Ubiquitin S65 phosphorylation engenders a pH-sensitive conformational switch

Abstract

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...

An invisible ubiquitin conformation is required for efficient phosphorylation by PINK 1

“…Experimentally, the phosphoUb‐CR conformation was supported by large (> 1.5 ppm) chemical shift perturbations and by determination of the hydrogen bonding patterns for the β‐sheet, using long‐range HNCO‐based NMR analysis (Wauer et al , ). A recent NMR structure of the phosphoUb‐CR conformation confirmed our findings (Dong et al , ).…”

“…Our previous work showed that the Ub-CR conformation was stabilised in Ser65 phosphoUb (Wauer et al, 2015a), which was recently confirmed by an NMR solution structure (Dong et al, 2017). We hence set out to identify stable versions of phosphoUb-CR for further study.…”

“…This phosphoUb conformation was confirmed in a crystal structure (Wauer et al , ) and more recently by an NMR structure (Dong et al , ). More striking was a second conformation, in which the entire last β‐strand slipped by two amino acids, extending the Ser65 loop, and simultaneously shortening the Ub C‐terminal tail (hereafter referred to as the Ub‐CR conformation for C‐terminally retracted) (Wauer et al , ; Dong et al , ). This change is facilitated by a Leu‐repeat pattern in the β5‐strand: Leu67, Leu69 and Leu71 occupy complementary Leu pockets in the Ub core, whereas Leu73 is mostly solvent exposed.…”

“…The first state resembles the common Ub conformation observed in all reported crystal structures to date. This phos-phoUb conformation was confirmed in a crystal structure (Wauer et al, 2015a) and more recently by an NMR structure (Dong et al, 2017). More striking was a second conformation, in which the entire last b-strand slipped by two amino acids, extending the Ser65 loop, and simultaneously shortening the Ub C-terminal tail (hereafter referred to as the Ub-CR conformation for C-terminally retracted) (Wauer et al, 2015a;Dong et al, 2017).…”

“…This loop conformation has also been observed in a subset of crystal structures, such as in Lys6‐linked triUb (3ZLZ). See Hospenthal et al () for further analysis. Two additional views of Ub‐CR structures, highlighting the differences in the Ser65‐containing loop. Comparison of phosphoUb TVLN with NMR structures of phosphoUb‐CR (5XK4; Dong et al , ). Comparison of phosphoUb TVLN and phosphoUb crystal structures (4WZP; Wauer et al , ) with NMR structures of phosphoUb (5XK5; Dong et al , ).…”