Comparison between TRF2 and TRF1 of their telomeric DNA-bound structures and DNA-binding activities

Abstract

Mammalian telomeres consist of long tandem arrays of double-stranded telomeric TTAGGG repeats packaged by the telomeric DNA-binding proteins TRF1 and TRF2. Both contain a similar C-terminal Myb domain that mediates sequence-specific binding to telomeric DNA. In a DNA complex of TRF1, only the single Myb-like domain consisting of three helices can bind specifically to double-stranded telomeric DNA. TRF2 also binds to double-stranded telomeric DNA. Although the DNA binding mode of TRF2 is likely identical to that of TRF1, TRF2 plays an important role in the t-loop formation that protects the ends of telomeres. Here, to clarify the details of the double-stranded telomeric DNA-binding modes of TRF1 and TRF2, we determined the solution structure of the DNA-binding domain of human TRF2 bound to telomeric DNA; it consists of three helices, and like TRF1, the third helix recognizes TAGGG sequence in the major groove of DNA with the N-terminal arm locating in the minor groove. However, small but significant differences are observed; in contrast to the minor groove recognition of TRF1, in which an arginine residue recognizes the TT sequence, a lysine residue of TRF2 interacts with the TT part. We examined the telomeric DNA-binding activities of both DNA-binding domains of TRF1 and TRF2 and found that TRF1 binds more strongly than TRF2. Based on the structural differences of both domains, we created several mutants of the DNA-binding domain of TRF2 with stronger binding activities compared to the wild-type TRF2.

Figure 1.

Domain structures and amino acid sequences of the TRF2 and TRF1 DNA-binding domains with base sequence of the telomeric DNA used. (A) Comparison of domain structures of hTRF1, hTRF2, and c-Myb. basic, basic domain; acidic, acidic domain; TRFH, TRF homology domain; Myb, Myb domain; R1, R2, and R3, the first, second, and third repeats of the c-Myb DNA-binding domain, respectively; activation, transcription activation domain; negative regulation, transcription negative regulation domain. (B) Sequence alignment of DNA-binding domains of hTRF1, hTRF2, and three repeats of the DNA-binding domains of mouse c-Myb. Well conserved residues involved in the hydrophobic core are boxed in yellow. Two pairs of polar residues that form an intramolecular salt bridge are connected with broken lines. Three helical regions are underlined. Amino acids that interact with DNA bases are indicated by closed circles; amino acids that interact with the sugar-phosphate backbone are indicated by open triangles, and amino acids that interact with both of them are indicated by closed triangles. (C) Sequence of the DNA duplex. The numbering scheme used in this paper is shown.

Figure 2.

Structures of the DNA-binding domain of hTRF2. (A) Stereo view of the superposition of 25 NMR structures of the DNA-binding domain of hTRF2 in its DNA-free state with three tryptophan residues that form a hydrophobic core. The backbone atoms (N, Cα, C′) of the DNA-binding domain, amino acids Lys447–Asn500, are shown in blue, and three tryptophan residues are shown in green. (B) Stereo view of the lowest-energy structure of the 25 structures of the DNA-free form. (C) Stereo view of the superposition of 20 NMR structures of the DNA-binding domain bound to the 13mer DNA. The backbone atoms (N, Cα, C′) of the DNA-binding domain, amino acids Lys447–Asn500, are shown in green, and all heavy atoms of DNA are shown in blue. (D) Stereo view of the lowest-energy structure of the 25 structures of the hTRF2 complex with DNA.

Figure 3.

DNA recognition modes of DNA-binding domains of hTRF1 and hTRF2. (A) Summary of the intermolecular contacts observed in the hTRF2-DNA complex. Black broken lines indicate hydrophilic contacts, and red broken lines indicate hydrophobic contacts. For amino acids that interact with DNA, contact frequencies over 70% in the 20 NMR structures are shown in blue, and the frequencies between 20% and 70% are shown in light blue. The criteria of the hydrogen bond were set as N-H••D (O or N): N••D distance < 3.5 Å; N-H-D angle > 90°. (B) Electrostatics potential surface of the DNA-binding domain of hTRF2 bound to DNA. The amino acids colored in red differ between hTRF1 and hTRF2. (C) Summary of the intermolecular contacts observed in the hTRF1-DNA complex. Black broken lines indicate hydrophilic contacts, and red broken lines indicate hydrophobic contacts. For amino acids that interact with DNA, contact frequencies over 70% in the 20 NMR structures are shown in red, and the frequencies between 20% and 70% are shown in light red. The criteria of the hydrogen bond were set as N-H-D (O or N): N••D distance < 3.5 Å; N-H-D angle > 90°. (D) Electrostatics potential surface of the DNA-binding domain of hTRF1 bound to DNA. The amino acids colored in red differ between hTRF1 and hTRF2.

Figure 4.

Comparison of DNA recognition modes between hTRF1 and hTRF2. (A) DNA recognition of the DNA-binding domain of hTRF2. The red circle indicates hydrophobic contact containing methyl groups of Ala484, Val485, and T3. Black broken lines indicate hydrophilic contacts between Asp489 and C7′, C8′. (B) The interaction modes of Ser404/Ser417, Ala471/Ala484, and the phosphate group of T3. Black broken lines indicate hydrophilic contacts. (C) The interaction modes in the minor groove of DNA. Sidechains of Arg380 of hTRF1 and Lys447 of hTRF2, and DNA are shown. In hTRF1, the average distances over 20 structures between NH1 of Arg380 and O2 of T9, NH2 of Arg380 and O2 of T9, NH1 of Arg380 and N3 of A6′, and NH2 of Arg380 and N3 of A6′ are shown. In hTRF2, the average distances over 20 structures between NZ of Lys447 and O2 of T9, and NZ of Lys447 and N3 of A6′ are shown. (D) The number of hydrogen bonds in the determined structures of the hTRF1 complex and the hTRF2 complex, respectively. The criteria of the hydrogen bonds were set as N-H••D (O or N): N••D distance < 3.5 Å; N-H-D angle > 90°.

Figure 5.

NMR titration experiments of the wild-type, K447R, A471S, A484S, R496K, DM (A471S/A484S), and QM (K447R/A471S/A484S/R496K) to the telomeric double-stranded DNA with the sequence GT-TAGGGTTAGGG.

Figure 6.

The chemical shift changes of the imino proton signals of each mutant from the corresponding signals of the wild-type hTRF2. (A) K447R, (B) A471S, (C) A484S, (D) R496K, (E) DM (A471S/A484S), (F) QM (K447R/A471S/A484S/R496K).

Figure 7.

The results of SPR analyses for equilibrium dissociation constant (Kd) values between DNA binding domains (TRF1, TRF2, K447R, A471S, A484S, R496K, DM, and QM) and the 13mer telomeric DNA in the buffer containing 10 mM HEPES-KOH, 3 mM EDTA, 180 mM KCl, and 0.003% Triton X-100 (v/v) (pH 6.8).