dHax3 DNA-bound and DNA-free State

X-ray crystallography analysis on the DNA-bound dHax3 revealed that there are 2 complexes in each asymmetrical unit (Molecule A and B). The two protein molecules are highly identical and almost completely superimposable for most of the repeat. The only difference observed between these two molecules is in the RVD Loop of 6th repeat in Molecule A and of 5th repeat in Molecule B. Thus, only molecule A was studied.

Figure 3.1 The two complexes of DNA-bound dHax3. Both Molecule A (left) and Molecule B (right) are shown.

dHax3 appears to have different confirmation for when it is bound and not bound to DNA. When dHax3 is bound to the B form DNA target 17 base pairs, the helical dHax3 tracks along the major groove and the super helical pitch of the protein reduces from 60Å to 35Å.

Figure 3.2 The different pitch size of dHax3 in DNA bound and DNA free state

In the DNA free and bound state the main chains, being the first 22 amino acids, of each dHax3 are able to accurately overlap with each other. The confirmation change starts to accumulate from residues 23 to 34 causing a visible difference in the positions of the Cα atoms in Gly³⁴. This difference is gradually amplified as the number of TALE repeats in the protein increases causing compression of the superhelical structure in the DNA bound state. The ability to undergo conformational changes is important for the function of TALE’s and causes the dHax3 pitch change in the DNA bound and free state.

Figure 3.3 The 3rd repeat of dHax3 in DNA-free and DNA-bound state overlapped. Circled in yellow are the changes from residue 23 to 34.