Computational Investigation Reveals the Autoinhibitory Mechanism of the SERCA2b C-Terminal Extension

The sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) maintains cellular calcium homeostasis by transporting Ca²⁺ from the cytoplasm to the ER lumen. Among SERCA isoforms, SERCA2b is unique in possessing a C-terminal extension (CTE) composed of loop L10/11, a transmembrane helix M11, and a luminal extension (LE). The CTE inhibits activity, but the mechanism remains unclear because the structure of L10/11 is unresolved. Using integrative structural modeling and molecular dynamics simulations, we find that L10/11, which contains a short helix (Helix₁₀₀₃), docks into a native pocket formed by loops L8/9 and L6/7 and remains engaged across transport intermediates. This engagement stabilizes specific conformations and slows transitions required for Ca²⁺ transport via a “pull and block” dual mechanism. Mutational analyses that disrupt L10/11 contacts relieve inhibition, supporting this model. We propose that LE anchors M11 to stabilize L10/11, enabling Helix₁₀₀₃ to wedge between L8/9 and L6/7, restricts central core tilting, and impedes conformational transitions across the transport cycle. When Helix₁₀₀₃ docking is lost, inhibition shifts to L10/11-phosphorylation domain contacts, partially suppressing activity. These results define an autoinhibitory mechanism in SERCA2b and nominate L10/11 as a therapeutic target for Darier’s disease.