Molecular basis of Climp63-mediated ER lumen spacing.

The width of cisternal structures in the endoplasmic reticulum (ER) is maintained by ER-resident protein Climp63. Self-association of the Climp63 luminal domain (LD), even though moderate, plays a key role in shaping ER sheets. However, the molecular basis of luminal spacing remains elusive. Here, we analyze the homotypic interactions of the Climp63 LD using AI-predicted structures. The LD is highly alpha-helical, with a flexible leading helix followed by a five-helix bundle (5HB). Charge-based trans associations are formed between the tip of the 5HB and the C-terminus of the LD, consistent with generating a width of ∼50 nm for ER sheets. The leading helix of the LD is dispensable for homotypic interactions, but packing of the 5HB regulates self-association. The density of Climp63, likely reflecting the strength of cis interactions, influences the ER width, which is maintained by trans interactions. These results indicate that a general principle in maintaining membrane tethering is multi-modular self-association.