Identification of Two Secondary Ligand Binding Sites in 14-3-3 Proteins Using Fragment Screening

Abstract

Proteins typically interact with multiple binding partners, and often different parts of their surfaces are employed to establish these protein–protein interactions (PPIs). Members of the class of 14-3-3 adapter proteins bind to several hundred other proteins in the cell. Multiple small molecules for the modulation of 14-3-3 PPIs have been disclosed; however, they all target the conserved phosphopeptide binding channel, so that selectivity is difficult to achieve. Here we report on the discovery of two individual secondary binding sites that have been identified by combining nuclear magnetic resonance-based fragment screening and X-ray crystallography. The two pockets that these fragments occupy are part of at least three physiologically relevant and structurally characterized 14-3-3 PPI interfaces, including those with serotonin N-acetyltransferase and plant transcription factor FT. In addition, the high degree of conservation of the two sites implies their relevance for 14-3-3 PPIs. This first identification of secondary sites on 14-3-3 proteins bound by small molecule ligands might facilitate the development of new chemical tool compounds for more selective PPI modulation.

Structural characterization of 14-3-3ζ in complex with the human Son of sevenless homolog 1 (SOS1)

“…In this framework, the complex structure 14-3-3ζ/SOS1pSer 1161 would provide an initial first step for investigating and manipulating the binding mechanisms of binding of SOS1 to 14-3-3, in particular with small molecules identified, for example, by fragment-based approaches (Sijbesma et al, 2017) to stabilize the 14-3-3ζ/SOS1 interface. Because of the structural rigidity 14-3-3 proteins, X-ray crystallography is an useful method for the design and the optimization of new small molecules modulators (Mori et al, 2013).…”

“…2012; Molzan et al, 2013;Anders et al, 2013, Stevers et al 2016, can induce the desired effect on the activity, dimerization, or cellular localization of the binding partner. In addition, the chemical toolbox to target 14-3-3s has been extended by modified peptides (Glas et al, 2014;Milroy et al, 2015), supramolecular ligands (Bier et al, 2013(Bier et al, , 2017, and fragments (Sijbesma et al, 2017). Since the stabilization of the negative regulation of 14-3-3 to oncogenic SOS1 could be a valuable new therapeutic approach in certain cancer, this study could represent the starting point towards a pharmacological intervention in tumor cells with aberrant Ras-MAPK signaling.…”

14-3-3 proteins activatePseudomonasexotoxins-S and –T by chaperoning a hydrophobic surface

“…2c). 33 We found that STO1704, a compound related to NV-1, inhibited K-Ras modification by ExoS 233-453 and CRK modification by ExoT 235-457 in a dose dependent manner ( Fig. 2d and Supplementary Fig.…”

A new soaking procedure for X-ray crystallographic structural determination of protein–peptide complexes

“…For example, the 14-3-3 isoform (but not other isoforms) specifically targets p53 and is associated with tumour suppression (Benzinger et al, 2005), making it invaluable to determine crystal structures of the target peptide with the correct isoform (Benzinger et al, 2005). These 14-3-3-peptide co-crystal structures have provided the basis for early drug-discovery projects, representing structural proofs from a druggability perspective and leading to the identification of starting-point modulators for PPIs (Sijbesma et al, 2017(Sijbesma et al, , 2019. So far, promising starting points have been achieved.…”