Phosphorylation-dependent conformational switch in spin-labeled phospholamban bound to SERCA

Area of Science:

• Biochemistry
• Molecular Biology
• Cardiovascular Physiology

Background:

• Phospholamban (PLB) is a key regulator of the sarcoplasmic reticulum Ca-ATPase (SERCA) in cardiac muscle.
• PLB's transmembrane domain inhibits SERCA activity at low calcium concentrations.
• The cytoplasmic domain of PLB modulates SERCA inhibition, with phosphorylation at Ser16 reversing this effect.

Purpose of the Study:

• To investigate the functional dynamics of phospholamban (PLB) using chemical synthesis and electron paramagnetic resonance (EPR).
• To elucidate the molecular mechanisms by which PLB regulates SERCA activity.
• To differentiate the effects of phosphorylation versus calcium on PLB dynamics and SERCA inhibition.

Main Methods:

• Chemical synthesis of monomeric PLB incorporating a TOAC spin label to report backbone dynamics.
• Functional reconstitution of TOAC-PLB into membranes with or without SERCA.
• Electron paramagnetic resonance (EPR) spectroscopy to analyze PLB conformational states and dynamics.

Main Results:

• Phosphorylation of PLB shifts its cytoplasmic domain from an ordered (T state) to a disordered, extended (R state).
• This phosphorylation-induced disordering relieves SERCA inhibition without dissociating the PLB-SERCA complex.
• Micromolar calcium levels affect SERCA inhibition but do not alter PLB dynamics, indicating distinct regulatory mechanisms.

Conclusions:

• PLB phosphorylation relieves SERCA inhibition via an order-to-disorder transition in its cytoplasmic domain, promoting SERCA structural changes.
• This mechanism is distinct from calcium-mediated relief of PLB inhibition.
• A synthetic lipid anchor prevents phosphorylation-induced disordering, highlighting the importance of PLB's cytoplasmic domain dynamics.