Linkage via K27 Bestows Ubiquitin Chains with Unique Properties among Polyubiquitins

Castañeda, Carlos; Dixon, Emma K.; Walker, Olivier; Chaturvedi, Apurva; Nakasone, Mark A.; Curtis, Joseph E.; Reed, Megan R.; Krueger, Susan; Cropp, T. Ashton; Fushman, David

doi:10.1016/j.str.2016.01.007

Cited by 56 publications

(69 citation statements)

References 49 publications

(86 reference statements)

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“…For example these ncAAs have also been exploited to characterize the effects of ubiquitination and histone methylation on gene regulation. 71, 270 Finally, E. coli has been engineered for the recombinant production of ribosomally synthesized posttranslationally modified peptides (RIPPs) that contain ncAAs, and analogues of the lanthipeptide nisin with altered ring structures and sizes have been produced. 271–275 …”

Section: Applications Of Non-canonical Amino Acidsmentioning

confidence: 99%

Playing with the Molecules of Life

2018

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Our understanding of the complex processes of living organisms at the molecular level is growing exponentially. This knowledge, together with a powerful arsenal of tools for manipulating the structures of macromolecules, is allowing chemists to harness and reprogram the cellular machinery. Here we review one example in which the genetic code itself has been expanded with new building blocks that allow us to probe and manipulate the structures and functions of proteins in ways previously unimaginable

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Section: Applications Of Non-canonical Amino Acidsmentioning

confidence: 99%

Playing with the Molecules of Life

2018

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“…Given the advances in chemical synthesis of Ub and Ub molecules containing thiolysine, we were able to generate distally labeled diUbs and studied the interactions between the two Ub moieties from the perspective of distal Ub. The synthesis of Ub chains by genetic incorporation of protected lysine residues using modified tRNA synthetases followed by selective chemical ligation and deprotection has also enabled generating diUb molecules of all linkages which were then analyzed by NMR spectroscopy (Castañeda et al, 2016b). Both approaches have demonstrated the advantages of using chemoenzymatic procedures to make diUb molecules to study their structural dynamics related to functionality.…”

Section: Discussionmentioning

confidence: 99%

Diubiquitin-Based NMR Analysis: Interactions Between Lys6-Linked diUb and UBA Domain of UBXN1

et al. 2020

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Ubiquitination is a process in which a protein is modified by the covalent attachment of the C-terminal carboxylic acid of ubiquitin (Ub) to the ε-amine of lysine or N-terminal methionine residue of a substrate protein or another Ub molecule. Each of the seven internal lysine residues and the N-terminal methionine residue of Ub can be linked to the C-terminus of another Ub moiety to form 8 distinct Ub linkages and the resulting differences in linkage types elicit different Ub signaling pathways. Cellular responses are triggered when proteins containing ubiquitin-binding domains (UBDs) recognize and bind to specific polyUb linkage types. To get more insight into the differences between polyUb chains, all of the seven lysine-linked di-ubiquitin molecules (diUbs) were prepared and used as a model to study their structural conformations in solution using NMR spectroscopy. We report the synthesis of diUb molecules, fully 15 N-labeled on the distal (N-terminal) Ub moiety and revealed their structural orientation with respect to the proximal Ub. As expected, the diUb molecules exist in different conformations in solution, with multiple conformations known to exist for K6-, K48-, and K63-linked diUb molecules. These multiple conformations allow structural flexibility in binding with UBDs thereby inducing unique responses. One of the well-known but poorly understood UBD-Ub interaction is the recognition of K6 polyubiquitin by the ubiquitin-associated (UBA) domain of UBXN1 in the BRCA-mediated DNA repair pathway. Using our synthetic 15 N-labeled diUbs, we establish here how a C-terminally extended UBA domain of UBXN1 confers specificity to K6 diUb while the non-extended version of the domain does not show any linkage preference. We show that the two distinct conformations of K6 diUb that exist in solution converge into a single conformation upon binding to this extended form of the UBA domain of the UBXN1 protein. It is likely that more of such extended UBA domains exist in nature and can contribute to linkage-specificity in Ub signaling. The isotopically labeled diUb compounds described here and the use of NMR to study their interactions with relevant partner molecules will help accelerate our understanding of Ub signaling pathways.

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“…Fushman et al. generated K27 diUb using the genetically encoded orthogonal protection and activated ligation (GOPAL) method and reported its NMR structure . An interesting finding of both studies was that the K27 isopeptide bond is rigidly buried, such that the inter‐domain mobility of K27 Ubs is restricted.…”

Section: Figurementioning

confidence: 99%

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

et al. 2019

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New synthetic strategies that exploited the strengths of both chemoselective ligation and recombinant protein expression were developed to prepare K27 di-ubiquitins (diUb), which enabled mechanistic studies on the molecular recognition of K27-linked Ubs by single-molecule Fçrster resonance energy transfer (smFRET) and X-ray crystallography.The results revealed that free K27 diUb adopted acompact conformation, whereas upon binding to UCHL3, K27 diUb was remodeled to an open conformation. The K27 isopeptide bond remained rigidly buried inside the diUb moiety during binding,a ni nteresting unique structural feature that may explain the distinctive biological function of K27 Ub chains.

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Linkage via K27 Bestows Ubiquitin Chains with Unique Properties among Polyubiquitins

Cited by 56 publications

References 49 publications

Playing with the Molecules of Life

Playing with the Molecules of Life

Diubiquitin-Based NMR Analysis: Interactions Between Lys6-Linked diUb and UBA Domain of UBXN1

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

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