Fragment-based exploration of the 14-3-3/Amot-p130 interface

Abstract

The modulation of protein-protein interactions (PPIs) has developed into a well-established field of drug discovery. Despite the advances achieved in the field, many PPIs are still deemed as 'undruggable' targets and the design of PPIs stabilizers remains a significant challenge. The application of fragment-based methods for the identification of drug leads and to evaluate the 'tractability' of the desired protein target has seen a remarkable development in recent years. In this study, we explore the molecular characteristics of the 14-3-3/Amot-p130 PPI and the conceptual possibility of targeting this interface using X-ray crystallography fragment-based screening. We report the first structural elucidation of the 14-3-3 binding motif of Amot-p130 and the characterization of the binding mode and affinities involved. We made use of fragments to probe the 'ligandability' of the 14-3-3/Amot-p130 composite binding pocket. Here we disclose initial hits with promising stabilizing activity and an early-stage selectivity toward the Amot-p130 motifs over other representatives 14-3-3 partners. Our findings highlight the potential of using fragments to characterize and explore proteins' surfaces and might provide a starting point toward the development of small molecules capable of acting as molecular glues.

Keywords: 14-3-3 /protein-protein interactions stabilizers; AIP4, Atrophin-1 interacting protein 4; Amot, Angiomotin; Amot-p130; AmotL1/2, Angiomotin-like 1/2; FBDD, Fragment-based drug discovery; FP, Fluorescence polarization; Fragment-based drug discovery; Lats 1/2, Large tumor suppressor 1/2; Ligandability; MST, Microscale thermophoresis; PPI, Protein-protein interaction; PTMs, post-translational modifications; X-ray crystallography; YAP1, Yes-associated protein 1.

Graphical abstract

Fig. 1

Characterization of the interaction between 14-3-3 and Amot-p130. (A) Domain organization of Amot-p130 including LPTY and PPEY binding motifs for YAP1 interaction, the conserved coiled-coil domains (CC), Angiostatin binding domain, and the C-terminal PDZ-binding domain. The 14-3-3 binding site and its sequence are indicated. (B) Schematic illustration of the interaction between 14-3-3, Amot-p130, and YAP1. The binding of 14-3-3 to Amot-p130 is induced by phosphorylation of S175 by the Lats 1/2 kinase. 14-3-3, Amot-p130, and YAP1 are recruited into a common complex that can interact with the AIP4 E3 ubiquitin ligase. The ubiquitination induced by AIP4 results in YAP1's degradation by the proteasome system. The sequestration of YAP1 to the cytosol and its ubiquitin-induced degradation prevents YAP1 from entering the nucleus to promote the transcription of pro-proliferative genes (Adler et al., 2013a). (C) Fluorescence Polarization data (mean ​± ​SD; triplicates) of the 14-3-3 human isoforms titration against the FITC-labelled Amot-p130 peptide. Background polarization was subtracted from all values. SD error bars are smaller than data point symbols. (D) High resolution crystal structure of 13-mer Amot-p130 peptide in complex with 14-3-3σΔC (PDB ID: 7NMA). The crystals belong to the space group C2221 with one 14-3-3 monomer in the asymmetric unit. Front view of 14-3-3σ monomer (white surface) bound to the Amot-p130 phosphopeptide (cyan vdw spheres). (E) Close-up view of the binding groove. Polar contacts are indicated by dashed lines and hydrophobic interactions are depicted as a wireframe. The peptide sequence is ¹⁶⁹GHVRSLpSERLMQM¹⁸¹. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Crystal structures of fragments binding to the 14-3-3σΔC/Amot-p130 peptide complex. (A–B) Front and side view of the pocket formed at the 14-3-3/Amot-p130 interface. 14-3-3σΔC protein is depicted with white cartoons and surface. The Amot-p130 peptide is represented with cyan sticks and surface. (C-D-E) Crystal structures of fragments binding to the 14-3-3σΔC protein/Amot-p130 complex. The fragments are depicted with colored sticks: 40 green (PDB ID: 7NMW), 12 pink (PDB ID: 7NMX), and 41 orange (PDB ID: 7NN2). The 2Fo-Fc electron density maps for both the peptide and fragments are displayed at 1σ. No direct contact with the peptide and the ligands were observed. (F–G–H) Close up of the interactions and distances in Å between the fragments and 14-3-3. 14-3-3 protein is depicted with white ribbons. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Crystal structures of fragments 09 and 22 in complex with 14-3-3σΔC and the Amot-p130 peptide. (A–B) Crystal structures of fragments 09 (PDB ID: 7NND) and 22 (PDB ID: 7NNE) binding at the protein/peptide interface and stabilizing the C-terminus of the peptide allowing the fitting of Met179 in the electron density. The 2F₀-F_C electron density map for the Amot-p130 peptide and ligands is contoured at 1σ. Contacts between the fragments, Glu14, and the water network are depicted with black dashed lines. 14-3-3σΔC is shown with a white surface and cartoon. Amot peptide (cyan), fragment 09 (magenta), and fragment 22 (purple) are represented with colored sticks. (C) Close up of the interactions (dashed lines) established by fragment 09 with 14-3-3 and Amot-p130. Distances in Å are reported. (D) Overlay of the Amot-p130 peptide in presence of fragments 09 and 22 (cyan sticks) and absence of the fragments (pink sticks). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Measurement of stabilization of fragments 09 and 22. (A) Chemical structures of fragments 22 and 09. (B) MST data (mean; duplicates) of fragment 22 titration (1:1 dilutions starting from 5 ​mM) against 14-3-3η/Amot-p130 complex. (C) MST data (mean; duplicates) of 14-3-3η titration against Cy5 labelled Amot-p130 peptide in presence of 1 ​mM 09 (red circles) or 22 (green circles). 1% DMSO control is represented with light blue circles. Because of the binding-dependent decrease in initial fluorescence upon protein titration, the raw fluorescence data were analyzed to generate binding curves (Fig. S6). (D) FP data (mean; triplicates) of 14-3-3η titration against FITC Amot-p130 peptide in presence of 1 ​mM 09 (pink squares) or 22 (purple triangle). 1% DMSO control is represented with black circles. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Comparison of hit fragment 22 activity over different 14-3-3 binding epitopes. (A) Overview of the selected 14-3-3 partner and their binding epitopes observed in the crystal structures. (B) Overlay of the crystal structures of the 14-3-3 binding partners (cartoon representation). The phosphorylated site and C-terminus of Amot-p130 and Ataxin are highlighted with stick representations. Fragment 22 is depicted with purple lines and surface. 14-3-3σΔC protein is always depicted as a white surface. (C–E) Crystal structures of fragment 22 (purple sticks and surface) bound to 14-3-3σΔC in complex with Amot-p130 peptide (cyan sticks and surface) PDB ID: 7NNE or in complex with p53 peptide (green sticks and surface) PDB ID: 6RM7 (Guillory et al., 2020). (D) Binding mode of the Ataxin peptide (orange sticks and surface) PDB ID: 6QIU (Leysen et al., 2021). The polar contacts between Arg781 of the peptide and Glu14 of 14-3-3 are depicted as black dashed lines. Glu14 of 14-3-3 is highlighted with red sticks and surface. (F) FP data (mean; triplicates) of 14-3-3η titration in presence and absence of 1 ​mM 22 and TAMRA-labelled Amot-p130, Ataxin, or p53 peptides. Background polarization was subtracted from all values. DMSO in the assay was 1%. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)