The Molecular Tweezer CLR01 Stabilizes a Disordered Protein-Protein Interface

Abstract

Protein regions that are involved in protein-protein interactions (PPIs) very often display a high degree of intrinsic disorder, which is reduced during the recognition process. A prime example is binding of the rigid 14-3-3 adapter proteins to their numerous partner proteins, whose recognition motifs undergo an extensive disorder-to-order transition. In this context, it is highly desirable to control this entropy-costly process using tailored stabilizing agents. This study reveals how the molecular tweezer CLR01 tunes the 14-3-3/Cdc25CpS216 protein-protein interaction. Protein crystallography, biophysical affinity determination and biomolecular simulations unanimously deliver a remarkable finding: a supramolecular "Janus" ligand can bind simultaneously to a flexible peptidic PPI recognition motif and to a well-structured adapter protein. This binding fills a gap in the protein-protein interface, "freezes" one of the conformational states of the intrinsically disordered Cdc25C protein partner and enhances the apparent affinity of the interaction. This is the first structural and functional proof of a supramolecular ligand targeting a PPI interface and stabilizing the binding of an intrinsically disordered recognition motif to a rigid partner protein.

Figure 1

(A) Structure of the molecular tweezer CLR01. (B) The Janus-type molecular tweezer CLR01 offers two separate recognition sites for proteins: the aromatic cavity with flanking hydrogen phosphate anions accommodates a lysine or argine chain, whereas the convex exterior presents an apolar aromatic surface able to dock onto hydrophobic clefts on the target protein.

Figure 2

Binding of FAM-labeled Cdc25CpS216 to 14-3-3ζ. Titration of 14-3-3ζ to FAM-Cdc25CpS216 resulted in the determination of a K_d of 30.4 μM, the same measurement in the presence of 250 μM CLR01 produced an approximately 20-fold lower apparent K_d of 1.4 μM.

Figure 3

Binding of the Cdc25CpS216 peptide to 14-3-3ζ measured by ITC. (A) Binding isotherms of titrating Cdc25CpS216 into 14-3-3ζ in the absence and presence of CLR01. (B) Thermodynamic parameters of the ITC titrations shown in A.

Figure 4

Binding of CLR01 to the 14-3-3ζ/Cdc25CpS216 interface. (A) Electron density (2F_o–F_c, blue mesh) of the Cdc25CpS216 peptide bound to 14-3-3ζ (white solid surface). (B) Electron density as in (A) after soaking the crystals with CLR01. (C) Overlay of the Cdc25CpS216 peptide bound to 14-3-3ζ (white surface) in the absence (magenta sticks) and the presence (green sticks) of CLR01 (yellow sticks).

Figure 5

Crystal structure of Cdc25CpS216 bound to 14-3-3ζ and CLR01. (A) Details of the interface of Cdc25CpS216 (green sticks) and 14-3-3ζ (white cartoon), stabilized by CLR01 (yellow sticks). Residues of 14-3-3ζ that are important for direct contacts with the Cdc25CpS216 peptide are shown as sticks, residues that contribute van-der-Waals contacts are shown as transparent spheres. Dotted lines indicate hydrogen bonds, water molecules are shown as red spheres. (B) Composite omit map (contoured at 1σ) electron density of CLR01 (yellow sticks) bound to 14-3-3ζ (white cartoon and semitransparent surface) and Cdc25CpS216 (green sticks). (C) Details of the binding surface of the molecular tweezer accommodated in the amphipathic groove of 14-3-3ζ.

Figure 6

Binding of CLR01 to K74 of 14-3-3. (A) 14-3-3ζ dimer (white cartoon) in complex with Cdc25CpS216 (20mer, green sticks) and three molecules of CLR01 (yellow spheres). (B) Unbiased F_o–F_c electron density map (green mesh, contoured at 2.5σ) previous to include CLR01 in the model. (C) Final 2F_o–F_c electron density map (blue mesh, contoured at 1σ). (D) Environment of CLR01 binding to K74 of one monomer of the 14-3-3ζ dimer. (E) Stabilization of CLR01 binding to K74 by contacts established with a symmetry-related 14-3-3 molecule (cyan cartoon and sticks).

Figure 7

Binding of CLR01 to R208_Cdc25C in solution. (A) CLR01 acts as a molecular “glue” between Cdc25C and 14-3-3ζ. (B) The addition of CLR01 results in additional hydrophobic contacts between Cdc25C and 14-3-3ζ (left, beginning of the simulation; right, hydrophobic collapse as the simulation progresses).