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.

Installing stable, functional mimics of phosphorylated amino acids into proteins offers a powerful strategy to study protein regulation. Previously, a genetic code expansion (GCE) system was developed to translationally install non-hydrolyzable phosphoserine (nhpSer), with the γ-oxygen replaced with carbon, but it has seen limited usage. Here, we achieve a 40-fold improvement in this system by engineering into Escherichia coli a biosynthetic pathway that produces nhpSer from the central metabolite phosphoenolpyruvate. Using this "PermaPhosSer" system — an autonomous 21-amino acid E. coli expression system for incorporating nhpSer into target proteins — we show that nhpSer faithfully mimics the effects of phosphoserine in three stringent test cases: promoting 14-3-3/client complexation, disrupting 14-3-3 dimers, and activating GSK3-β phosphorylation of the SARS-CoV-2 nucleocapsid protein. This facile access to nhpSer containing proteins should allow nhpSer to replace Asp and Glu as the go-to pSer phosphomimetic for proteins produced in E. coli.

Multivalent complexes formed between the cancer-promoting transcriptional co-activator, Yes-associated protein (YAP), and proteins containing short linear motifs of type PPxY modulate cell proliferation and are attractive therapeutic targets. However, challenges producing PPxY polypeptides containing the full binding domain has limited understanding of the assembly process. Here, we successfully produced a polypeptide containing the complete set of three PPxY binding sites of Angiomotin-like 1 (AMOTL1), a scaffolding protein that regulates the nucleo-cytoplasmic shuttling of YAP via WW-PPxY interactions. Using an array of biophysical techniques including isothermal titration calorimetry, size-exclusion chromatography coupled to multi-angle light scattering, and solution nuclear magnetic resonance spectroscopy, we show that the AMOTL1 polypeptide is partially disordered, and binds the YAP WW domains to form an ensemble of complexes of varying stabilities. The binding process is initiated by the binding of one YAP WW domain to one AMOTL1 PPxY motif and is completed by transient interactions of the second YAP WW domain with a second AMOTL1 PPxY motif to form an equilibrating mixture composed of various species having two YAP sites bound to two conjugate AMOTL1 sites. We rationalize that the transient interactions fine-tune the stability of the complex for rapid assembly and disassembly in response to changes in the local cellular environment.

Multivalent interactions of intrinsically disordered segments of proteins are essential in many cellular functions, but challenges in recombinant protein production has limited understanding of how the complexes assemble. One example is the interaction between Angiomotin-like 1 (AMOTL1), Yesassociated protein (YAP), and kidney and brain expressed protein (KIBRA), three multivalent proteins with roles in protein scaffolding, cell polarity, memory performance, and cell proliferation. YAP and KIBRA each contain two tandem WW domains, a 40-residue interaction module composed of three antiparallel b-strands which recognize the Leucine/Proline-Proline-any amino acid-Tyrosine (L/PPXY) motif, three of which are present in a primarily disordered segment of AMOTL1. While several studies support co-localization of AMOTL1 with YAP and/or KIBRA in cultured cells, the structural basis of assembly in the context of all binding-competent domains is not well understood. Here, we show by multiple approaches including circular dichroism (CD), isothermal titration calorimetry (ITC), analytical size exclusion chromatography (SEC), and solution NMR that YAP and KIBRA bind AMOTL1 with micromolar affinity and have mapped the binding interfaces to the N and C-terminal L/PPXY motifs, respectively. The two KIBRA WW domains synergize to enhance binding to the AMOTL1 motifs but only one WW domain was required for assembly of the YAP-AMOTL1 complex. Our findings provide novel, molecular-level insights that extend understanding of intrinsically disordered regions and multivalent binding modules in protein complexes.

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