Senoo, A.; Guillen-Poza, P.; Fujishima, K.; Kosuge, H.; Doumoto, T.; Kasahara, K.; Tanihara, T.; Yoshida, Y.; Yanaka, S.; Nakakido, M.; Nagatoishi, S.; Maenaka, K.; Ohdo, S.; Matsunaga, N.; Hervas, R.; Tsumoto, K.; Caaveiro, J.

The intestinal brush border (BB), composed of densely packed microvilli on enterocytes, is essential for nutrient absorption and host defense. Its organization relies on the intermicrovillar adhesion complex (IMAC), mediated by protocadherins CDHR2 and CDHR5. Despite their clinical relevance in inflammatory bowel disease and several carcinomas, structural details of IMAC assemblies have remained elusive. Herein, we report the Cryo-EM structure of the adhesive complex at 3.4 [A] resolution, revealing a heterotetrameric ensemble composed of a dimer of CDHR2 and a dimer of CDHR5. This assembly ensures uniform adhesive strength between neighboring microvilli, and facilitates hexagonal packing of microvilli. Biophysical analyses and molecular dynamics simulations revealed a kinked, Ca2+-free linker between domains EC3 and EC4 of CDHR5 conferring the necessary flexibility to withstand the shear stress caused during intestinal peristalsis. Collectively, these findings provide a structural framework for understanding BB organization and suggest strategies for therapeutics targeting IMAC in intestinal disorders.