Facile synthesis of Fmoc-protected phosphonate pSer mimetic and its application in assembling a substrate peptide of 14-3-3 ζ

Kang, Jie; Chen, Hong-Xue; Huang, Siqi; Zhang, Yun‐Lai; Chang, Rong Seng; Li, Fangyi; Li, Yanmei; Chen, Yong-Xiang

doi:10.1016/j.tetlet.2017.05.037

Cited by 17 publications

(18 citation statements)

References 31 publications

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“…For example, proteins Histone 2A 30 and AANAT 28 with nhpSer (but not Asp/Glu) installed at their N-termini by EPL were able to pull down 14-3-3, a pSer/pThr specific binding protein, from cellular extracts. Consistent with this, a nhpSer containing peptide bound 14-3-3 with comparable affinity to the equivalent pSer-peptide (0.86 vs 0.46 μM, respectively) 24 .…”

Section: Introductionsupporting

confidence: 63%

“…Because the electronegative F-atoms of F2-nhpSer (i) can be H-bond acceptors like the pSer -oxygen and (ii) lower the phosphonate pKa2 from in the 7 to 8 range to near 5.5 so that its charge at physiological pH is ~ -2 (like pSer), it is generally considered a better analog than nhpSer. In fact, reviews give the impression that nhpSer is not a useful mimic for these reasons 5,23 , yet the effectiveness of both has been established in the few studies in which they were incorporated into synthetic peptides 12,[24][25][26][27] and into proteins via semi-synthetic methods such as expressed protein ligation (EPL) [28][29][30][31] . For example, proteins Histone 2A 30 and AANAT 28 with nhpSer (but not Asp/Glu) installed at their N-termini by EPL were able to pull down 14-3-3, a pSer/pThr specific binding protein, from cellular extracts.…”

Section: Introductionmentioning

confidence: 99%

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

Zhu

Franklin

Vogel

et al. 2021

Preprint

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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.

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Section: Introductionsupporting

confidence: 63%

Section: Introductionmentioning

confidence: 99%

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

Zhu

Franklin

Vogel

et al. 2021

Preprint

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

Section: Resultsmentioning

confidence: 99%

“…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.…”

Section: Methodsmentioning

confidence: 99%

“…1c). This approach was inspired by effective imitation of phosphoserine by nhpSer in previous 14-3-3 binding studies 32,34 . On SEC, human 14-3-3γ co-eluted with SARS-CoV-2 N nhpS197, but not with unphosphorylated SARS-CoV-2 N, confirming the formation of the phosphorylation-dependent complex (Fig.…”

Section: Identification Of Alternative 14-3-3-binding Sites Within Sa...mentioning

confidence: 99%

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

Tugaeva

Sysoev

Smith

et al. 2021

Preprint

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The SARS-CoV-2 nucleocapsid protein (N) is responsible for viral genome packaging and virion assembly. Being highly abundant in the host cell, N interacts with numerous human proteins and undergoes multisite phosphorylation in vivo. When phosphorylated within its Ser/Arg-rich region, a tract highly prone to mutations as exemplified in the Omicron and Delta variants, N recruits human 14-3-3 proteins, potentially hijacking their functions. Here, we show that in addition to phosphorylated Ser197, an alternative, less conserved phosphosite at Thr205 not found in SARS-CoV N binds 14-3-3 with micromolar affinity and is in fact preferred over pS197. Fluorescence anisotropy reveals a distinctive pT205/pS197 binding selectivity towards the seven human 14-3-3 isoforms. Crystal structures explain the structural basis for the discovered selectivity towards SARS-CoV-2 N phosphopeptides, and also enable prediction for how N variants interact with 14-3-3, suggesting a link between the strength of this interaction and replicative fitness of emerging coronavirus variants.

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Phosphorylated and Phosphonated Low‐Complexity Protein Segments for Biomimetic Mineralization and Repair of Tooth Enamel

et al. 2022

Self Cite

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Biomimetic mineralization based on self‐assembly has made great progress, providing bottom‐up strategies for the construction of new organic–inorganic hybrid materials applied in the treatment of hard tissue defects. Herein, inspired by the cooperative effects of key components in biomineralization microenvironments, a new type of biocompatible peptide scaffold based on flexibly self‐assembling low‐complexity protein segments (LCPSs) containing phosphate or phosphonate groups is developed. These LCPSs can retard the transformation of amorphous calcium phosphate into hydroxyapatite (HAP), leading to merged mineralization structures. Moreover, the application of phosphonated LCPS over phosphorylated LCPS can prevent hydrolysis by phosphatases that are enriched in extracellular mineralization microenvironments. After being coated on the etched tooth enamel, these LCPSs facilitate the growth of HAP to generate new enamel layers comparable to the natural layers and mitigate the adhesion of Streptococcus mutans. In addition, they can effectively stimulate the differentiation pathways of osteoblasts. These results shed light on the potential biomedical applications of two LCPSs in hard tissue repair.

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Facile synthesis of Fmoc-protected phosphonate pSer mimetic and its application in assembling a substrate peptide of 14-3-3 ζ

Cited by 17 publications

References 31 publications

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

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

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

Phosphorylated and Phosphonated Low‐Complexity Protein Segments for Biomimetic Mineralization and Repair of Tooth Enamel

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Facile synthesis of Fmoc-protected phosphonate pSer mimetic and its application in assembling a substrate peptide of 14-3-3 ζ

Cited by 17 publications

References 31 publications

PermaPhosSer: autonomous synthesis of functional, permanently phosphorylated proteins

PermaPhosSer: autonomous synthesis of functional, permanently phosphorylated proteins

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

Phosphorylated and Phosphonated Low‐Complexity Protein Segments for Biomimetic Mineralization and Repair of Tooth Enamel

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Product

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

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