High Conformational Flexibility of the E2F1/DP1/DNA Complex

Highlights

• The structure and dynamics of the E2F1/DP1/DNA complex is modelled.

• E2F1 and DP1 play a different role in the dynamics of the system.

• DP1 flexibility may be relevant for protein–protein and protein-DNA interactions.

Abstract

The E2F1 transcription factor is a master regulator of cell-cycle progression whose uncontrolled activation contributes to tumor cells growth. E2F1 binds DNA as a heterodimer with DP partners, resulting in a multi-domain quaternary-structure complex composed of DNA binding domains, a coiled coil domain and a marked box domain separated by short linkers. Building on the 3D knowledge of the single domains of E2F and DPs, we characterized the structure and dynamics of the complete E2F1/DP1/DNA complex by a combination of small-angle X-ray scattering and molecular dynamics simulations. It shows an asymmetric contribution of the dynamics of the two proteins. Namely, the coiled-coil domain leans toward the DP1 side of the complex; the DP1 loop between α2 and α3 of the DBD partially populates a helical structure leaning far from the DNA and in the same direction of the coiled-coil domain; and the N-terminal disordered region of DP1, rich in basic residues, contributes to DNA binding stabilization. Intriguingly, tumor mutations in the flexible regions of the complex suggest that perturbation of protein dynamics could affect protein function in a context-dependent way. Our data suggest fundamental contributions of DP proteins in distinct aspects of E2F biology.

Introduction

RNA production is regulated by a plethora of sequence-specific Transcription Factors -TFs- that recognize short DNA sequences in enhancers and promoters of genes, forming modules dictated by the arrangement of the DNA sites. Binding of TFs to regulatory regions leads to the recruitment of cofactors endowed with chromatin remodeling and histone modification activities, which make chromatin accessible for RNA Polymerase processivity.¹ TFs typically require a minimum of a sequence-specific DNA-binding domain (DBD) and a Trans-Activation domain (TAD).

E2Fs constitute a family of eight evolutionarily conserved TFs, whose founding member -E2F1- was originally identified as a master regulator of cell-cycle progression.2, 3 In particular, E2F1 is essential for the G1/S transition and S-phase progression⁴; E2F4, as part of the DREAM complex, is also involved in repression of G2/M genes in G1/S, and cell-cycle exit during terminal differentiation,⁵ but it is an activator in mES cells.⁶ In addition to the regulation of cell-cycle genes, E2F1 has a global role in the regulation of apoptosis, the DNA-damage response, metabolism, and differentiation.⁷ E2F1 is usually catalogued as an activator, but depending on the cellular context, it can be also a repressor in differentiation.⁸ E2F1, among others, is targeted by the Retinoblastoma -Rb- and by other “pocket” proteins, as part of repressive complexes that include other corepressors: genetic mutations in Rb, as found in tumors, eliminate or weaken interaction, leading to uncontrolled E2F1 activation, cell cycle progression and cell growth.⁹ Somatic mutations in the E2F1 gene are relatively rare in cancer, but, as many other family members, the gene expression is often upregulated in transformed cells.¹⁰

E2Fs bind DNA as heterodimers with the structurally related DP1/2/3 subunits. In addition to DBDs, E2Fs have a coiled-coil domain (CC) involved in dimerization with the partner DPs, a marked-box (MB) domain responsible for the interaction with pocket proteins, and a C-terminal TAD. A similar structural scheme is shared by DP proteins, except that they lack a TAD. The DBD and CC domains are typically connected by a short linker.³ From the structural viewpoint, two parts have been characterized by crystallography: the minimal DBDs of E2F4/DP2, and of E2F8 in complex with “canonical” DNA sites11, 12; the CC-MB domains of E2F1/DP1 and of E2F5/DP1, including in complex with Rb pocket proteins peptides.13, 14 Such structures refer to DBDs separated from CC-MB: a coherent view of the relative topology of the linked domains is not available to date (Figure 1). For this reason, we set out to produce and characterize, both biochemically and structurally, the ensembles of E2F1 and DP1 DBD, CC, and MB domains. In so doing, we provide evidence for a high level of flexibility of the heterodimer, including a newly identified region stabilizing DNA contacts.