PermaPhos^Ser: autonomous synthesis of functional, permanently phosphorylated proteins

Abstract

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.

Human 14-3-3 proteins site-selectively bind the mutational hotspot region of SARS-CoV-2 nucleoprotein modulating its phosphoregulation

“…1c). This approach was inspired by effective imitation of phosphoserine by nhpSer in previous 14-3-3 binding studies 32,34 .…”

“…The wild-type SARS-CoV-2 N plasmid was used as template to PCR amplify the S197L mutant using the S197L reverse mutagenic primer (5’-CTGGAGT TAA ATTTCTTGAACTG-3’, the mutated codon underlined). For site-specific translational incorporation of phosphoserine or its non-hydrolyzable analog phosphono-methyl-alanine (nhpSer) at positions 197 or 205 of SARS-CoV-2 N using amber codon suppression in autonomous E. coli systems 32,33 , we created the 197TAG and 205TAG mutants using the reverse mutagenic primers (5’-CTGGAGT CTA ATTTCTTGAACTG-3’ for 197TAG or 5’-CAGGAGA CTA TCCCCTACTGCTG-3’ for 205TAG, the mutated codons are underlined) and DNA template being the SARS-CoV-2 N plasmid with a C-terminal His 6 -tag cleavable by 3C protease. The resulting PCR products including the C-terminal cleavable His 6 -tag were treated with NdeI/HindIII restriction endonucleases and then cloned into a pRBC_14-3-3γ/His 6 SUMO_mBAD 43-204 vector 33 pretreated with the same endonucleases.…”

“…The site-specifically phosphorylated SARS-CoV-2 N protein pS197 was expressed in BL21(DE3) Δ serB cells tailored to co-translational phosphoserine incorporation 49 . This yielded partially phosphorylated purified protein, therefore we also produced the nhpSer197 protein based on the same plasmid containing a TAG 197 codon (ampicillin resistance) in BL21(DE3) Δ serC cells specifically engineered to autonomously biosynthesize nhpSer from the central metabolite phosphoenolpyruvate 32 . It was reported that phosphonomethylalanine (nhpSer) effectively imitates phosphoserine in previous 14-3-3 binding studies 32,34 .…”

“…This yielded partially phosphorylated purified protein, therefore we also produced the nhpSer197 protein based on the same plasmid containing a TAG 197 codon (ampicillin resistance) in BL21(DE3) Δ serC cells specifically engineered to autonomously biosynthesize nhpSer from the central metabolite phosphoenolpyruvate 32 . It was reported that phosphonomethylalanine (nhpSer) effectively imitates phosphoserine in previous 14-3-3 binding studies 32,34 . Likewise, we also produced the SARS-CoV-2 N protein containing only nhpSer205 imitating site-specifically phosphorylated protein at position 205.…”

“…1b), resulting in heterogeneously phosphorylated N forms when incubated with kinases 26,31 . To ensure specific phosphorylation at selected residues, here we utilized the genetic code expansion approach 32,33 . We identified two phosphorylated pseudo-repeats centered at Ser197 or Thr205 in SARS-CoV-2 N (Fig.…”