NMR Lineshape Analysis of Intrinsically Disordered Protein Interactions

Waudby, Christopher A.; Christodoulou, John

doi:10.1007/978-1-0716-0524-0_24

Cited by 11 publications

(12 citation statements)

References 70 publications

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“…These conditions preclude us from considering CSPs as simple weighted averages of free and bound species, as we are no longer under fast exchange conditions (Williamson, 2013). Instead, we used TITAN 2D lineshape analysis (Waudby et al, 2016), which accounts for both CSP and peak broadening during the titration, to obtain K d values. Assuming single-site binding between UBQLN2 and each Ub unit in Ub4, we obtained binding affinities of 3 μM for both Ub and K63-Ub4, and a lower affinity of 25 μM for K48-Ub4 (Fig 2F; S1).…”

Section: Nmr and Fluorescence Anisotropy Titrations Reveal Difference...mentioning

confidence: 99%

“…Concentrations of Ub ligand were adjusted by the number of Ub units in the polyUb chain such that 50 μM Ub4 was equal to 200 μM Ub. For TITAN (Waudby et al, 2016) lineshape analysis, only SOFAST-HMQC spectra for titrations involving 15 N-labeled protein (UBQLN2 450-624) were used, following the NMR processing protocol in (Waudby & Christodoulou, 2020). TITAN software was used within NMRbox (Maciejewski et al, 2017).…”

Section: Nmr Titration Experiments and K D Determinationmentioning

confidence: 99%

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Mechanistic insights into enhancement or inhibition of phase separation by different polyubiquitin chains

et al. 2022

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Ubiquitin‐binding shuttle UBQLN2 mediates crosstalk between proteasomal degradation and autophagy, likely via interactions with K48‐ and K63‐linked polyubiquitin chains, respectively. UBQLN2 comprises self‐associating regions that drive its homotypic liquid–liquid phase separation (LLPS). Specific interactions between one of these regions and ubiquitin inhibit UBQLN2 LLPS. Here, we show that, unlike ubiquitin, the effects of multivalent polyubiquitin chains on UBQLN2 LLPS are highly dependent on chain types. Specifically, K11‐Ub4 and K48‐Ub4 chains generally inhibit UBQLN2 LLPS, whereas K63‐Ub4, M1‐Ub4 chains, and a designed tetrameric ubiquitin construct significantly enhance LLPS. We demonstrate that these opposing effects stem from differences in chain conformations but not in affinities between chains and UBQLN2. Chains with extended conformations and increased accessibility to the ubiquitin‐binding surface promote UBQLN2 LLPS by enabling a switch between homotypic to partially heterotypic LLPS that is driven by both UBQLN2 self‐interactions and interactions between multiple UBQLN2 units with each polyubiquitin chain. Our study provides mechanistic insights into how the structural and conformational properties of polyubiquitin chains contribute to heterotypic LLPS with ubiquitin‐binding shuttles and adaptors.

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Section: Nmr and Fluorescence Anisotropy Titrations Reveal Difference...mentioning

confidence: 99%

Section: Nmr Titration Experiments and K D Determinationmentioning

confidence: 99%

Mechanistic insights into enhancement or inhibition of phase separation by different polyubiquitin chains

et al. 2022

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“…Concentrations of Ub ligand were adjusted by the number of Ub units in the polyUb chain such that 50 µM Ub4 was equal to 200 µM Ub. For TITAN (Waudby et al, 2016) lineshape analysis, only SOFAST-HMQC spectra for titrations involving 15 N-labeled protein (UBQLN2 450-624) were used, following the NMR processing protocol in (Waudby and Christodoulou, 2020). For all cases, the single-site binding model (two-state ligand binding in TITAN) was used.…”

Section: Nmr Titration Experiments and Kd Determinationmentioning

confidence: 99%

Mechanistic insights into the enhancement or inhibition of phase separation by polyubiquitin chains of different lengths or linkages

