A Structural Code for Assembly Specificity in GID/CTLH-Type E3 Ligases
Curation statements for this article:-
Curated by eLife
eLife Assessment
This structural biology study provides insights into the assembly of the GID/CTLH E3 ligase complex. The multi-subunit complex forms unique, ring shaped assemblies and the findings presented here describe a "specificity code" regulates formation of subunit interfaces. The data supporting the conclusions are convincing, both in thoroughness and rigor. This study will be valuable to biochemists, structural biologists, and could lay foundation for novel designed protein assemblies.
This article has been Reviewed by the following groups
Discuss this preprint
Start a discussion What are Sciety discussions?Listed in
- Evaluated articles (eLife)
Abstract
GID/CTLH-type E3 ligases assemble into conserved ring-shaped architectures built from repeating LisH-CTLH-CRA modules, yet the molecular rules that enforce their highly specific subunit arrangement have remained unknown. Here, we decode the structural assembly specificity code that governs CRA-CRA pairing. Using crystal structures of multiple CTLH-CRA domains, including the RanBP9-muskelin heterodimer, integrated with quantitative binding analyses, we show that several interfaces operate with exceptionally high affinity, reaching the picomolar range, and that conserved sequence and geometric features enable each subunit to only select cognate partners. Strikingly, targeted perturbations of these features are sufficient to reprogram pairing preferences, enabling engineered subunits such as RanBP10 or Twa1 to adopt non-native interaction partners. These findings reveal the molecular logic that preserves the architecture of GID/CTLH-type E3 ligases and demonstrate that their assembly code is both decipherable and engineerable, providing a conceptual foundation for reconfiguring these ring-shaped E3 ligases.
Article activity feed
-
eLife Assessment
This structural biology study provides insights into the assembly of the GID/CTLH E3 ligase complex. The multi-subunit complex forms unique, ring shaped assemblies and the findings presented here describe a "specificity code" regulates formation of subunit interfaces. The data supporting the conclusions are convincing, both in thoroughness and rigor. This study will be valuable to biochemists, structural biologists, and could lay foundation for novel designed protein assemblies.
-
Reviewer #1 (Public review):
Summary:
GID/CTLH-type RING ligases are huge multi-protein complexes that play an important role in protein ubiquitylation. The subunits of its core complex are distinct and form a defined structural arrangement, but there can be variations in subunit composition, such as exchange of RanBP9 and RanBP10. In this study, van gen Hassend and Schindelin provide new crystal structures of (parts of) key subunits and use those structures to elucidate the molecular details of the pairwise binding between those subunits. They identify key residues that mediate binding partner specificity. Using in vitro binding assays with purified protein, they show that altering those residues can switch specificity to a different binding partner.
Strengths:
This is a technically demanding study that sheds light on an interesting …
Reviewer #1 (Public review):
Summary:
GID/CTLH-type RING ligases are huge multi-protein complexes that play an important role in protein ubiquitylation. The subunits of its core complex are distinct and form a defined structural arrangement, but there can be variations in subunit composition, such as exchange of RanBP9 and RanBP10. In this study, van gen Hassend and Schindelin provide new crystal structures of (parts of) key subunits and use those structures to elucidate the molecular details of the pairwise binding between those subunits. They identify key residues that mediate binding partner specificity. Using in vitro binding assays with purified protein, they show that altering those residues can switch specificity to a different binding partner.
Strengths:
This is a technically demanding study that sheds light on an interesting structural biology problem in residue-level detail. The combination of crystallization, structural modeling, and binding assays with purified mutant proteins is elegant and, in my eyes, convincing.
Weaknesses:
I mainly have some suggestions for further clarification, especially for a broad audience beyond the structural biology community.
(1) The authors establish what they call an 'engineering toolkit' for the controlled assembly of alternative compositions of the GID complex. The mutagenesis results are great for the specific questions asked in this manuscript. It would be great if they could elaborate on the more general significance of this 'toolkit' - is there anything from a technical point of view that can be generalized? Is there a biological interest in altering the ring composition for functional studies?
(2) Along the same lines, the mutagenesis required to rewire Twa1 binding was very complex (8 mutations). While this is impressive work, the 'big picture conclusion' from this part is not as clear as for the simpler RanBP9/10. It would be great if the authors could provide more context as to what this is useful for (e.g., potential for in vivo or in vitro functional studies, maybe even with clinical significance?)
(3) For many new crystal structures, the authors used truncated, fused, or otherwise modified versions of the proteins for technical reasons. It would be helpful if the authors could provide reasoning why those modifications are unlikely to change the conclusions of those experiments compared to the full-length proteins (which are challenging to work with for technical reasons). For instance, could the authors use folding prediction (AlphaFold) that incorporates information of their resolved structures and predicts the impact of the omitted parts of the proteins? The authors used AlphaFold for some aspects of the study, which could be expanded.
