Mechanism of cognate sequence discrimination by the ETS-family transcription factor ETS-1

Kenneth Huang,Suela Xhani,Amanda V Albrecht,Van L.T Ha,Shingo Esaki,Gregory M.K Poon

doi:10.1074/jbc.ra119.007866

Abstract

Functional evidence increasingly implicates low-affinity DNA recognition by transcription factors as a general mechanism for the spatiotemporal control of developmental genes. Although the DNA sequence requirements for affinity are well-defined, the dynamic mechanisms that execute cognate recognition are much less resolved. To address this gap, here we examined ETS1, a paradigm developmental transcription factor, as a model for which cognate discrimination remains enigmatic. Using molecular dynamics simulations, we interrogated the DNA-binding domain of murine ETS1 alone and when bound to high-and low-affinity cognate sites or to nonspecific DNA. The results of our analyses revealed collective backbone and side-chain motions that distinguished cognate versus nonspecific as well as high- versus low-affinity cognate DNA binding. Combined with binding experiments with site-directed ETS1 mutants, the molecular dynamics data disclosed a triad of residues that respond specifically to low-affinity cognate DNA. We found that a DNA-contacting residue (Gln-336) specifically recognizes low-affinity DNA and triggers the loss of a distal salt bridge (Glu-343/Arg-378) via a large side-chain motion that compromises the hydrophobic packing of two core helices. As an intact Glu-343/Arg-378 bridge is the default state in unbound ETS1 and maintained in high-affinity and nonspecific complexes, the low-affinity complex represents a unique conformational adaptation to the suboptimization of developmental enhancers.

Highlights

Functional evidence increasingly implicates low-affinity DNA recognition by transcription factors as a general mechanism for the spatiotemporal control of developmental genes
The low-affinity complex was uniquely characterized by concerted side-chain dynamics in which contact by Gln-336 with the DNA backbone adjacent to the core consensus was allosterically coupled to the distal Glu-343/Arg-378 salt bridge
A conserved triad of residues mediates low-affinity binding by ETS1

Summary

Results

To maximize the relevance of the simulations to experiments, a binary co-crystal structure (PDB code 1K79) that most closely corresponded to the C-terminal ETS domain (⌬N331) used in experimental studies [15, 17, 21, 35,36,37] was used to template the simulational models. The high- and low-affinity cognate sequences (termed in the literature SC1 and SC12) were established substrates originally identified in SELEX (systematic evolution of ligands by exponential enrichment) screening against this same ETS1 construct [15]. Cognate complexes of ⌬N331 differed in stability by 13-fold (⌬⌬G° ϭ ϩ6.40 kJ/mol) and that the nonspecific complex bound to a randomized sequence was an additional 28-fold weaker (⌬⌬G° ϭ ϩ8.27 kJ/mol) than the low-affinity complex. The established secondary structure assignment for ETS domains, a winged helix–loop– helix motif, is H1–S1–S2–H2– loop–H3–S3–wing–S4 (H, ␣-helix; S, ␤-strand). ⌬N331 did not include additional N-terminal helices (termed HI-1 and HI-2) that are extrinsic to the winged helix–loop– helix motif and negatively regulate DNA binding. We reserve the label “low-affinity” for suboptimal consensus-bearing sequences but not nonspecific DNA

Validation of simulations against experiments

Discussion

Molecular dynamics simulation

MD trajectory analysis

Molecular cloning and recombinant protein preparation