Wntless (WLS), an evolutionarily conserved multi-pass transmembrane protein, is essential for secretion of Wnt proteins. Wnt-triggered signaling pathways control many crucial life events, whereas aberrant Wnt signaling is tightly associated with many human diseases including cancers. Here, we report the cryo-EM structure of human WLS in complex with Wnt3a, the most widely studied Wnt, at 3.8 Å resolution. The transmembrane domain of WLS bears a GPCR fold, with a conserved core cavity and

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... opening. A β-hairpin of Wnt3a containing a conserved palmitoleoylation site has extensive interactions with the WLS luminal domain, and the tip region is accommodated in the WLS cavity. The flexibility of Wnt3a loop/hairpin regions involved in the multiple binding sites indicates a possible sequential release mechanism when Wnts are transferred to their receptors. We found Wnt3a palmitoleoylation site mutant has similar binding capability with WLS compared with wild type Wnt3a, suggesting palmitoleoylation is not essential for Wnt-WLS association. Our findings provide important insights into the molecular mechanism of Wnt palmitoleoylation, secretion and signaling.