Wnts are a large family of hydrophobic, secreted signalling proteins that regulate embryogenesis, stem-cell fate, cell polarity, and adult tissue homeostasis. Aberrant Wnt signalling is implicated in cancer, one of the leading causes of mortality in Australia. Wnts act at short range, being secreted from their producing cell to interact with Frizzled (FZD) receptors on neighbouring cells, thereby activating several downstream signalling cascades, and modifying gene expression. Prior to secretion, Wnt proteins undergo processing in the endoplasmic reticulum (ER) by an integral membrane enzyme, Porcupine (PORCN). PORCN catalyses the addition of a cis-monounsaturated palmitoylate lipid onto a conserved serine on PORCN. This O-palmitoylation is essential for the secretion and activity of Wnt as it forms part of the binding interface with its receptor. Targeting the maturation of Wnt via PORCN shows significant promise in pre-clinical models of Wnt-dependent cancers. Multiple PORCN inhibitors have progressed to clinical trials, including the potent inhibitor C59.
Here we present the cryo-EM structure of PORCN bound to C59 to a resolution of 2.4 Å. The structure provides molecular insights into the mechanism of enzymatic activity and how small molecules such as C59 can induce inhibition. PORCN represented a challenging target for structure solution by cryoEM, due to its size (55 kDa), and its encapsulation almost entirely within a detergent micelle. Despite this, we were able to determine the structure to high resolution without the use of a rigid fusion or antibody fragment to increase mass. This makes this PORCN structure one of the smallest yet determined by cryoEM and shows the potential for cryoEM to provide high-resolution, inhibitor-bound structural information for small target membrane proteins.