The dynamic conformational landscape of the protein methyltransferase SETD8
- Memorial Sloan Kettering Cancer Center, United States
- McMaster University, Canada
- Icahn School of Medicine at Mount Sinai, United States
- University of Toronto, Canada
- The University of Georgia, United States
- Takeda, United States
- Shanghai Institute of Materia Medica, China
Abstract
Elucidating the conformational heterogeneity of proteins is essential for understanding protein function and developing exogenous ligands. With the rapid development of experimental and computational methods, it is of great interest to integrate these approaches to illuminate the conformational landscapes of target proteins. SETD8 is a protein lysine methyltransferase (PKMT), which functions in vivo via the methylation of histone and nonhistone targets. Utilizing covalent inhibitors and depleting native ligands to trap hidden conformational states, we obtained diverse X-ray structures of SETD8. These structures were used to seed distributed atomistic molecular dynamics simulations that generated a total of six milliseconds of trajectory data. Markov state models, built via an automated machine learning approach and corroborated experimentally, reveal how slow conformational motions and conformational states are relevant to catalysis. These findings provide molecular insight on enzymatic catalysis and allosteric mechanisms of a PKMT via its detailed conformational landscape.
Data availability
The molecular dynamics datasets generated and analyzed in this study are available via the Open Science Framework at https://osf.io/2h6p4.The code used for the generation and analysis of the molecular dynamics data is available via a Github repository at https://github.com/choderalab/SETD8-materials.PDB files: 6BOZ for BC-Inh1, 5W1Y for BC-Inh2, 4IJ8 for BC-SAM, and 5V2N for APO.
Article and author information
Author details
Funding
National Cancer Institute
- Jian Jin
- John D Chodera
- Minkui Luo
K. C. Wong Education Foundation
- Cheng Luo
Chinese Academy of Sciences
- Cheng Luo
National Natural Science Foundation of China
- Cheng Luo
the Tri-Institutional PhD Program in Chemical Biology
- Shi Chen
- Rafal P Wiewiora
Peer Reviewed Cancer Research Program of the Department of Defense
- Rafal P Wiewiora
AbbVie
- Peter J Brown
Bayer Pharma AG
- Peter J Brown
Boehringer Ingelheim
- Peter J Brown
Eshelman Institute for Innovation
- Peter J Brown
Genome Canada
- Peter J Brown
National Institute of General Medical Sciences
- Yujun George Zheng
- Jian Jin
- John D Chodera
- Minkui Luo
Innovative Medicines Initiative
- Peter J Brown
Canada Foundation for Innovation
- Peter J Brown
Janssen
- Peter J Brown
Merck & Co.
- Peter J Brown
Novartis Pharma AG
- Peter J Brown
Ontario Ministry of Economic Development and Innovation
- Peter J Brown
Pfizer
- Peter J Brown
São Paulo Research Foundation-FAPESP
- Peter J Brown
Takeda
- Hua Zou
- Robert J Skene
- Peter J Brown
the Wellcome Trust
- Peter J Brown
Eunice Kennedy Shriver National Institute of Child Health and Human Development
- Jian Jin
Starr Cancer Consortium
- John D Chodera
- Minkui Luo
MSKCC Functional Genomics Initiative
- John D Chodera
- Minkui Luo
The Sloan Kettering Institute
- Kyle A Beauchamp
- John D Chodera
- Minkui Luo
Mr. William H. Goodwin and Mrs. Alice Goodwin Commonwealth Foundation for Cancer Research, and the Experimental Therapeutics Center of Memorial Sloan Kettering Cancer Center
- Minkui Luo
Tri-Institutional Therapeutics Discovery Institute
- Minkui Luo
Louis V. Gerstner Young Investigator Award
- John D Chodera
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2019, Chen et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 6,293
- views
-
- 933
- downloads
-
- 43
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
The dynamic conformational landscape of the protein methyltransferase SETD8
eLife 8:e45403.
https://doi.org/10.7554/eLife.45403
Further reading
-
- Biochemistry and Chemical Biology
- Cell Biology
Activation of the Wnt/β-catenin pathway crucially depends on the polymerization of dishevelled 2 (DVL2) into biomolecular condensates. However, given the low affinity of known DVL2 self-interaction sites and its low cellular concentration, it is unclear how polymers can form. Here, we detect oligomeric DVL2 complexes at endogenous protein levels in human cell lines, using a biochemical ultracentrifugation assay. We identify a low-complexity region (LCR4) in the C-terminus whose deletion and fusion decreased and increased the complexes, respectively. Notably, LCR4-induced complexes correlated with the formation of microscopically visible multimeric condensates. Adjacent to LCR4, we mapped a conserved domain (CD2) promoting condensates only. Molecularly, LCR4 and CD2 mediated DVL2 self-interaction via aggregating residues and phenylalanine stickers, respectively. Point mutations inactivating these interaction sites impaired Wnt pathway activation by DVL2. Our study discovers DVL2 complexes with functional importance for Wnt/β-catenin signaling. Moreover, we provide evidence that DVL2 condensates form in two steps by pre-oligomerization via high-affinity interaction sites, such as LCR4, and subsequent condensation via low-affinity interaction sites, such as CD2.
-
- Biochemistry and Chemical Biology
The development of proteolysis targeting chimeras (PROTACs), which induce the degradation of target proteins by bringing them into proximity with cellular E3 ubiquitin ligases, has revolutionized drug development. While the human genome encodes more than 600 different E3 ligases, current PROTACs use only a handful of them, drastically limiting their full potential. Furthermore, many PROTAC development campaigns fail because the selected E3 ligase candidates are unable to induce degradation of the particular target of interest. As more and more ligands for novel E3 ligases are discovered, the chemical effort to identify the best E3 ligase for a given target is exploding. Therefore, a genetic system to identify degradation-causing E3 ligases and suitable target/E3 ligase pairs is urgently needed. Here, we used the well-established dimerization of the FKBP12 protein and FRB domain by rapamycin to bring the target protein WDR5 into proximity with candidate E3 ligases. Strikingly, this rapamycin-induced proximity assay (RiPA) revealed that VHL, but not Cereblon, is able to induce WDR5 degradation - a finding previously made by PROTACs, demonstrating its predictive power. By optimizing the steric arrangement of all components and fusing the target protein with a minimal luciferase, RiPA can identify the ideal E3 for any target protein of interest in living cells, significantly reducing and focusing the chemical effort in the early stages of PROTAC development.
