid at position 13 is the one which is specific to a DNA base and recognises it to bind to.(Deng et al., 2012)

There are 20 known combinations of the hypervariable residues at positions 12 and 13. dHax3 has a combination of RVDs which are specific for each DNA base. Some examples are, His/Asp which recognises Cytosine, Asn/Gly which recognises Thymine and Asn/Ser which recognises Adenine, and Asn/Asn which recognises Guanine. The two residues are adjacent to the major groove in the sense strand of the DNA. Residue 12 (His or Asn) has no direct interaction with the DNA, instead the side chain faces away and forms a hydrogen bond with the carbonyl oxygen of the conserved alanine at residue 8. Thus, the underlying function of residue 12 is to provide structural support for the local conformation, which indirectly promotes DNA binding.(Deng et al., 2012) (Bogdanove, Voytas 2011)

The DNA binding properties of the residues at position 13 are explained by the structure of DNA bound dHax3. In the case of His/Asp recognising Cytosine, the amine of Cytosine can form a hydrogen bond with the carboxylate oxygen of Asp. For Asn/Ser recognising Adenine, the 7^th nitrogen of Adenine forms a hydrogen bond with the hydroxyl group in the side chain of Ser. Guanine also has a 7^th Nitrogen analogous to Adenine, therefore allowing Asn/Ser to bind to Guanine in similar manner. The explanation for Asn/Gly is less straightforward; it is possible that Gly is required as the 13 residue to avoid steric clashes with the 5-methyl group of thymine. (Deng et al., 2012)

 

Figure 3. An illustration to represent the hydrogen bond
between the Nitrogen 7 in Adenine and hydroxyl group
from Ser.

 

 

The structural information gained from studying the DNA bound dHax3 provides an explanation for how some of the residues combinations in the RVD loop recognise DNA, however exact mechanism for DNA recognition is still unknown for other residues. It is important to note that the binding of TALEs to DNA can contain mismatches, so the mechanism of recognition using the RVDs is not perfect. Though it is known that the binding of TALEs is modular, which is an example of how the structure of TALEs have evolved for DNA binding. (Deng et al., 2012)(Bogdanove, Voytas 2011)

A structural feature of the DNA sequence which the TALE binds is that there is a Thymine adjacent/upstream to the binding sequence. This has been proposed to be a signal to the TALE that a target gene activiation sequence is downstream of the Thymine. (Bogdanove, Voytas 2011)

When DNA is bound to dHax3 there are 12 repeats rather than the 11.5 repeats when DNA is not bound, the extra 0.5 amino acids are “unconserved” amino acids. The DNA sequence dHax3 binds is 17 base pairs long; 5’-TGTCCCTTTATCTCTCT-3’. (Deng et al., 2012)

 

Figure 4. This image shows the structure of DNA bound dHax3, this image includes two TALEs.






The TAL repeats come together in to a superhelical structure with 11 helices (so 5.5 TAL repeats) per turn of the super-helix. Positive amino acid residues line the superhelical assembly of TAL repeats, this attracts the negative DNA to them, which is another example of how structure is evolved for function. (Deng et al., 2012)