Biophysical characterization of the ETV6 PNT domain polymerization interfaces

Chloe A.N Gerak,Sophia Y Cho,Maxim Kolesnikov,Mark Okon,Michael E.P Murphy,Richard B Sessions,Michel Roberge,Lawrence P Mcintosh

doi:10.1016/j.jbc.2021.100284

Chloe A.N Gerak, Sophia Y Cho + Show 6 more

Open Access

https://doi.org/10.1016/j.jbc.2021.100284

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Abstract

ETV6 is an E26 transformation specific family transcriptional repressor that self-associates by its PNT domain to facilitate cooperative DNA binding. Chromosomal translocations frequently generate constitutively active oncoproteins with the ETV6 PNT domain fused to the kinase domain of one of many protein tyrosine kinases. Although an attractive target for therapeutic intervention, the propensity of the ETV6 PNT domain to polymerize via the tight head-to-tail association of two relatively flat interfaces makes it challenging to identify suitable small molecule inhibitors of this protein–protein interaction. Herein, we provide a comprehensive biophysical characterization of the ETV6 PNT domain interaction interfaces to aid future drug discovery efforts and help define the mechanisms by which its self-association mediates transcriptional repression. Using NMR spectroscopy, X-ray crystallography, and molecular dynamics simulations, along with amide hydrogen exchange measurements, we demonstrate that monomeric PNT domain variants adopt very stable helical bundle folds that do not change in conformation upon self-association into heterodimer models of the ETV6 polymer. Surface plasmon resonance–monitored alanine scanning mutagenesis studies identified hot spot regions within the self-association interfaces. These regions include both central hydrophobic residues and flanking salt-bridging residues. Collectively, these studies indicate that small molecules targeted to these hydrophobic or charged regions within the relatively rigid interfaces could potentially serve as orthosteric inhibitors of ETV6 PNT domain polymerization.

Highlights

Domain self-associates in a head-to-tail fashion to form an open-ended, left-handed helical polymer [4, 5]
Amide chemical shifts are highly sensitive to even subtle environmental changes, and an interaction with the unlabeled species can usually be detected through chemical shift perturbations (CSPs) of the labeled protein
Amides exhibiting CSPs typically cluster around the protein–ligand interface, yet may be distal if binding causes longer range structural changes [20]

Summary

Results

The NMR spectroscopy can give insights into the thermodynamic, kinetic, and structural mechanisms of protein–ligand interactions. Residues in the 15N-labeled A93D-PNT and V112E-PNT domains that experienced the greatest spectral perturbations cluster within the EH- and ML-surfaces, respectively (Fig. 2) This confirms that, as seen by X-ray crystallography, the two monomerized PNT domains associate in solution through their wild-type interfaces. E112, the monomers assembled in the crystal lattice via their ML- and EH-surfaces to form an extended helical polymer with an approximate 65 screw symmetry (Fig. 3A) They determined the structure of a heterodimer composed of a A93D-PNT domain bound to a V112R-PNT domain via their complementary wild-type interfaces (PDB: 1LKY, P1 space group, 3 heterodimers in the asymmetric unit) [5]. The latter serves as a reliable experimental structure of the ETV6 PNT domain polymer

A93 E112 V112E-PNT domain Asymmetric units

D PNT Domain Complexed Comparison

16 V112E-PNT domain

Discussion

Experimental procedures