Dao

Yang

Presti

et al. 2021

Preprint

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SummaryUbiquitin-binding shuttle UBQLN2 mediates crosstalk between proteasomal degradation and autophagy, likely via interactions with K48- and K63-linked polyubiquitin chains, respectively. UBQLN2 is recruited to stress granules in cells and undergoes liquid-liquid phase separation (LLPS) in vitro. However, interactions with ubiquitin or multivalent K48-linked chains eliminate LLPS. Here, we found that, although some polyubiquitin chain types (K11-Ub4 and K48-Ub4) did generally inhibit UBQLN2 LLPS, others (K63-Ub4, M1-Ub4 and a designed tetrameric ubiquitin construct) significantly enhanced LLPS. Using nuclear magnetic resonance (NMR) spectroscopy and complementary biophysical techniques, we demonstrated that these opposing effects stem from differences in chain conformations, but not in affinities between chains and UBQLN2. Chains with extended conformations and increased accessibility to the ubiquitin binding surface significantly promoted UBQLN2 LLPS by enabling a switch between homotypically to partially heterotypically-driven phase separation. Our study provides mechanistic insights into how the structural and conformational properties of polyubiquitin chains contribute to heterotypic phase separation with ubiquitin-binding shuttles and adaptors.HighlightsUbiquitin or short polyubiquitin chains bind to phase separation-driving stickers on UBQLN2 and inhibit its phase separation whereas longer chains provide the multivalency needed to enhance UBQLN2 phase separation.Compact K11- and K48-linked Ub4 chains destabilized UBQLN2 phase separation, while extended M1- and K63-linked Ub4 promoted UBQLN2 phase separation.Chain conformation and accessibility of the Ub interacting surface is a driving factor of UBQLN2/polyUb co-phase separation.UBQLN2 condensates assemble during in vitro enzymatic assembly of K63-linked polyUb chains as free ubiquitin is depleted.

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“…To discriminate between these two models, we carried out two-dimensional NMR line shape analysis of our NMR titration data for the variant CITED2 peptides using the program TITAN ( 25 , 26 ). A simple two-state binding model ( Scheme 1 ) is unable to reproduce the simultaneous changes in chemical shift and resonance intensity that are observed in our spectra as a function of increasing CITED2 peptide concentration ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…For two-dimensional NMR line shape analysis, the 1 H- 15 N HSQC spectra were processed using exponential window functions with 4-Hz line broadening in the direct dimension and 8-Hz line broadening in the indirect dimension. The spectra were imported into TITAN ( 25 , 26 ) and analyzed using the “two-state” and “ligand exchange via ternary complex” binding models. Line shape analysis was carried out in two steps, in which the chemical shifts and line widths of the 15 N-TAZ1:HIF-1α bound state were determined first, followed by subsequent fitting of the chemical shifts and line widths of the intermediate and bound states and kinetic parameters for the binding models.…”

Section: Methodsmentioning

confidence: 99%

Multivalency enables unidirectional switch-like competition between intrinsically disordered proteins

Berlow

Dyson

Wright

2022

Proc. Natl. Acad. Sci. U.S.A.

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Intrinsically disordered proteins must compete for binding to common regulatory targets to carry out their biological functions. Previously, we showed that the activation domains of two disordered proteins, the transcription factor HIF-1α and its negative regulator CITED2, function as a unidirectional, allosteric molecular switch to control transcription of critical adaptive genes under conditions of oxygen deprivation. These proteins achieve transcriptional control by competing for binding to the TAZ1 domain of the transcriptional coactivators CREB-binding protein (CBP) and p300 (CREB: cyclic-AMP response element binding protein). To characterize the mechanistic details behind this molecular switch, we used solution NMR spectroscopy and complementary biophysical methods to determine the contributions of individual binding motifs in CITED2 to the overall competition process. An N-terminal region of the CITED2 activation domain, which forms a helix when bound to TAZ1, plays a critical role in initiating competition with HIF-1α by enabling formation of a ternary complex in a process that is highly dependent on the dynamics and disorder of the competing partners. Two other conserved binding motifs in CITED2, the LPEL motif and an aromatic/hydrophobic motif that we term ϕC, function synergistically to enhance binding of CITED2 and inhibit rebinding of HIF-1α. The apparent unidirectionality of competition between HIF-1α and CITED2 is lost when one or more of these binding regions is altered by truncation or mutation of the CITED2 peptide. Our findings illustrate the complexity of molecular interactions involving disordered proteins containing multivalent interaction motifs and provide insight into the unique mechanisms by which disordered proteins compete for occupancy of common molecular targets within the cell.

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NMR Lineshape Analysis of Intrinsically Disordered Protein Interactions

Cited by 11 publications

References 70 publications

Mechanistic insights into enhancement or inhibition of phase separation by different polyubiquitin chains

Mechanistic insights into enhancement or inhibition of phase separation by different polyubiquitin chains

Mechanistic insights into the enhancement or inhibition of phase separation by polyubiquitin chains of different lengths or linkages

Multivalency enables unidirectional switch-like competition between intrinsically disordered proteins

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