Polyubiquitin ligand-induced phase transitions are optimized by spacing between ubiquitin units
Edited by Ned Wingreen, Princeton University, Princeton, NJ; received April 25, 2023; accepted September 1, 2023 by Editorial Board Member Paul Chaikin
Significance
Biomolecular condensates, implicated in many cellular processes, can assemble via phase transitions of a few key driver macromolecules. Condensate formation is further modulated by the interactions with ligands such as polyubiquitin chains, a common protein posttranslational modification. Using a library of designed polyubiquitin ligand hubs with decreasing or increasing spacings between binding sites and altered binding affinities with drivers, we employ theory and experiments to determine key properties that govern how ligand hubs affect driver phase transitions. Importantly, linear (M1-linked) polyubiquitin chains are already optimized (in terms of spacing between binding sites) to maximize phase separation of driver macromolecules such as proteasomal shuttle factor UBQLN2. Polyubiquitin chains likely use their multivalent architecture to dynamically regulate condensate formation and other properties.
Abstract
Biomolecular condensates form via multivalent interactions among key macromolecules and are regulated through ligand binding and/or posttranslational modifications. One such modification is ubiquitination, the covalent addition of ubiquitin (Ub) or polyubiquitin chains to target macromolecules. Specific interactions between polyubiquitin chains and partner proteins, including hHR23B, NEMO, and UBQLN2, regulate condensate assembly or disassembly. Here, we used a library of designed polyubiquitin hubs and UBQLN2 as model systems for determining the driving forces of ligand-mediated phase transitions. Perturbations to either the UBQLN2-binding surface of Ub or the spacing between Ub units reduce the ability of hubs to modulate UBQLN2 phase behavior. By developing an analytical model based on polyphasic linkage principles that accurately described the effects of different hubs on UBQLN2 phase separation, we determined that introduction of Ub to UBQLN2 condensates incurs a significant inclusion energetic penalty. This penalty antagonizes the ability of polyUb hubs to scaffold multiple UBQLN2 molecules and cooperatively amplify phase separation. The extent to which polyubiquitin hubs promote UBQLN2 phase separation is encoded in the spacings between Ub units. This spacing is modulated by chains of different linkages and designed chains of different architectures, thus illustrating how the ubiquitin code regulates functionality via the emergent properties of the condensate. The spacing in naturally occurring linear polyubiquitin chains is already optimized to promote phase separation with UBQLN2. We expect our findings to extend to other condensates, emphasizing the importance of ligand properties, including concentration, valency, affinity, and spacing between binding sites in studies and designs of condensates.
Data, Materials, and Software Availability
SAXS data for HT6-Ub & M1-Ub4 ligand hubs are deposited in SASBDB: https://www.sasbdb.org/project/1831/ (56). Raw data for figures and scripts are deposited at https://doi.org/10.5281/zenodo.8199908 (57). All other data supporting the findings of this study are included in this article and/or supporting information.
Acknowledgments
This work was supported by NIH R01GM136946 (all protein purifications, turbidity assays, structural modeling, and NMR experiments) to C.A.C. and NIH R01GM141235 (theoretical modeling) to J.D.S. NMR data were acquired on an 800 MHz NMR spectrometer funded by NIH-shared instrumentation grant 1S10OD012254. This research used resources of the Advanced Photon Source (APS), a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. This project was supported by NIH grant P30 GM138395. Use of the Pilatus 3 1 M detector was provided by grant 1S10OD018090-01 from NIGMS. We acknowledge support and assistance from Dr. Jesse Hopkins and Dr. Maxwell Watkins on collecting SEC-MALS-SAXS data at APS. Suzanne Enos and Antara Chaudhuri were supported by NSF REU grant CHE1950802. We acknowledge Yiran Yang for assistance in purifying some of the proteins for this project. We thank Susan Krueger, Tanja Mittag, and the Condensate Colloquium series for scientific discussions on this project.
Author contributions
S.K.K.G., T.P.D., and C.A.C. designed research; S.K.K.G., T.P.D., S.E.E., A.C., and C.A.C. performed research; J.D.S. contributed new reagents/analytic tools; S.K.K.G., T.P.D., J.D.S., and C.A.C. analyzed data; and S.K.K.G., T.P.D., J.D.S., and C.A.C. wrote the paper.
Competing interests
The authors declare no competing interest.
Supporting Information
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Copyright © 2023 the Author(s). Published by PNAS. This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).
Data, Materials, and Software Availability
SAXS data for HT6-Ub & M1-Ub4 ligand hubs are deposited in SASBDB: https://www.sasbdb.org/project/1831/ (56). Raw data for figures and scripts are deposited at https://doi.org/10.5281/zenodo.8199908 (57). All other data supporting the findings of this study are included in this article and/or supporting information.
Submission history
Received: April 25, 2023
Accepted: September 1, 2023
Published online: October 12, 2023
Published in issue: October 17, 2023
Keywords
Acknowledgments
This work was supported by NIH R01GM136946 (all protein purifications, turbidity assays, structural modeling, and NMR experiments) to C.A.C. and NIH R01GM141235 (theoretical modeling) to J.D.S. NMR data were acquired on an 800 MHz NMR spectrometer funded by NIH-shared instrumentation grant 1S10OD012254. This research used resources of the Advanced Photon Source (APS), a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. This project was supported by NIH grant P30 GM138395. Use of the Pilatus 3 1 M detector was provided by grant 1S10OD018090-01 from NIGMS. We acknowledge support and assistance from Dr. Jesse Hopkins and Dr. Maxwell Watkins on collecting SEC-MALS-SAXS data at APS. Suzanne Enos and Antara Chaudhuri were supported by NSF REU grant CHE1950802. We acknowledge Yiran Yang for assistance in purifying some of the proteins for this project. We thank Susan Krueger, Tanja Mittag, and the Condensate Colloquium series for scientific discussions on this project.
Author contributions
S.K.K.G., T.P.D., and C.A.C. designed research; S.K.K.G., T.P.D., S.E.E., A.C., and C.A.C. performed research; J.D.S. contributed new reagents/analytic tools; S.K.K.G., T.P.D., J.D.S., and C.A.C. analyzed data; and S.K.K.G., T.P.D., J.D.S., and C.A.C. wrote the paper.
Competing interests
The authors declare no competing interest.
Notes
This article is a PNAS Direct Submission. N.W. is a guest editor invited by the Editorial Board.
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Polyubiquitin ligand-induced phase transitions are optimized by spacing between ubiquitin units, Proc. Natl. Acad. Sci. U.S.A.
120 (42) e2306638120,
https://doi.org/10.1073/pnas.2306638120
(2023).
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