Sequence specific DNA binding of Ets-1 transcription factor: molecular dynamics study on the Ets domain--DNA complexes

Molecular dynamics (MD) simulations for Ets-1 ETS domain-DNA complexes were performed to investigate the mechanism of sequence-specific recognition of the GGAA DNA core by the ETS domain. Employing the crystal structure of the Ets-1 ETS domain-DNA complex as a starting structure we carried out MD simulations of: (i). the complex between Ets-1 ETS domain and a 14 base-pair DNA containing GGAA core sequence (ETS-GGAA); (ii). the complex between the ETS domain and a DNA having single base-pair mutation, GGAG sequence (ETS-GGAG); and (iii). the 14 base-pair DNA alone (GGAA). Comparative analyses of the MD structures of ETS-GGAA and ETS-GGAG reveal that the DNA bending angles and the ETS domain-DNA phosphate interactions are similar in these complexes. These results support that the GGAA core sequence is distinguished from the mutated GGAG sequence by a direct readout mechanism in the Ets-1 ETS domain-DNA complex. Further analyses of the direct contacts in the interface between the helix-3 region of Ets-1 and the major groove of the core DNA sequence clearly show that the highly conserved arginine residues, Arg391 and Arg394, play a critical role in binding to the GGAA core sequence. These arginine residues make bidentate contacts with the nucleobases of GG dinucleotides in GGAA core sequence. In ETS-GGAA, the hydroxyl group of Tyr395 is hydrogen bonded to N7 nitrogen of A(3) (the third adenosine in the GGAA core), while the hydroxyl group makes a contact with N4 nitrogen of C(4') (the complementary nucleotide of the fourth guanosine G(4) in the GGAG sequence) in the ETS-GGAG complex. We have found that this difference in behavior of Tyr395 results in the relatively large motion of helix-3 in the ETS-GGAG complex, causing the collapse of bidentate contacts between Arg391/Arg394 and the GG dinucleotides in the GGAG sequence.     

Precise alignment of sites required for mu enhancer activation in B cells.

The lymphocyte-specific immunoglobulin mu heavy-chain gene intronic enhancer is regulated by multiple nuclear factors. The previously defined minimal enhancer containing the muA, muE3, and muB sites is transactivated by a combination of the ETS-domain proteins PU.1 and Ets-1 in nonlymphoid cells. The core GGAAs of the muA and muB sites are separated by 30 nucleotides, suggesting that ETS proteins bind to these sites from these same side of the DNA helix. We tested the necessity for appropriate spatial alignment of these elements by using mutated enhancers with altered spacings. A 4- or 10-bp insertion between muE3 and muB inactivated the mu enhancer in S194 plasma cells but did not affect in vitro binding of Ets-1, PU.1, or the muE3-binding protein TFE3, alone or in pairwise combinations. Circular permutation and phasing analyses demonstrated that PU.1 binding but not TFE3 or Ets-1 bends mu enhancer DNA toward the major groove. We propose that the requirement for precise spacing of the muA and muB elements is due in part to a directed DNA bend induced by PU.1.