Sequence Specific DNA Binding of Ets-1 Transcription Factor: Molecular Dynamics Study on the Ets Domain–DNA Complexes

doi:10.1016/S0022-2836(03)00726-5

Journal of Molecular Biology

Volume 331, Issue 2, 8 August 2003, Pages 345-359

https://doi.org/10.1016/S0022-2836(03)00726-5 Get rights and content

Abstract

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 A₃ (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 G₄ 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.

Introduction

The ETS protein family contains more than 45 eukaryotic transcription activators and inhibitors, such as Ets-1, PU.1, Fli-1, GABPα, SAP-1, TEL and Elk-1.1., 2., 3. Members of this family play an important role in normal cell proliferation and differentiation. The DNA rearrangement and/or overexpression of ets gene have been known to lead to tumorigenesis.⁴ In order to regulate gene expression, the ETS family of proteins bind to a consensus DNA sequence centered on the core sequence 5′-GGA(A/T)-3′ through the highly conserved DNA-binding domain.³ The DNA-binding domain for ETS proteins, termed ETS domain, is about 85 amino acid residues in length and forms a winged helix-turn-helix motif consisting of three α-helices and four β-strands. The recent X-ray5., 6., 7., 8., 9., 10. and NMR11., 12., 13. studies of the ETS domain–DNA complexes have shown that the helix-3 in the winged helix-turn-helix motif binds in the major groove of the consensus DNA sequence.

In the crystal structure of Ets-1 ETS domain–DNA [d(TAGTGCCGGAAATGT)₂] complex (PDB code: 1K79), two arginine residues, Arg391 and Arg394, which are in the helix-3 region and conserved among the ETS family, make bidentate interactions with G₁ and G₂, respectively (Figure 1).⁵ However, the pattern of these hydrogen bonds is not maintained in the crystal structures of other ETS domain–DNA complexes.7., 9., 10. In addition, the interaction between the arginine residues, Arg391 and Arg394, and the consensus DNA sequence is not observed in the NMR study on Ets-1 ETS domain–DNA complex.12., 13.

On the other hand, a few studies have proposed direct contacts of the amino acid residues in ETS domain with the AA region (+3 and +4 positions) in the GGAA core. For example, an X-ray study of the Ets-1 ETS domain–DNA complex indicated what would be a vital role of hydrophobic interaction between the phenyl ring of Tyr395 and 5-methyl group of T_4′ or T_5′.⁵ However, this type of interaction was not observed in other ETS domain–DNA complexes,⁶ nor is the tyrosine residue conserved in other ETS family proteins such as PU.1 and TEL.9., 10. Thus, the precise molecular mechanism that clearly explains the sequence-specific GGAA recognition by ETS domain is still lacking.

The phosphate groups of DNA adjacent to the core sequence GGAA have contacts to the winged segment and the turn region between helix-2 and helix-3 of the ETS domain. It was reported that the neutralization of anionic phosphate charges on one face of DNA resulted in the DNA bending, probably due to the electrostatic repulsions of the remaining anionic charges.14., 15., 16. In fact, DNA bending was observed in the crystal structures of the ETS domain–DNA complexes.5., 6., 7., 8., 9., 10. It was also supposed that the conformational change of DNA caused by the DNA bending would serve to provide effective GGAA core recognition by the helix-3 of the ETS domain. However, the bending angles of DNA previously reported in the X-ray crystallographic analyses of ETS domain–DNA complexes vary from one system to another.5., 6., 7., 8., 9., 10. Thus, additional investigation is required in order to clarify a common role of the DNA bending in the sequence-specific binding of the ETS domain.

Significant developments have been made in the last few years in the procedures of molecular dynamics (MD). Improvements in AMBER,¹⁷ CHARMM18., 19. and GROMOS²⁰ force fields and effective treatment of long-range electrostatic interactions by using particle mesh Ewald (PME) method,²¹ explicit inclusion of solvent and ions have opened the possibilities for accurate determination of protein and DNA structures.²² Besides availability of supercomputers have enabled to undertake simulations on a nanosecond (ns) time-scale which expanded conformational sampling and eventually to elucidate biomolecular interactions. Further understanding of the molecular mechanism of sequence-specific DNA binding of the ETS domain is likely to provide novel clues for the design of drugs that bind to inhibit the interaction between the ETS domain and DNA. We report here 3.5–3.9 ns MD simulations of two Ets-1 ETS domain–DNA complex systems. The amino acid sequence of Ets-1 ETS domain and the DNA sequences used in this study are shown in Figure 2. The binding activity of the ETS domain is known to be higher than that of the whole ETS-1 protein.23., 24., 25., 26., 27. Therefore, the ETS domain–DNA complex would be a good model system for MD simulation. The first system has the ETS domain (103 amino acid residues) of Ets-1 protein (Figure 2(a)) and the high affinity 14 base-pair DNA containing GGAA core (+1 to +4, Figure 2(b)) sequence, while the second one has a low affinity DNA involving a mutation of a single base-pair, GGAG sequence. In the text, we refer to these complexes as ETS–GGAA and ETS–GGAG, respectively. In addition, results from the MD simulation of the 14 base-pair DNA having the GGAA core sequence (referred as GGAA) are also discussed for comparison.