-
Reviewer #2 (Public review):
Summary:
This is a very interesting study focusing on a remarkable oligomerization domain, the LisH-CTLH-CRA module. The module is found in a diverse set of proteins across evolution. The present manuscript focuses on the extraordinary elaboration of this domain in GID/CTLH RING E3 ubiquitin ligases, which assemble into a gigantic, highly ordered, oval-shaped megadalton complex with strict subunit specificity. The arrangement of LisH-CTLH-CRA modules from several distinct subunits is required to form the oval on the outside of the assembly, allowing functional entities to recruit and modify substrates in the center. Although previous structures had shown that data revealed that CTLH-CRA dimerization interfaces share a conserved helical architecture, the molecular rules that govern subunit pairing have not …
Reviewer #2 (Public review):
Summary:
This is a very interesting study focusing on a remarkable oligomerization domain, the LisH-CTLH-CRA module. The module is found in a diverse set of proteins across evolution. The present manuscript focuses on the extraordinary elaboration of this domain in GID/CTLH RING E3 ubiquitin ligases, which assemble into a gigantic, highly ordered, oval-shaped megadalton complex with strict subunit specificity. The arrangement of LisH-CTLH-CRA modules from several distinct subunits is required to form the oval on the outside of the assembly, allowing functional entities to recruit and modify substrates in the center. Although previous structures had shown that data revealed that CTLH-CRA dimerization interfaces share a conserved helical architecture, the molecular rules that govern subunit pairing have not been explored. This was a daunting task in protein biochemistry that was achieved in the present study, which defines this "assembly specificity code" at the structural and residue-specific level.
The authors used X-ray crystallography to solve high-resolution structures of mammalian CTLH-CRA domains, including RANBP9, RANBP10, TWA1, MAEA, and the heterodimeric complex between RANBP9 and MKLN. They further examined and characterized assemblies by quantitative methods (ITC and SEC-MALS) and qualitatively using nondenaturing gels. Some of their ITC measurements were particularly clever and involved competitive titrations and titrations of varying partners depending on protein behavior. The experiments allowed the authors to discover that affinities for interactions between partners is exceptionally tight, in the pM-nM range, and to distill the basis for specificity while also inferring that additional interactions beyond the LisH-CTLH-CRA modules likely also contribute to stability. Beyond discovering how the native pairings are achieved, the authors were able to use this new structural knowledge to reengineer interfaces to achieve different preferred partnerings.
Strengths:
Nearly everything about this work is exceptionally strong.
(1) The question is interesting for the native complexes, and even beyond that, has potential implications for the design of novel molecular machines.
(2) The experimental data and analyses are quantitative, rigorous, and thorough.
(3) The paper is a great read - scholarly and really interesting.
(4) The figures are exceptional in every possible way. They present very complex and intricate interactions with exquisite clarity. The authors are to be commended for outstanding use of color and color-coding throughout the study, including in cartoons to help track what was studied in what experiments. And the figures are also outstanding aesthetically.
Weaknesses:
There are no major weaknesses of note, but I can make a few recommendations for editing the text.
-
Reviewer #3 (Public review):
Summary:
Protein complexes, like the GID/CTLH-type E3 ligase, adopt a complex three-dimensional structure, which is of functional importance. Several domains are known to be involved in shaping the complexes. Structural information based on cryo-EM is available, but its resolution does not always provide detailed information on protein-protein interactions. The work by van gen Hassend and Schindelin provides additional structural data based on crystal structures.
Strengths:
The work is solid and very carefully performed. It provides high-resolution insights into the domain architecture, which helps to understand the protein-protein interactions on a detailed molecular level. They also include mutant data and can thereby draw conclusions on the specificity of the domain interactions. These data are probably …
Reviewer #3 (Public review):
Summary:
Protein complexes, like the GID/CTLH-type E3 ligase, adopt a complex three-dimensional structure, which is of functional importance. Several domains are known to be involved in shaping the complexes. Structural information based on cryo-EM is available, but its resolution does not always provide detailed information on protein-protein interactions. The work by van gen Hassend and Schindelin provides additional structural data based on crystal structures.
Strengths:
The work is solid and very carefully performed. It provides high-resolution insights into the domain architecture, which helps to understand the protein-protein interactions on a detailed molecular level. They also include mutant data and can thereby draw conclusions on the specificity of the domain interactions. These data are probably very helpful for others who work on a functional level with protein complexes containing these domains.
Weaknesses:
The manuscript contains a lot of useful, very detailed information. This information is likely very helpful to investigate functional and regulatory aspects of the protein complexes, whose assembly relies on the LisH-CTLH-CRA modules. However, this goes beyond the scope of this manuscript.
-
