Chimeric 14-3-3 proteins for unraveling interactions with intrinsically disordered partners

In eukaryotes, several "hub" proteins integrate signals from different interacting partners that bind through intrinsically disordered regions. The 14-3-3 protein hub, which plays wide-ranging roles in cellular processes, has been linked to numerous human disorders and is a promising target for therapeutic intervention. Partner proteins usually bind via insertion of a phosphopeptide into an amphipathic groove of 14-3-3. Structural plasticity in the groove generates promiscuity allowing accommodation of hundreds of different partners. So far, accurate structural information has been derived for only a few 14-3-3 complexes with phosphopeptide-containing proteins and a variety of complexes with short synthetic peptides. To further advance structural studies, here we propose a novel approach based on fusing 14-3-3 proteins with the target partner peptide sequences. Such chimeric proteins are easy to design, express, purify and crystallize. Peptide attachment to the C terminus of 14-3-3 via an optimal linker allows its phosphorylation by protein kinase A during bacterial co-expression and subsequent binding at the amphipathic groove. Crystal structures of 14-3-3 chimeras with three different peptides provide detailed structural information on peptide-14-3-3 interactions. This simple but powerful approach, employing chimeric proteins, can reinvigorate studies of 14-3-3/phosphoprotein assemblies, including those with challenging low-affinity partners, and may facilitate the design of novel biosensors

The renaissance of Ras

Increased signalling by the small G protein Ras is found in many human cancers and is often caused by direct mutation of this protein. Hence, small-molecule attenuation of pathological Ras activity is of utmost interest in oncology. However, despite nearly three decades of intense drug discovery efforts, no clinically viable option for Ras inhibition has been developed. Very recently, reports of a number of new approaches of addressing Ras activity have led to the revival of this molecular target with the prospect of finally fulfilling the therapy promises associated with this important protein

Dual-input regulation and positional control in hybrid oligonucleotide/discotic supramolecular wires

\u3cp\u3eThe combination of oligonucleotides and synthetic supramolecular systems allows for novel and long-needed modes of regulation of the self-assembly of both molecular elements. Discotic molecules were conjugated with short oligonucleotides and their assembly into responsive supramolecular wires studied. The self-assembly of the discotic molecules provides additional stability for DNA-duplex formation owing to a cooperative effect. The appended oligonucleotides allow for positional control of the discotic elements within the supramolecular wire. The programmed assembly of these hybrid architectures can be modulated through the DNA, for example, by changing the number of base pairs or salt concentration, and through the discotic platform by the addition of discotic elements without oligonucleotide handles. These hybrid supramolecular-DNA structures allow for advanced levels of control over 1D dynamic platforms with responsive regulatory elements at the interface with biological systems.\u3c/p\u3

Mutually exclusive cellular uptake of combinatorial supramolecular copolymers

\u3cp\u3eThe cellular uptake of self-assembled biological and synthetic matter results from their multicomponent properties. However, the interplay of the building block composition of self-assembled materials and uptake mechanisms urgently requires addressing. It is shown here that supramolecular polymers that self-assemble in aqueous media, are a modular and controllable platform to modulate cellular delivery by the introduction of small ligands or cationic moieties, with concomitantly different cellular uptake kinetics and valence dependence. A library of supramolecular copolymers revealed stringent mutually exclusive uptake behavior in which either of the uptake pathways dominated, with sharp compositional transition. Supramolecular biomaterial engineering thus provides for adaptive platforms with great potential for efficient tuning of multivalent and multicomponent systems interfacing with biological matter.\u3c/p\u3

Supramolecular chemistry targeting proteins

\u3cp\u3eThe specific recognition of protein surface elements is a fundamental challenge in the life sciences. New developments in this field will form the basis of advanced therapeutic approaches and lead to applications such as sensors, affinity tags, immobilization techniques, and protein-based materials. Synthetic supramolecular molecules and materials are creating new opportunities for protein recognition that are orthogonal to classical small molecule and protein-based approaches. As outlined here, their unique molecular features enable the recognition of amino acids, peptides, and even whole protein surfaces, which can be applied to the modulation and assembly of proteins. We believe that structural insights into these processes are of great value for the further development of this field and have therefore focused this Perspective on contributions that provide such structural data.\u3c/p\u3

