Site-specific ubiquitination affects protein energetics and proteasomal degradation

Carroll, Emma C.; Greene, Eric R.; Martin, Andreas; Marqusee, Susan

doi:10.1038/s41589-020-0556-3

“…S8C). These results provide an exciting molecular explanation for the lack of destabilization observed in this non-sensitive variant, and this increased stability at the C terminus may prevent barstarK78 from undergoing proteasomal degradation (21).…”

Section: Thermodynamic Basis For Site-specific Effects Of Ubiquitinationmentioning

confidence: 84%

Mechanistic basis for ubiquitin modulation of a protein energy landscape

Carroll

¹

,

Latorraca

²

,

Lindner

³

et al. 2020

Preprint

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0

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Ubiquitin is a common posttranslational modification canonically associated with targeting proteins to the 26S proteasome for degradation and also plays a role in numerous other non-degradative cellular processes. Ubiquitination at certain sites destabilizes the substrate protein, with consequences for proteasomal processing, while ubiquitination at other sites has little energetic effect. How this site specificity—and, by extension, the myriad effects of ubiquitination on substrate proteins—arises remains unknown. Here, we systematically characterize the atomic-level effects of ubiquitination at various sites on a model protein, barstar, using a combination of NMR, hydrogen-deuterium exchange mass spectrometry, and molecular dynamics simulation. We find that, regardless of the site of modification, ubiquitination does not induce large structural rearrangements in the substrate. Destabilizing modifications, however, increase fluctuations from the native state resulting in exposure of the substrate’s C terminus. Both of the sites occur in regions of barstar with relatively high conformational flexibility. Destabilization, however, appears to occur through different thermodynamic mechanisms, involving a reduction in entropy in one case and a loss in enthalpy in another. By contrast, ubiquitination at a non-destabilizing site protects the substrate C terminus through intermittent formation of a structural motif with the last three residues of ubiquitin. Thus, the biophysical effects of ubiquitination at a given site depend greatly on local context. Taken together, our results reveal how a single post-translational modification can generate a broad array of distinct effects, providing a framework to guide the design of proteins and therapeutics with desired degradation and quality-control properties. (248 words)Significance StatementFluctuations on a protein energy landscapes encode the mechanistic basis for vital biological processes not always evident from static structures alone. Ubiquitination, a key posttranslational modification, can affect a protein’s energy landscape with consequences for proteasomal degradation, but the molecular mechanisms driving ubiquitin-induced energetic changes remain elusive. Here, we systematically characterize the energetic effects of ubiquitination at three sites on a model protein. We find that distinct thermodynamic mechanisms can produce the same outcome of ubiquitin-induced destabilization at sensitive sites. At a non-sensitive site, we observe formation of a substrate–ubiquitin interaction that may serve to protect against destabilization. This work will enable development of predictive models of the effect of ubiquitin at any given site on a protein with implications for understanding and engineering regulated ubiquitin signaling and protein quality control in vivo.

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“…Some proteins have an inherent flexible/loosely folded segment, which when compounded with ubiquitination render them efficient proteasome substrates [ 54 , 67 ]. Notably, even in a fully globular protein, partial unfolding may be triggered by ubiquitination [ 68 ], contributing to substrate engagement. However, the presence of an unstructured region alone without ubiquitination does not ensure high affinity of substrate binding nor efficient degradation by 26S proteasomes [ 54 ].…”