Section snippets

Results

The root-mean-square deviations (RMSD) of the protein backbone and DNA heavy-atoms with respect to the minimized structures of ETS domain–DNA complexes and DNA (GGAA) are given in Figure 3. During the simulation, the RMSD values of the protein fluctuated around 1.16–2.12 Å in ETS–GGAA and 0.92–1.55 Å in ETS–GGAG (Figure 3(a)), while those of DNA in ETS–GGAA and ETS–GGAG are from 1.06 Å to 2.18 Å and from 1.12 Å to 1.96 Å, respectively (Figure 3(b)). The plots indicate the stability at about 900 ps,

Comparison of the MD structures to the experimental data

MD simulations on ETS–GGAA and ETS–GGAG clearly show the presence of meta-stable states of hydrogen bonding in which the conserved residues, Arg391 and Arg394, are participating (Figure 7). In the low affinity ETS–GGAG complex, both arginine residues change hydrogen bonding partners after 2 ns MD simulation. In contrast, Arg394 in ETS–GGAA changes only its side-chain conformation preference at around 1.7 ns to form more stable bidentate hydrogen bonds with the same partner (Figure 8, Figure 8).

Conclusions

We conducted 3.5–3.9 ns MD simulations of Ets-1 ETS domain–14 base-pair DNA complexes with PME treatment of electrostatic interactions. These MD simulations have provided us a good deal of information on the sequence specific interaction between ETS domain and the consensus DNA, as schematically shown in Figure 10. Two conserved arginine residues, Arg391 and Arg394, play an important role in binding with the GGAA core sequence. Although these arginine residues show certain flexibility in

Modeling of initial structures

The crystal structure (PDB code: 1K79) of Ets-1 ETS domain–15 base-pair DNA complex⁵ was used for preparation of the starting structure of ETS–GGAA. Although the crystal structure involved two complexes in the asymmetric unit, these two structures are essentially identical. So, the first complex was considered in our study. In the crystal structure, the 15 base-pair DNA has a 5′-overhang structure, so the 5′-terminal nucleotide in each strand was deleted. An unusual hydrogen bond pattern was

Acknowledgements

We thank Sun Hur (UCSB) for helpful discussions. This work was supported by NIH grant 5R37DK0917136. We acknowledge computer time on UCSB's SGI Origin 2000.

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Journal of Molecular Biology

Sequence Specific DNA Binding of Ets-1 Transcription Factor: Molecular Dynamics Study on the Ets Domain–DNA Complexes

Abstract

Introduction

Section snippets

Results

Comparison of the MD structures to the experimental data

Conclusions

Modeling of initial structures

Acknowledgements

Int. J. Biochem. Cell. Biol

Int. J. Biochem. Cell. Biol

Biochim. Biophys. Acta Rev. Cancer

Mol. Cell

Mol. Cell

J. Biol. Chem

Cell

J. Biol. Chem

Biochem. Biophys. Res. Commun

J. Mol. Biol

J. Mol. Biol

Nature Struct. Biol

J. Biol. Chem

J. Mol. Graph

J. Mol. Graph

The Ets family of transcription factors

Eur. J. Biochem

Structure of the Elk-1–DNA complex reveals how DNA-distal residues affect ETS domain recognition of DNA

Nature Struct. Biol

The structure of GABP alpha/beta: an ETS domain ankyrin repeat heterodimer bound to DNA

Science

A new pattern for helix-turn-helix recognition revealed by the PU.1 ETS-domain–DNA complex

Nature

Solution structure of the Ets domain of Fli-1 when bound to DNA

Nature Struct. Biol

Correction of the NMR structure of the ETS1/DNA complex

J. Biomol. NMR

Bending of DNA by asymmetric charge neutralization: all-atom energy simulations

J. Am. Chem. Soc

DNA bending by asymmetric phosphate neutralization

Science

A modified version of the Cornell et al. Force field with improved sugar pucker phases and helical repeat

J. Biomol. Struct. Dynam

All-atom empirical force field for nucleic acids: I. Parameter optimization based on small molecule and condensed phase macromolecular target data

J. Comput. Chem

All-atom empirical force field for nucleic acids: II. Application to molecular simulations of DNA and RNA in solution

J. Comput. Chem