Modulators of protein-protein interactions

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A study on the effect of synthetic α-to-β3-amino acid mutations on the binding of phosphopeptides to 14-3-3 proteins

\u3cp\u3eHere we describe the synthesis of a series of α,β-phosphopeptides, based on the phosphoepitope site on YAP1 (yes-associated protein 1), and the biochemical, biophysical and structural characterization of their binding to 14-3-3 proteins. The impact of systematic mono- and di-substitution of α → β3 amino acid residues around the phosphoserine residue are discussed. Our results confirm the important role played by the +2 proline residue in the thermodynamics and structure of the phosphoepitope/14-3-3 interaction.\u3c/p\u3

Relationship between side-chain polarity and the self-assembly characteristics of perylene diimide derivatives in aqueous solution

\u3cp\u3ePerylene-3,4,9,10-tetracarboxylic acid diimides (PDIs) have recently gained considerable interest for water-based biosensing applications. PDIs have been studied intensively in the bulk state, but their physical properties in aqueous solution in interplay with side-chain polarity are, however, poorly understood. Therefore, three perylene diimide based derivatives were synthesized to study the relationship between side-chain polarity and their self-assembly characteristics in water. The polarity of the side chains was found to dictate the size and morphology of the formed aggregates. Side-chain polarity rendered the self-assembly and photophysical properties of the PDIs—both important for imminent water-based applications—and these were revealed to be especially responsive to changes in solvent composition.\u3c/p\u3

Inhibition of 14-3-3/Tau by hybrid small-molecule peptides operating via two different binding modes

\u3cp\u3eCurrent molecular hypotheses have not yet delivered marketable treatments for Alzheimer's disease (AD), arguably due to a lack of understanding of AD biology and an overreliance on conventional drug modalities. Protein-protein interactions (PPIs) are emerging drug targets, which show promise for the treatment of, e.g., cancer, but are still underexploited for treating neurodegenerative diseases. 14-3-3 binding to phosphorylated Tau is a promising PPI drug target based on its reported destabilizing effect on microtubules, leading to enhanced neurofibrillary tangle formation as a potential cause of AD-related neurodegeneration. Inhibition of 14-3-3/Tau may therefore be neuroprotective. Previously, we reported the structure-guided development of modified peptide inhibitors of 14-3-3/Tau. Here, we report further efforts to optimize the binding mode and activity of our modified Tau peptides through a combination of chemical synthesis, biochemical assays, and X-ray crystallography. Most notably, we were able to characterize two different high-affinity binding modes, both of which inhibited 14-3-3-binding to full-length PKA-phosphorylated Tau protein in vitro as measured by NMR spectroscopy. Our findings, besides producing useful tool inhibitor compounds for studying 14-3-3/Tau, have enhanced our understanding of the molecular parameters for inhibiting 14-3-3/Tau, which are important milestones toward the establishment of our 14-3-3 PPI hypothesis.\u3c/p\u3

A binary bivalent supramolecular assembly platform based on Cucurbit[8]uril and Dimeric adapter protein 14-3-3

\u3cp\u3eInteractions between proteins frequently involve recognition sequences based on multivalent binding events. Dimeric 14-3-3 adapter proteins are a prominent example and typically bind partner proteins in a phosphorylation-dependent mono- or bivalent manner. Herein we describe the development of a cucurbit[8]uril (Q8)-based supramolecular system, which in conjunction with the 14-3-3 protein dimer acts as a binary and bivalent protein assembly platform. We fused the phenylalanine-glycine-glycine (FGG) tripeptide motif to the N-terminus of the 14-3-3-binding epitope of the estrogen receptor α (ERα) for selective binding to Q8. Q8-induced dimerization of the ERα epitope augmented its affinity towards 14-3-3 through a binary bivalent binding mode. The crystal structure of the Q8-induced ternary complex revealed molecular insight into the multiple supramolecular interactions between the protein, the peptide, and Q8.\u3c/p\u3