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

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 cellularlike environments, but a previous genetic code expansion (GCE) system to translationally install nonhydrolyzable phosphoserine (nhpSer), with the γ-oxygen replaced with CH 2 , 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.

Genetic Encoding of Phosphorylated Amino Acids into Proteins

“…The tendency of a target protein to be dephosphorylated in E. coli depends on the protein of interest and site of encoding and is likely higher when the site of phosphorylation resides within a disordered or flexible region of a protein easily accessible to phosphatases. Shorter expression times, lower expression temperatures, or use of nonhydrolyzable mimics (discussed more below) can mitigate unwanted dephosphorylation . In mammalian cells, target proteins with pSer encoded into them were purified with serine at the site of encoding, caused by the elaborate network of endogenous protein phosphatases, necessitating the need to encode a nonhydrolyzable mimic of pSer …”

“…Charged amino acids do not cross the cellular membranes well, explaining why it is important to use Δ serB expression strains that generate high concentrations of intracellular pSer amino acid via biosynthesis for pSer protein expression (Figure B). Thus, to address the low nhpSer encoding efficiency that resulted when nhpSer ( 20 ) was supplemented to the media, Zhu et al sought to engineer its biosynthesis . To do this, they built on work by Johannes et al., who predicted that nhpSer ( 20 ) was an intermediate along the 10-step Streptomyces rubellomurinus pathway that converted phosphoenolpyruvate ( 24 ) into the antimalarial natural product FR-900098 .…”

“…With the collective efforts invested into their development, pSer ( 2 )/nhpSer ( 20 ) GCE systems are increasingly being used to address important biological questions about phospho-protein function, as can be seen in Table and Figure , that summarize aspects of each of the 73 studies to date that have developed or used these technologies. As adopting these GCE technologies has not always been trivial, motivating factors are often articulated, and these largely include: (i) technical challenges associated with using the needed kinase such as incomplete and/or off-target phosphorylation (16 reports) (refs , , , , − ), (ii) inadequate knowledge about the required kinase (7 reports) (refs , , − ), and (iii) uncertainty about the ability of Asp/Glu to mimic pSer function (31 reports) (, , , , , , , , , , , , , − ). In at least 13 of the latter cases, GCE encoding of pSer was used because Asp/Glu mutations failed to recapitulate pSer function (, , , , , , , , , , , , ).…”

“…As adopting these GCE technologies has not always been trivial, motivating factors are often articulated, and these largely include: (i) technical challenges associated with using the needed kinase such as incomplete and/or off-target phosphorylation (16 reports) (refs , , , , − ), (ii) inadequate knowledge about the required kinase (7 reports) (refs , , − ), and (iii) uncertainty about the ability of Asp/Glu to mimic pSer function (31 reports) (, , , , , , , , , , , , , − ). In at least 13 of the latter cases, GCE encoding of pSer was used because Asp/Glu mutations failed to recapitulate pSer function (, , , , , , , , , , , , ). Although the phosphorylation-dependent pathways and processes studied span many areas, as one would expect, four notably well-represented foci are the areas of (i) ubiquitin and protein degradation, (ii) kinase activation, (iii) 14–3–3/client complexes, and (iv) histone function.…”

“…In separate work, TRIM9 phosphorylated at Ser79 was able to bind to and block the activity of the E2/E3 ligase β-TrCP-SCF, preventing it from ubiquitinating target proteins IκBα and p100 . Also, pSer GCE aided the discovery of several pSer-degrons that recruit target proteins to E2/E3 ligase complexes in a phosphorylation-dependent manner: the target proteins discovered include centromeric protein A (CENP-A), master transcriptional regulators TFE3 and MITF, and potentially 14–3–3ζ. , In the case of 14–3–3ζ, nhpSer encoded at Ser58 pulled down the E3 ligase adaptor protein Cereblon from cell-extracts, implying that phosphorylation serves to promote ubiquitination of 14–3–3 as well as the bound clients of 14–3–3ζ …”