Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jun 9;292(23):9493-9504.
doi: 10.1074/jbc.M117.785287. Epub 2017 Apr 25.

Structure and energetics of pairwise interactions between proteasome subunits RPN2, RPN13, and ubiquitin clarify a substrate recruitment mechanism

Ryan T VanderLinden  1 , Casey W Hemmis  1 , Tingting Yao  2 , Howard Robinson  3 , Christopher P Hill  4
Affiliations

Structure and energetics of pairwise interactions between proteasome subunits RPN2, RPN13, and ubiquitin clarify a substrate recruitment mechanism

Ryan T VanderLinden et al. J Biol Chem. .

Abstract

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. 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.

Keywords: RPN13; RPN2; crystallography; fluorescence polarization; proteasome; protein-protein interaction; surface plasmon resonance (SPR); ubiquitin.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Examination of the RPN2-RPN13 interaction. A, GFP retention assays used to visualize stable association of various RPN2 peptides with RPN13. Complex formation is visualized by retention of green GFP-RPN2 on the resin. Microcentrifuge tubes outlined for clarity. B, representative SPR sensorgrams illustrating full-length RPN13 binding to RPN2 peptides corresponding to residues 940–953 (top) and 932–953 (bottom). C, representative fluorescence polarization binding curves of full-length RPN13 (black) and RPN13PRU (magenta) binding to fluorescently labeled RPN2(940–952). D, representative fluorescence polarization competition curves of RPN2(940–952) (blue) and RPN2(940–953) (black) competing with fluorescently labeled RPN2(940–952) for binding of RPN13PRU.
Figure 2.
Figure 2.
Structure of the RPN13-RPN2-ubiquitin complex. A, schematic of human RPN13 domain architecture. B, two orthogonal views of the ternary complex of RPN13PRU (magenta), RPN2(940–953) (cyan), and ubiquitin (gray) in space-filling and ribbon representations. C, overview of the RPN13-RPN2 interface. Surface of RPN13 residues within 3.5 Å of RPN2, white. Ubiquitin removed for clarity. D, detailed view of RPN2 residues (Pro-945, Phe-948, and Tyr-950) within the binding groove of RPN13. RPN13 colored as in C. RPN2 residues labeled in black. Fo − Fc omit map (cyan mesh, 2× r.m.s.d.) within 1.6 Å of RPN2 peptide. The map was calculated by removing RPN2 peptides from the model, introducing random shifts to all atoms in remaining chains, and performing a single round of refinement. E, detailed view of RPN13 residues at RPN13-RPN2 interface. RPN13 residues within 4 Å of RPN2, magenta. RPN2 residues, cyan.
Figure 3.
Figure 3.
Sequence alignments of RPN13, RPN2, and ubiquitin from higher eukaryotes. A, RPN13 alignments. Stars indicate residues not conserved between mouse and human. Blue triangles indicate residues at the RPN13-RPN2 interface. Gray triangles indicate residues at the RPN13-ubiquitin interface. B, RPN2 sequence alignment. Magenta triangles indicate residues at the RPN13-RPN2 interface. C, ubiquitin sequence alignment; magenta triangles indicate residues at the RPN13-ubiquitin interface.
Figure 4.
Figure 4.
RPN13-ubiquitin interaction. A, overview of the RPN13-ubiquitin interface. RPN13 residues within 4 Å of ubiquitin, white. B, view of ubiquitin residues at RPN13 interface upon ∼180° rotation around y axis of Fig. 3A. RPN13 removed for clarity. C, zoomed view of RPN13 residues at ubiquitin interface as visualized in A. Ubiquitin removed for clarity. D, electrostatic interactions at the RPN13-ubiquitin interface. E, disparity in ubiquitin positioning between crystallographic (gray) and NMR docking (wheat, PDB code 2Z59) models. F, differences in positioning of key residues (shown as sticks) between crystallographic (gray) and NMR docking (wheat) models upon alignment on RPN13.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Pickart C. M., and Cohen R. E. (2004) Proteasomes and their kin: proteases in the machine age. Nat. Rev. Mol. Cell Biol. 5, 177–187 - PubMed
    1. Finley D. (2009) Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu. Rev. Biochem. 78, 477–513 - PMC - PubMed
    1. Rosenzweig R., Bronner V., Zhang D., Fushman D., and Glickman M. H. (2012) Rpn1 and Rpn2 coordinate ubiquitin processing factors at proteasome. J. Biol. Chem. 287, 14659–14671 - PMC - PubMed
    1. Gomez T. A., Kolawa N., Gee M., Sweredoski M. J., and Deshaies R. J. (2011) Identification of a functional docking site in the Rpn1 LRR domain for the UBA-UBL domain protein Ddi1. BMC Biol. 9, 33. - PMC - PubMed
    1. Fatimababy A. S., Lin Y. L., Usharani R., Radjacommare R., Wang H. T., Tsai H. L., Lee Y., and Fu H. (2010) Cross-species divergence of the major recognition pathways of ubiquitylated substrates for ubiquitin/26S proteasome-mediated proteolysis. FEBS J. 277, 796–816 - PubMed

MeSH terms