Chemical Protein Synthesis Enabled Mechanistic Studies on the Molecular Recognition of K27‐linked Ubiquitin Chains

Pan, Man; Zheng, Qin; Ding, Shan; Zhang, Lujia; Qu, Qian; Hong, Danning; Ren, Yujing; Liang, Lujun; Chen, Chunlai; Mei, Ziqing; Liu, Lei

doi:10.1002/ange.201810814

Cited by 17 publications

(12 citation statements)

References 40 publications

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“…This way we hoped to maximize the synthetic yield of the peptide segments and thus the synthetic protein. An extra C-terminal Cys residue was included in the expression plasmid for the first segment—Phe–Gly64, which was supposed to be able to transform to the corresponding thioester via a reported hydrazinolysis procedure ( Adams et al, 2013 ; Pan et al, 2019 ). However, the effect of hydrazinolysis is not satisfactory, giving only minor product (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Chemical Synthesis of the Sec-To-Cys Homologue of Human Selenoprotein F and Elucidation of Its Disulfide-pairing Mode

Liao

2021

Front. Chem.

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Herein, we document a highly optimized synthesis of the Sec-to-Cys homologue of the human selenoprotein F (SelF) through a three-segment two-ligation semisynthesis strategy. Highlighted in this synthetic route are two one-pot manipulations, i.e. the first ligation followed by a desulfurization and the second ligation followed by the protein refolding. This way multi-milligrams of the folded synthetic protein was obtained, which set the stage for the synthesis of the natural selenoprotein. Moreover, the disulfide pairing mode of the SelF was elucidated through a combination of site-directed mutagenesis and LC-MS study. It provides not only a criterion to judge the viability of the synthetic protein, and more importantly, useful structural insights into the previously unresolved UGGT-binding domain of SelF.

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Section: Resultsmentioning

confidence: 99%

Chemical Synthesis of the Sec-To-Cys Homologue of Human Selenoprotein F and Elucidation of Its Disulfide-pairing Mode

Liao

2021

Front. Chem.

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“…The plasmid containing yeast (Saccharomyces cerevisiae) Ubr1 was purchased from Addgene (plasmid # 24506) 41 . DNA sequence of yeast (Saccharomyces cerevisiae) Ubc2 was synthesized and codon optimized for E. coli overexpression by Genscript.…”

Section: Cloning and Plasmid Constructionmentioning

confidence: 99%

Structural Insights Into the Initiation and Elongation of Ubiquitination by Ubr1

Pan¹,

Zheng

Wang

et al. 2021

Preprint

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The N-end rule pathway was one of the first ubiquitin (Ub)-dependent degradation pathways to be identified. Ubr1, a single-chain E3 ligase, targets proteins bearing a destabilizing residue at the N-terminus (N-degron) for rapid K48-linked ubiquitination and proteasome-dependent degradation. How Ubr1 catalyses the initiation of ubiquitination on the substrate and elongation of the Ub chain in a linkage-specific manner through a single E2 ubiquitin-conjugating enzyme (Ubc2) remains unknown. Here, we report the cryo-electron microscopy structures of two complexes representing the initiation and elongation intermediates of Ubr1 captured using chemical approaches. In these two structures, Ubr1 adopts different conformations to facilitate the transfer of Ub from Ubc2 to either an N-degron peptide or a monoubiquitinated degron. These structures not only reveal the architecture of the Ubr1 complex but also provide mechanistic insights into the initiation and elongation steps of ubiquitination catalyzed by Ubr1.

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“…The methylene-to-sulfur atom displacement of this analog has been previously established and shown to have no effect on either the structures nor biochemical activities of the Ub chains. [29][30][31] Figure 2 | (a) Synthesis of phosphorylated diUbs. 3: Ub-hydrazide segment; 3p: phosphorylated (65Ser) Ub-hydrazide segment; 4: Ub-auxiliary segment; 4p: phosphorylated (65Ser) Ub-auxiliary segment.…”

Section: Synthesis Of K6-linked Diub and Its Selectively Phosphorylated Analogsmentioning

confidence: 99%

Chemical Synthesis of Structurally Defined Phosphorylated Ubiquitins Suggests Impaired Parkin Activation by Phosphorylated Ubiquitins with a Non-Phosphorylated Distal Unit

et al. 2019

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Mutations in genes encoding PINK1 (PTEN-induced kinase 1) and Parkin (E3 ubiquitin ligase) are identified in familial Parkinson's disease. However, it remains unclear whether the phosphorylated Ub chains activate wildtype Parkin (w-Parkin) or phosphorylated Parkin (p-Parkin), with the consequent expulsion of the damaged mitochondria. To address this problem, we developed a chemical protein synthesis strategy that integrates hydrazide-based native chemical ligation, dehydroalanine conjugation, and chemoenzymatic modification to enable us to access the precise phosphorylated side-chain K6-linked diUbs, namely: (1) Ub P K6 Ub, which carries out phosphorylation at the distal Ub. (2) Ub K6 Ub P , which carries out phosphorylation at the proximal Ub. (3) Ub P K6 Ub P , which carries out phosphorylation at both distal and proximal Ub units. The structure of Ub P K6 Ub P was validated by X-ray crystallographic analysis at 2.2 Å resolution. Using these structurally defined phosphorylated diUbs to activate Parkin, we found that Ub K6 Ub P could activate p-Parkin but not w-Parkin. Subsequent biochemical studies showed that Ub K6 Ub P interfered with the transfer of Ub by the Ub-E2 enzyme conjugate with the thioester-linked intermediate of w-Parkin. Thus our results suggested that w-Parkin and p-Parkin are activated via two different mechanisms. Collectively, our study exemplifies the utilization of a novel synthesis strategy for the generation of structurally defined proteins bearing post-translational modifications and would be useful in elucidating ubiquitin chain signaling and pathways regulation studies in modern biochemistry and biophysics.

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Chemical Protein Synthesis Enabled Mechanistic Studies on the Molecular Recognition of K27‐linked Ubiquitin Chains

Cited by 17 publications

References 40 publications

Chemical Synthesis of the Sec-To-Cys Homologue of Human Selenoprotein F and Elucidation of Its Disulfide-pairing Mode

Chemical Synthesis of the Sec-To-Cys Homologue of Human Selenoprotein F and Elucidation of Its Disulfide-pairing Mode

Structural Insights Into the Initiation and Elongation of Ubiquitination by Ubr1

Chemical Synthesis of Structurally Defined Phosphorylated Ubiquitins Suggests Impaired Parkin Activation by Phosphorylated Ubiquitins with a Non-Phosphorylated Distal Unit

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