Structure and energetics of pairwise interactions between proteasome subunits RPN2, RPN13, and ubiquitin clarify a substrate recruitment mechanism
Abstract
This work presents that the 26S proteasome is a large cellular assembly that mediates the selective degradation of proteins in the nucleus and cytosol and is an established target for anticancer therapeutics. Protein substrates are typically targeted to the proteasome through modification with a polyubiquitin chain, which can be recognized by several proteasome-associated ubiquitin receptors. One of these receptors, RPN13/ADRM1, is recruited to the proteasome through direct interaction with the large scaffolding protein RPN2 within the 19S regulatory particle. To better understand the interactions between RPN13, RPN2, and ubiquitin, we used human proteins to map the RPN13-binding epitope to the C-terminal 14 residues of RPN2, which, like ubiquitin, binds the N-terminal pleckstrin-like receptor of ubiquitin (PRU) domain of RPN13. We also report the crystal structures of the RPN13 PRU domain in complex with peptides corresponding to the RPN2 C terminus and ubiquitin. Through mutational analysis, we validated the RPN2-binding interface revealed by our structures and quantified binding interactions with surface plasmon resonance and fluorescence polarization. In contrast to a previous report, we find that RPN13 binds ubiquitin with an affinity similar to that of other proteasome-associated ubiquitin receptors and that RPN2, ubiquitin, and the deubiquitylase UCH37 bind to RPN13 withmore »
- Authors:
-
- Univ. of Utah, Salt Lake City, UT (United States). Department of Biochemistry
- Colorado State Univ., Fort Collins, CO (United States). Department of Biochemistry and Molecular Biology
- Brookhaven National Lab. (BNL), Upton, NY (United States). Biology Department
- Publication Date:
- Research Org.:
- Brookhaven National Laboratory (BNL), Upton, NY (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Basic Energy Sciences (BES)
- OSTI Identifier:
- 1376133
- Report Number(s):
- BNL-114068-2017-JA
Journal ID: ISSN 0021-9258
- Grant/Contract Number:
- SC0012704; AC02-76SF00515
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Journal of Biological Chemistry
- Additional Journal Information:
- Journal Volume: 292; Journal Issue: 23; Journal ID: ISSN 0021-9258
- Publisher:
- American Society for Biochemistry and Molecular Biology
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; proteasome; RPN13; RPN2; ubiquitin; crystallography; surface plasmon resonance; fluorescence polarization
Citation Formats
VanderLinden, Ryan T., Hemmis, Casey W., Yao, Tingting, Robinson, Howard, and Hill, Christopher P. Structure and energetics of pairwise interactions between proteasome subunits RPN2, RPN13, and ubiquitin clarify a substrate recruitment mechanism. United States: N. p., 2017.
Web. doi:10.1074/jbc.M117.785287.
VanderLinden, Ryan T., Hemmis, Casey W., Yao, Tingting, Robinson, Howard, & Hill, Christopher P. Structure and energetics of pairwise interactions between proteasome subunits RPN2, RPN13, and ubiquitin clarify a substrate recruitment mechanism. United States. https://doi.org/10.1074/jbc.M117.785287
VanderLinden, Ryan T., Hemmis, Casey W., Yao, Tingting, Robinson, Howard, and Hill, Christopher P. Tue .
"Structure and energetics of pairwise interactions between proteasome subunits RPN2, RPN13, and ubiquitin clarify a substrate recruitment mechanism". United States. https://doi.org/10.1074/jbc.M117.785287. https://www.osti.gov/servlets/purl/1376133.
@article{osti_1376133,
title = {Structure and energetics of pairwise interactions between proteasome subunits RPN2, RPN13, and ubiquitin clarify a substrate recruitment mechanism},
author = {VanderLinden, Ryan T. and Hemmis, Casey W. and Yao, Tingting and Robinson, Howard and Hill, Christopher P.},
abstractNote = {This work presents that the 26S proteasome is a large cellular assembly that mediates the selective degradation of proteins in the nucleus and cytosol and is an established target for anticancer therapeutics. Protein substrates are typically targeted to the proteasome through modification with a polyubiquitin chain, which can be recognized by several proteasome-associated ubiquitin receptors. One of these receptors, RPN13/ADRM1, is recruited to the proteasome through direct interaction with the large scaffolding protein RPN2 within the 19S regulatory particle. To better understand the interactions between RPN13, RPN2, and ubiquitin, we used human proteins to map the RPN13-binding epitope to the C-terminal 14 residues of RPN2, which, like ubiquitin, binds the N-terminal pleckstrin-like receptor of ubiquitin (PRU) domain of RPN13. We also report the crystal structures of the RPN13 PRU domain in complex with peptides corresponding to the RPN2 C terminus and ubiquitin. Through mutational analysis, we validated the RPN2-binding interface revealed by our structures and quantified binding interactions with surface plasmon resonance and fluorescence polarization. In contrast to a previous report, we find that RPN13 binds ubiquitin with an affinity similar to that of other proteasome-associated ubiquitin receptors and that RPN2, ubiquitin, and the deubiquitylase UCH37 bind to RPN13 with independent energetics. In conclusion, these findings provide a detailed characterization of interactions that are important for proteasome function, indicate ubiquitin affinities that are consistent with the role of RPN13 as a proteasomal ubiquitin receptor, and have major implications for the development of novel anticancer therapeutics.},
doi = {10.1074/jbc.M117.785287},
journal = {Journal of Biological Chemistry},
number = 23,
volume = 292,
place = {United States},
year = {Tue Apr 25 00:00:00 EDT 2017},
month = {Tue Apr 25 00:00:00 EDT 2017}
}
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