Universität zu Köln

Hameed, Shahul Dharjath, van Tilburg, Gabrielle B. A., Merkx, Remco, Flierman, Dennis, Wienk, Hans ORCID: 0000-0001-9577-0064, El Oualid, Farid, Hofmann, Kay ORCID: 0000-0002-2289-9083, Boelens, Rolf ORCID: 0000-0002-6939-8913 and Ovaa, Huib (2020). Diubiquitin-Based NMR Analysis: Interactions Between Lys6-Linked diUb and UBA Domain of UBXN1. Front. Chem., 7. LAUSANNE: FRONTIERS MEDIA SA. ISSN 2296-2646

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Abstract

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 epsilon-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 N-15-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 N-15-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.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Hameed, Shahul Dharjath UNSPECIFIED UNSPECIFIED UNSPECIFIED van Tilburg, Gabrielle B. A. UNSPECIFIED UNSPECIFIED UNSPECIFIED Merkx, Remco UNSPECIFIED UNSPECIFIED UNSPECIFIED Flierman, Dennis UNSPECIFIED UNSPECIFIED UNSPECIFIED Wienk, Hans UNSPECIFIED orcid.org/0000-0001-9577-0064 UNSPECIFIED El Oualid, Farid UNSPECIFIED UNSPECIFIED UNSPECIFIED Hofmann, Kay UNSPECIFIED orcid.org/0000-0002-2289-9083 UNSPECIFIED Boelens, Rolf UNSPECIFIED orcid.org/0000-0002-6939-8913 UNSPECIFIED Ovaa, Huib UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-348423
DOI:	10.3389/fchem.2019.00921
Journal or Publication Title:	Front. Chem.
Volume:	7
Date:	2020
Publisher:	FRONTIERS MEDIA SA
Place of Publication:	LAUSANNE
ISSN:	2296-2646
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Biology > Institute for Genetics
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language UBIQUITIN CHAINS; CHEMICAL-SYNTHESIS; CRYSTAL-STRUCTURES; STRUCTURAL BASIS; RECOGNITION; LINKAGE; LIGASE; SPECIFICITY; VALIDATION; MONOMER Multiple languages Chemistry, Multidisciplinary Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/34842