Dong X, Gong Z, Lu Y B, Liu K, Qin L Y, Ran M L, Zhang C L, Liu Z, Zhang W P, and Tang C. Ubiquitin S65 phosphorylation engenders a pH-sensitive conformational switch. Proc Natl Acad Sci U S A, 2017 Jun 13.
Ubiquitin (Ub) is an important signaling protein. Recent studies showed that Ub can be enzymatically phosphorylated at S65, and the resulting pUb exhibits two conformational states—a relaxed state and a retracted state. In this paper, the authors presented the solution structures of pUb in both states obtained through refinement against statespecific 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 pKa value for the phosphoryl group in the relaxed state is higher by 1.4 units than that in the retracted state. Consequently, the 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 for phosphomimetic mutants to fully mimic pUb. The pH-sensitive conformational switch is likely preserved for polyubiquitin, as single-molecule FRET data indicated that pH change leads to quaternary rearrangement of a phosphorylated K63-linked diubiquitin. As cellular pH varies among compartments and changes upon pathophysiological insults, our finding suggests that pH and Ub phosphorylation confer additional target specificities and enable additional layer of modulation for Ub signals.
Ubiquitination and phosphorylation are the two most important protein post-translational modifications and cell signals. Ubiquitin can be specifically phosphorylated at S65, and the finding here suggests a general functional role for Ub phosphorylation. We show that subtle fluctuation near physiological and pH can affect the protonation status of S65 phosphoryl group, thus modulate the structure of ubiquitin monomer and of polyubiquitin. It is known that cellular pH varies among organelles and changes under physiological and pathological conditions. As ubiquitin is involved in myriad aspects of cell biology, a pH-sensitive conformational switch acquired upon S65 phosphorylation would allow the phosphorylated ubiquitin to interact with different target proteins at environmental cues. It would also enable crosstalks between ubiquitination and phosphorylation signals.