Locating the thapsigargin-binding site on Ca(2+)-ATPase by cryoelectron microscopy

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biophysics

Background:

• Thapsigargin (TG) is a known potent inhibitor of Ca(2+)-ATPase.
• Enzymatic studies suggest TG locks Ca(2+)-ATPase in a stable E2 conformation.
• Previous research indicates TG binds with high affinity (<1 nM).

Purpose of the Study:

• To investigate the structural impact of thapsigargin binding on Ca(2+)-ATPase.
• To determine the precise location of the thapsigargin-binding site.
• To understand the long-range conformational effects of thapsigargin binding.

Main Methods:

• Cryo-electron microscopy (cryo-EM) of tubular Ca(2+)-ATPase crystals.
• 3D reconstruction and comparison of Ca(2+)-ATPase in absence and presence of TG.
• Difference mapping to identify structural changes upon TG binding.

Main Results:

• Ca(2+)-ATPase molecular shape remains similar to the E2 conformation upon TG binding.
• A distinct binding site for TG was identified on the lumenal side, between transmembrane segments M3/M4 and M7/M8.
• A second difference density at the decavanadate-binding site suggests altered cytoplasmic domain affinity.

Conclusions:

• Thapsigargin binding stabilizes the E2 conformation of Ca(2+)-ATPase without major domain rearrangement.
• The identified binding site explains TG's inhibitory mechanism.
• Long-range conformational coupling influences ATP affinity and enzyme activity.