Ubiquitin S65 phosphorylation engenders a pH-sensitive conformational switch

Area of Science:

• Biochemistry
• Structural Biology
• Cell Signaling

Background:

• Ubiquitin (Ub) is a key signaling protein.
• Enzymatic phosphorylation of Ub at S65 yields pUb, which exists in relaxed and retracted states.
• Previous crystallization studies only captured the relaxed state structure.

Purpose of the Study:

• To determine the solution structures of pUb in both relaxed and retracted states.
• To elucidate the structural differences between the two pUb states.
• To investigate the pH-dependent equilibrium and its implications for Ub signaling.

Main Methods:

• Nuclear Magnetic Resonance (NMR) spectroscopy to obtain state-specific restraints.
• Computational refinement against NMR data to determine solution structures.
• Single-molecule Förster Resonance Energy Transfer (smFRET) to study polyubiquitin dynamics.

Main Results:

• Solution structures reveal distinct conformations: the retracted state features a retracted last β-strand and extended second α-helix compared to the relaxed state.
• The pK_a of the phosphoryl group differs by 1.4 units between states, establishing a pH-sensitive equilibrium.
• pUb exists in equilibrium between protonated/relaxed (acidic pH) and deprotonated/retracted (basic pH) forms.
• pH-induced conformational changes were observed in phosphorylated K63-linked diubiquitin.

Conclusions:

• The pH-sensitive conformational switch of pUb explains limitations of phosphomimetic mutants.
• This switch provides an additional layer of regulation for ubiquitin signaling, influenced by cellular compartment pH.
• Ubiquitin phosphorylation and pH act synergistically to modulate target specificity and signaling outcomes.