Crystal structure of a TALE protein reveals an extended N-terminal DNA binding region

Xanthomonas TALEs (transcription activator-like effectors) are modular proteins characterized by an N-terminal T3S signal (T3SS), a central tandem repeat domain, C-terminal nuclear localization signals (NLSs) and an acidic transcriptional activation domain (AD)^1,2,3. The central tandem repeats are nearly identical and are typically composed of 34 highly conserved amino acids, containing repeat-variable diresidues (RVDs) at the 12th and 13th positions that mediate their DNA binding specificity^1,2,3,4,5. More than 20 types of RVDs have been identified thus far, among which HD, NG, NI and NN are the four most common ones with a specificity for the nucleotides C, T, A and G/A, respectively^4,5. The codes of RVD-DNA associations have been broadly and successfully employed in a variety of genome editing applications^{6,7,8,9,10,11,12,13,14}. The recent crystal structures of the PthXo1-DNA and dHax3-DNA complexes show that the TALEs form a right-handed superhelix and wrap around the DNA major groove^15,16. The specific recognition of target DNAs by TALEs is achieved through the direct interaction of the 13th residue in the repeat with the base group of nucleotides^15,16.

Most of the customized TALE fusion proteins have been constructed with the full-length TALEs, which might interpose large unnecessary regions and could impair activity¹¹. Although the TALE-DNA specificity depends solely on the central repeat domain (CRD)^1,2,3,4,5,8, the CRD alone is insufficient to bind to the target sites and ensure the activity of the TALE fusion proteins in vivo^6,11,12,14. Consistently, the N-terminal region (NTR) immediately preceding the CRD has been shown to be crucial for the full activity of TALE fusion proteins^11,12,13,14, although the underlying mechanism remains less well understood. The structural study of the PthXo1-DNA complex has illustrated the presence and potential roles in DNA binding of two additional degenerate repeats from the NTR¹⁵; however, the exact boundaries of the NTR involved in DNA binding remain undefined. The TALE minimal DNA binding domain, which is essential for the rational design of customized TALE fusion proteins, needs to be precisely refined and optimized^6,8,10,15.

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