Conformational Dynamics of Wild-type Lys-48-linked Diubiquitin in Solution

Hirano, Takashi; Serve, Olivier; Yagi-Utsumi, Maho; Takemoto, Emi; Hiromoto, Takeshi; Satoh, Tadashi; Mizushima, Tsunehiro; Kato, Koichi

doi:10.1074/jbc.m111.256354

Cited by 55 publications

(102 citation statements)

References 37 publications

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“…Previous experimental data also suggested the high pH-dependence of the conformational equilibrium [20], [26], [27]. Despite the fact that lowering pH will increase the population of open state [26], it is still a matter of debate whether the open state is predominant at physiological conditions.…”

Section: Resultsmentioning

confidence: 99%

PolyUbiquitin Chain Linkage Topology Selects the Functions from the Underlying Binding Landscape

et al. 2014

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Ubiquitin (Ub) can generate versatile molecular signals and lead to different celluar fates. The functional poly-valence of Ub is believed to be resulted from its ability to form distinct polymerized chains with eight linkage types. To provide a full picture of ubiquitin code, we explore the binding landscape of two free Ub monomers and also the functional landscapes of of all eight linkage types by theoretical modeling. Remarkably, we found that most of the compact structures of covalently connected dimeric Ub chains (diUbs) pre-exist on the binding landscape. These compact functional states were subsequently validated by corresponding linkage models. This leads to the proposal that the folding architecture of Ub monomer has encoded all functional states into its binding landscape, which is further selected by different topologies of polymeric Ub chains. Moreover, our results revealed that covalent linkage leads to symmetry breaking of interfacial interactions. We further propose that topological constraint not only limits the conformational space for effective switching between functional states, but also selects the local interactions for realizing the corresponding biological function. Therefore, the topological constraint provides a way for breaking the binding symmetry and reaching the functional specificity. The simulation results also provide several predictions that qualitatively and quantitatively consistent with experiments. Importantly, the K48 linkage model successfully predicted intermediate states. The resulting multi-state energy landscape was further employed to reconcile the seemingly contradictory experimental data on the conformational equilibrium of K48-diUb. Our results further suggest that hydrophobic interactions are dominant in the functional landscapes of K6-, K11-, K33- and K48 diUbs, while electrostatic interactions play a more important role in the functional landscapes of K27, K29, K63 and linear linkages.

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Section: Resultsmentioning

confidence: 99%

PolyUbiquitin Chain Linkage Topology Selects the Functions from the Underlying Binding Landscape

et al. 2014

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“…pHsensitive conformational switches have been reported for many other proteins (25)(26)(27). It has also been reported that K48-linked diubiquitin switches from a closed conformation to a predominant open conformation at pH 4.5 (28,29). However, these examples of acid-base equilibrium all involve protein titratable side chains.…”

Section: Discussionmentioning

confidence: 97%

Ubiquitin S65 phosphorylation engenders a pH-sensitive conformational switch

Gong

et al. 2017

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

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Ubiquitin (Ub) is an important signaling protein. Recent studies have shown that Ub can be enzymatically phosphorylated at S65, and that the resulting pUb exhibits two conformational states-a relaxed state and a retracted state. However, crystallization efforts have yielded only the structure for the relaxed state, which was found similar to that of unmodified Ub. Here we present the solution structures of pUb in both states obtained through refinement against state-specific NMR restraints. We show that the retracted state differs from the relaxed state by the retraction of the last β-strand and by the extension of the second α-helix. Further, we show that at 7.2, the pK a value for the phosphoryl group in the relaxed state is higher by 1.4 units than that in the retracted state. Consequently, pUb exists in equilibrium between protonated and deprotonated forms and between retracted and relaxed states, with protonated/relaxed species enriched at slightly acidic pH and deprotonated/retracted species enriched at slightly basic pH. The heterogeneity of pUb explains the inability of phosphomimetic mutants to fully mimic pUb. The pHsensitive conformational switch is likely preserved for polyubiquitin, as single-molecule FRET data indicate that pH change leads to quaternary rearrangement of a phosphorylated K63-linked diubiquitin. Because cellular pH varies among compartments and changes upon pathophysiological insults, our finding suggests that pH and Ub phosphorylation confer additional target specificities and enable an additional layer of modulation for Ub signals., a 76-residue signaling protein, is found ubiquitously in cells. Two or more Ub molecules can be covalently linked to form a diubiquitin (diUb) and then a polyubiquitin (polyUb), as an isopeptide bond is formed between the carboxylate group of one Ub (called the distal Ub) and the amine group of another Ub (called the proximal Ub). Owing to the characteristic quaternary structures of polyUb and specific interactions between polyUb and its target proteins (1, 2), a polyUb with a specific linkage can be involved in a distinctive set of cellular functions (3).The heterogeneity of Ub also arises from other types of covalent modifications. Proteomics studies have indicated that Ub is phosphorylated at multiple sites (4, 5). However, PINK1 is the only Ub kinase known to date, which specifically phosphorylates Ub at S65 (6, 7). Under normal conditions, only a fraction of Ub is S65-phosphorylated. However, upon oxidative stress, neurodegeneration, or aging, the level of S65 phosphorylation increases significantly (4, 8). S65-phosphorylated Ub (pUb) in turn can activate PARKIN, a ubiquitin ligase, and induce mitophagy (9-12). However, no other pUb-specific targets have been clearly identified, and how phosphorylation affects Ub signaling in general remains unclear.Previously, Komander and coworkers showed that pUb gives two distinct sets of NMR peaks, which correspond to the two conformational states of pUb exchanging at a slow timescale (13). Based on NMR long-r...

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“…Furthermore, using single-molecule fluorescence resonance energy transfer, it was observed that Lys-48-linked di-Ub adopts predominantly compact conformations (35). However, additional conformations of Lys-48-tetra-Ub may also exist (36,37). Thus, although Lys-48-tetra-Ub overall is packed in a compact state, its topology is dynamic.…”

Section: Discussionmentioning

confidence: 99%

A SnapShot of Ubiquitin Chain Elongation

Kovacev

Spratt

et al. 2014

Journal of Biological Chemistry

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Background: Polyubiquitin chains are signaling polypeptides altering the fate of substrates. Results: A Lys-48-ubiquitin chain of a length greater than four, but not its Lys-63 linkage counterparts, slowed the rate of additional ubiquitin conjugation. Conclusion:The ubiquitin chain length and linkage may impact kinetic rates of chain elongation. Significance: Our findings suggest a self-restraining mechanism that limits Lys-48-polyubiquitination.

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Conformational Dynamics of Wild-type Lys-48-linked Diubiquitin in Solution

Cited by 55 publications

References 37 publications

PolyUbiquitin Chain Linkage Topology Selects the Functions from the Underlying Binding Landscape

PolyUbiquitin Chain Linkage Topology Selects the Functions from the Underlying Binding Landscape

Ubiquitin S65 phosphorylation engenders a pH-sensitive conformational switch

A SnapShot of Ubiquitin Chain Elongation

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