Autonomous Synthesis of Functional, Permanently Phosphorylated Proteins for Defining the Interactome of Monomeric 14-3-3ζ

Phillip Zhu; Stanislau Stanisheuski; Rachel Franklin; Amber Vogel; Cat Hoang Vesely; Patrick Reardon; Nikolai N Sluchanko; Joseph S Beckman; P Andrew Karplus; Ryan A Mehl; Richard B Cooley

doi:10.1021/acscentsci.3c00191

Autonomous Synthesis of Functional, Permanently Phosphorylated Proteins for Defining the Interactome of Monomeric 14-3-3ζ

ACS Cent Sci. 2023 Apr 10;9(4):816-835. doi: 10.1021/acscentsci.3c00191. eCollection 2023 Apr 26.

Affiliations

¹ Department of Biochemistry and Biophysics, Oregon State University, 2011 Agricultural and Life Sciences, Corvallis, Oregon 97331, United States.
² Department of Chemistry, Oregon State University, 153 Gilbert Hall, Corvallis, Oregon 97331, United States.
³ A.N. Bach Institute of Biochemistry, Federal Research Center of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia.
⁴ e-MSion Inc., 2121 NE Jack London St., Corvallis, Oregon 97330, United States.

Abstract

14-3-3 proteins are dimeric hubs that bind hundreds of phosphorylated "clients" to regulate their function. Installing stable, functional mimics of phosphorylated amino acids into proteins offers a powerful strategy to study 14-3-3 function in cellular-like environments, but a previous genetic code expansion (GCE) system to translationally install nonhydrolyzable phosphoserine (nhpSer), with the γ-oxygen replaced with CH₂, site-specifically into proteins has seen limited usage. Here, we achieve a 40-fold improvement in this system by engineering into Escherichia coli a six-step biosynthetic pathway that produces nhpSer from phosphoenolpyruvate. Using this autonomous "PermaPhos" expression system, we produce three biologically relevant proteins with nhpSer and confirm that nhpSer mimics the effects of phosphoserine for activating GSK3β phosphorylation of the SARS-CoV-2 nucleocapsid protein, promoting 14-3-3/client complexation, and monomerizing 14-3-3 dimers. Then, to understand the biological function of these phosphorylated 14-3-3ζ monomers (containing nhpSer at Ser58), we isolate its interactome from HEK293T lysates and compare it with that of wild-type 14-3-3ζ. These data identify two new subsets of 14-3-3 client proteins: (i) those that selectively bind dimeric 14-3-3ζ and (ii) those that selectively bind monomeric 14-3-3ζ. We discover that monomeric-but not dimeric-14-3-3ζ interacts with cereblon, an E3 ubiquitin-ligase adaptor protein of pharmacological interest.

Grants and funding

RM1 GM144227/GM/NIGMS NIH HHS/United States