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Structure of a TRIM21 - UBE2El complex reveals the specificity of E2 and ubiquitin recognition by TRIM E3 RINGs
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
Rheumatology Unit, Department of Medicine, Center for Molecular Medicine L8:04, Karolinska Institutet, Stockholm, Sweden.
Rheumatology Unit, Department of Medicine, Center for Molecular Medicine L8:04, Karolinska Institutet, Stockholm, Sweden.
European Molecular Biology Laboratory, Grenoble Outstation, Grenoble, France.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

TRIM21, a RlNG-containing E3 ubiquitin-ligase of the TRIM   protein family, is a major autoantigen in SLE and Sjögren's syndrome as well as a modifier of interferon regulatory factors, thereby regulating innate immune signalling. We herein report the 2.86 Å crystal structure ofhuman TRIM211-91 comprising the RING domain (residues 16-55), in complex with the human E2 conjugating UBE2El enzyme (also denoted UbcH6). The crystal structure, joint with analysis by NMR and SAXS as well as structure-directed mutations and functional assays provides a detailed view of the specificity-determining contacts that support specific E2 recognition in the TRIM family. A detailed comparison of our structure with known E2 bound ubiquitin complexes, supported by biochemical analyses, reveals the molecular basis for TRIM21 interactions with donor ubiquitin that activates catalytic ubiquitin transfer. Finally, our structure convincingly demonstrates the placement of the Ub-targeted Lys61 of the adjacent TRIM211- 91 close to the catalytically active UBE2El cysteine, and how the Lys61 amide is activated fora nucleophilic attack by hydrogen-bondeffected deshielding by conserved acidic residues at the E2 active site. In all, our structural findings provide molecular details ofthe selectivity involved in TRIM21 interactions with its cognate UBE2E1 enzyme and how TRIM21 positions ubiquitin in a catalytic conformation for ubiquitin transfer, and presents a snapshot of the Ub ligation step on a specific target residue of TRIM211-91 as an auto-ubiquitinated pseudo-substrate at high concentration. Increased structural and functional understanding of the TRIM mediated ubiquitination will aid development ofnovel therapeutic approaches in the entire TRIM family ofproteins.

National Category
Chemical Sciences Biochemistry and Molecular Biology
URN: urn:nbn:se:liu:diva-122466OAI: diva2:866610
Available from: 2015-11-03 Created: 2015-11-03 Last updated: 2015-11-13Bibliographically approved
In thesis
1. Structural insights into protein-protein interactions governing regulation in transcription initiation and ubiquitination
Open this publication in new window or tab >>Structural insights into protein-protein interactions governing regulation in transcription initiation and ubiquitination
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Virtually every aspect of the cellular processes in eukaryotes requires that the interactions between protein molecules are well coordinated in different regulatory pathways. Any protein dysfunction involved in these regulatory pathways might lead to various pathological conditions. Understanding the structural and functional peculiarities of these proteins molecular machineries will help in formulating structure-based drug design.

The first regulatory process studied here is the RNA polymerase-II mediated transcription of the eukaryotic protein-coding genes to produce mRNAs. This process requires the formation of the ‘transcription initiation’ by the assembly of Pre-Initiation Complex (PIC) formation at a core promoter region. Regulation at this initiation level is a key mechanism for the control of gene expression that governs cellular growth and differentiation. The transcription Factor IID (TFIID) is a conserved multiprotein general transcription factor with an essential role in  nucleating the PIC formation, composed of TATA Binding Protein (TBP) and about 14 TBP Associated Factors (TAFs). The here presented crystal structure (1.97Å) of TBP bound to TAND1 and TAND2 domains from TAF1 reveals a detailed molecular pattern of interactions involving both transcriptionally activating and repressing regions in TBP, thereby uncovering central principles for anchoring of TBP-binding motifs. Together with NMR and cellular analysis, this work provides the structural basis of competitive binding with TFIIA to modulate TBP in promoter recognition.

In eukaryotes, another fundamental mechanism in the regulation of cellular physiology is the posttranslational modification of substrate proteins by ubiquitin, termed ‘ubiquitination’. Important actors in this mechanism are the ubiquitin-ligases (E3s) that culminate the transfer of ubiquitin to the substrate and govern the specificity of this system. One E3 ligase in particular, TRIM21, defines a subgroup of the Tripartite Motif (TRIM) family, which belongs to the major RING-type of E3 ubiquitin ligases, and plays an important role in pathogenesis of autoimmunity by mediating ubiquitination of transcription factors. The crystal structure (2.86Å) of the RING domain from TRIM21 in complex with UBE2E1, an E2 conjugating enzyme, together with the NMR and SAXS analysis as well as biochemical functional analysis, reveals the molecular basis for the dynamic binding interfaces. The TRIM21 mode of ubiquitin recognition and activation for catalytic transfer of ubiquitin can be modeled onto the entire TRIM family.

Finally, we explored the concepts of conformational selection in proteins as a possible key component for protein-mediated transcriptional regulation. In this framework, MexR, a bacterial repressor of the MexAB-OprM efflux pump, and its mutant Arg21Trp were studied as an example for proteins presenting different conformations. The residue Arg21Trp mutation is clinically identified to cause of Multi-Drug Resistant (MDR) by attenuated DNA binding, and leads to the overexpression of the MexAB-OprM efflux pump. With the crystal structure (2.19Å) of MexR mutant Arg21Trp, in combination with MD-simulations and SAXS for both wild-type and mutant, we could unravel the atomic details of the wild-type conformations consisting in subsets of populations of DNA bound and unbound forms. Remarkably, the mutant Arg21Trp stabilize the DNA unbound state and shifts MexR in a pre-existing equilibrium, from a repressed to a derepressed state.

Taken together, these studies substantially broaden our knowledge at a molecular level in protein interactions that are involved in transcriptional regulation and ubiquitination, studied by a carefully selected combination of complementary structural methods spanning different resolutions and time scales.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2015. 73 p.
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1694
National Category
Chemical Sciences Biochemistry and Molecular Biology
urn:nbn:se:liu:diva-122468 (URN)10.3384/diss.diva-122468 (DOI)978-91-7685-984-1 (print) (ISBN)
Public defence
2015-12-04, Planck, Fysikhuset, Campus Valla, Linköping, 09:30 (English)
Available from: 2015-11-03 Created: 2015-11-03 Last updated: 2015-11-17Bibliographically approved

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