Solution structure of the arabidopsis thaliana telomeric repeat-binding protein DNA binding domain: A new fold with an additional C-terminal helix

The double-stranded telomeric repeat-binding protein (TRP) AtTRP1 is isolated from Arabidopsis thaliana. Using gel retardation assays, we defined the C-terminal 97 amino acid residues, Gln464 to Val560 (AtTRP1_464-560), as the minimal structured telomeric repeat-binding domain. This region contains a typical Myb DNA-binding motif and a C-terminal extension of 40 amino acid residues. The monomeric AtTRP1_464-560 binds to a 13-mer DNA duplex containing a single repeat of an A. thaliana telomeric DNA sequence (GGTTTAG) in a 1:1 complex, with a K_D∼10^-6-10^-7 M. Nuclear magnetic resonance (NMR) examination revealed that the solution structure of AtTRP1_464-560 is a novel four-helix tetrahedron rather than the three-helix bundle structure found in typical Myb motifs and other TRPs. Binding of the 13-mer DNA duplex to AtTRP1_464-560 induced significant chemical shift perturbations of protein amide resonances, which suggests that helix 3 (H3) and the flexible loop connecting H3 and H4 are essential for telomeric DNA sequence recognition. Furthermore, similar to that in hTRF1, the N-terminal arm likely contributes to or stabilizes DNA binding. Sequence comparisons suggested that the four-helix structure and the involvement of the loop residues in DNA binding may be features unique to plant TRPs.