Section: Signals For Degradation By 26s Proteasomesmentioning

confidence: 99%

Proteasome in action: substrate degradation by the 26S proteasome

Sahu

¹

,

Glickman

²

2021

Biochemical Society Transactions

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Ubiquitination is the major criteria for the recognition of a substrate-protein by the 26S proteasome. Additionally, a disordered segment on the substrate — either intrinsic or induced — is critical for proteasome engagement. The proteasome is geared to interact with both of these substrate features and prepare it for degradation. To facilitate substrate accessibility, resting proteasomes are characterised by a peripheral distribution of ubiquitin receptors on the 19S regulatory particle (RP) and a wide-open lateral surface on the ATPase ring. In this substrate accepting state, the internal channel through the ATPase ring is discontinuous, thereby obstructing translocation of potential substrates. The binding of the conjugated ubiquitin to the ubiquitin receptors leads to contraction of the 19S RP. Next, the ATPases engage the substrate at a disordered segment, energetically unravel the polypeptide and translocate it towards the 20S catalytic core (CP). In this substrate engaged state, Rpn11 is repositioned at the pore of the ATPase channel to remove remaining ubiquitin modifications and accelerate translocation. C-termini of five of the six ATPases insert into corresponding lysine-pockets on the 20S α-ring to complete 20S CP gate opening. In the resulting substrate processing state, the ATPase channel is fully contiguous with the translocation channel into the 20S CP, where the substrate is proteolyzed. Complete degradation of a typical ubiquitin-conjugate takes place over a few tens of seconds while hydrolysing tens of ATP molecules in the process (50 kDa/∼50 s/∼80ATP). This article reviews recent insight into biochemical and structural features that underlie substrate recognition and processing by the 26S proteasome.

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“…Ubiquitination at positions K2 and K60 (the sensitive sites) destabilizes barstar, while ubiquitination at K78 (the non-sensitive sites) has relatively little effect (Fig. 1A) (21); these effects appear sufficient to allow for proteasomal engagement and degradation. We used these same single lysine variants in this study.…”

Section: Mono-ubiquitination At All Three Sites Does Not Alter the Native Conformation Of Barstarmentioning

confidence: 99%

“…We next examined the HSQC spectra for the ubiquitin-modified variants. 15 N-labeled barstar variants were ubiquitinated with unlabeled, fully methylated ubiquitin (which increases yield of mono-ubiquitination and has been shown to induce the same energetic effects on barstar as non-methylated ubiquitin (21)). HSQC spectra reveal well-dispersed peaks characteristic of wellfolded proteins (Fig.…”

Section: Mono-ubiquitination At All Three Sites Does Not Alter the Native Conformation Of Barstarmentioning

confidence: 99%

Mechanistic basis for ubiquitin modulation of a protein energy landscape

Carroll

¹

,

Latorraca

²

,

Lindner

³

et al. 2021

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

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Ubiquitin is a common posttranslational modification canonically associated with targeting proteins to the 26S proteasome for degradation and also plays a role in numerous other nondegradative cellular processes. Ubiquitination at certain sites destabilizes the substrate protein, with consequences for proteasomal processing, while ubiquitination at other sites has little energetic effect. How this site specificity—and, by extension, the myriad effects of ubiquitination on substrate proteins—arises remains unknown. Here, we systematically characterize the atomic-level effects of ubiquitination at various sites on a model protein, barstar, using a combination of NMR, hydrogen–deuterium exchange mass spectrometry, and molecular dynamics simulation. We find that, regardless of the site of modification, ubiquitination does not induce large structural rearrangements in the substrate. Destabilizing modifications, however, increase fluctuations from the native state resulting in exposure of the substrate’s C terminus. Both of the sites occur in regions of barstar with relatively high conformational flexibility. Nevertheless, destabilization appears to occur through different thermodynamic mechanisms, involving a reduction in entropy in one case and a loss in enthalpy in another. By contrast, ubiquitination at a nondestabilizing site protects the substrate C terminus through intermittent formation of a structural motif with the last three residues of ubiquitin. Thus, the biophysical effects of ubiquitination at a given site depend greatly on local context. Taken together, our results reveal how a single posttranslational modification can generate a broad array of distinct effects, providing a framework to guide the design of proteins and therapeutics with desired degradation and quality control properties.

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Site-specific ubiquitination affects protein energetics and proteasomal degradation

Cited by 35 publications

References 66 publications

Mechanistic basis for ubiquitin modulation of a protein energy landscape

Mechanistic basis for ubiquitin modulation of a protein energy landscape

Proteasome in action: substrate degradation by the 26S proteasome

Mechanistic basis for ubiquitin modulation of a protein energy landscape

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