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

DOI: 10.1038/s41598-017-12214-9 DOI Help

Authors: Nikolai N. Sluchanko (A.N. Bach Institute of Biochemistry, Federal Research Center “Fundamentals of Biotechnology” of the Russian Academy of Sciences; Moscow State University) , Kristina V. Tugaeva (A.N. Bach Institute of Biochemistry, Federal Research Center “Fundamentals of Biotechnology” of the Russian Academy of Sciences; Moscow State University) , Sandra J. Greive (University of York) , Alfred A. Antson (University of York)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Scientific Reports , VOL 7

State: Published (Approved)
Published: September 2017
Diamond Proposal Number(s): 9948 , 13587

Open Access Open Access

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.

Journal Keywords: Biological fluorescence; Intracellular signalling peptides and proteins; Phosphorylation; Protein–protein interaction networks; X-ray crystallography

Subject Areas: Biology and Bio-materials, Medicine


Instruments: I02-Macromolecular Crystallography , I04-Macromolecular Crystallography

Added On: 11/10/2017 10:21

Documents:
s41598-017-12214-9.pdf

Discipline Tags:

Health & Wellbeing Structural biology Drug Discovery Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)