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; Wang, Tian; Hong, Danning; Ren, Yujing; Liang, Lujun; Chen, Chunlai; Mei, Ziqing; Liu, Lei

doi:10.1002/anie.201810814

Cited by 58 publications

(24 citation statements)

References 40 publications

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“…8,9 Its increasing capacity to address the production of proteins decorated by posttranslational modifications of much higher complexity stimulates the creativity of protein chemists worldwide. [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] In particular, chemical synthesis has been used to produce ubiquitin (Ub), 12, [25][26][27] polyubiquitin, [12][13][14]22,24,[28][29][30] small ubiquitin-like modifiers (SUMO) 19,20,31 and conjugates thereof 10,11,21,23,29,[32][33][34][35] (for reviews see 36,37 ) and as such has permitted to investigate in a new way how these modifications modulate protein structure and function.…”

Section: Introductionmentioning

confidence: 99%

The Role of the Conserved SUMO-2/3 Cysteine Residue on Domain Structure Investigated Using Protein Chemical Synthesis

et al. 2019

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While the semi or total synthesis of ubiquitin or polyubiquitin conjugates have attracted a lot of attention the last decade, the preparation of small-ubiquitin like modifier (SUMO) conjugates is much less developed. We describe hereinafter some important molecular features to consider when preparing SUMO-2/3 conjugates by chemical synthesis using the native chemical ligation and extended methods. In particular, we clarify the role of the conserved cysteine residue on SUMO-2/3 domain stability and properties. Our data reveal that SUMO-2 and 3 proteins behave differently to the CysAla modification with SUMO-2 being less impacted than SUMO-3 likely due to a stabilizing interaction occurring in SUMO-2 between its tail and the SUMO core domain. While the CysAla modification has no effect on the enzyme-catalyzed conjugation, it shows a deleterious effect on the enzyme-catalyzed deconjugation process, especially with the SUMO-3 conjugate. Whereas it is often stated that SUMO-2 and SUMO-3 are structurally and functionally indistinguishable, here we show that these proteins have specific structural and biochemical properties. This information is important to consider when designing and preparing SUMO-2/3 conjugates, and should help making progress in the understanding of the specific role of SUMO-2 and/or SUMO-3 modifications on protein structure and function.

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Section: Introductionmentioning

confidence: 99%

The Role of the Conserved SUMO-2/3 Cysteine Residue on Domain Structure Investigated Using Protein Chemical Synthesis

et al. 2019

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“…Wildtype Ub and Ub mutants were purified as previously described 41 . Briefly, plasmids for overexpression were transformed to E. coli BL21(DE3) competent cells.…”

Section: Protein Expression and Purificationmentioning

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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“…In addition, a Ub-hydrazide can be synthesized in a similar two-segment strategy, and used as a thioester equivalent. [51][52][53][54][55][56][57][58][59][60][61] Another approach for the synthesis of long peptides on the solid support is to incorporate "aggregation breakers" such as pseudoproline and dimethoxybenzyl dipeptides, 62 both of which disrupt the aggregation of the nascent peptide on the resin surface. Using this strategy, Ovaa et al successfully synthesized Ub thioesters and even SUMO2 (92 amino acids).…”

Section: Total Chemical Synthesis Of Ub Conjugate Modulementioning

confidence: 99%

Development and application of ubiquitin-based chemical probes

Sui

Wang

et al. 2020

Chem. Sci.

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

Cited by 58 publications

References 40 publications

The Role of the Conserved SUMO-2/3 Cysteine Residue on Domain Structure Investigated Using Protein Chemical Synthesis

The Role of the Conserved SUMO-2/3 Cysteine Residue on Domain Structure Investigated Using Protein Chemical Synthesis

Structural Insights Into the Initiation and Elongation of Ubiquitination by Ubr1

Development and application of ubiquitin-based chemical probes

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