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

Nikolai N Sluchanko,Kristina V Tugaeva,Alfred A Antson,Sandra J Greive

doi:10.1038/s41598-017-12214-9

Abstract

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.

Highlights

The [-3] family of eukaryotic proteins are abundant, medium sized proteins (~30 kDa subunit mass) endowed with a well-characterized phosphopeptide-binding ability[1]
The linker used for fusing the HSPB6 phosphopeptide to the C-terminal of 14-3-3σ∆C included: the ordered Thr residue at position 1 (Fig. 1B) that is always present in electron density maps, even for C-terminally truncated [-3] variants; the natural Leu residue preceding the [-3] binding motif of HSPB6 (RRApS16APL); and a GSGS segment designed to provide maximal flexibility to create the prototypical 14-3-3/HSPB6 chimera chimera with the HSPB6 peptide RRAS16APL (CH1) (Fig. 1B)
To achieve stoichiometric phosphorylation of peptides within the chimeras, we co-expressed them in E. coli with the catalytically active subunit of protein kinase A (PKA), known to phosphorylate [-3] binders in vivo[33,35,36]

Summary

Introduction

The [-3] family of eukaryotic proteins are abundant, medium sized proteins (~30 kDa subunit mass) endowed with a well-characterized phosphopeptide-binding ability[1]. Limited structural information prevents understanding of the molecular basis for function of this key regulatory node involved in many clinically important signal transduction pathways, decelerating the development of novel therapeutic approaches. Such information is vital for finding small molecule modulators of specific 14-3-3/target complexes[28,29,30,31,32] that won’t affect interactions of [-3] with other targets. The current lack of structural information prevents delineating a universal “14-3-3 binding law” and understanding molecular details of the selectivity for [-3] interaction with hundreds of competing partners

Methods

Results

Conclusion