Structure and Mechanism of Hedgehog Acyl Transferase

Abstract

SUMMARYThe iconic Sonic Hedgehog (SHH) morphogen pathway is a fundamental orchestrator of embryonic development and stem cell maintenance, and is implicated in cancers in various organs. A key step in signalling is transfer of a palmitate group to the N-terminal cysteine residue of SHH, catalysed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT) resident in the endoplasmic reticulum (ER). Here, we present the high-resolution cryo-EM structure of HHAT bound to substrate analogue palmityl-coenzyme A and a SHH mimetic megabody. Surprisingly, we identified a heme group bound to an HHAT cysteine residue and show that this modification is essential for HHAT structure and function. A structure of HHAT bound to potent small molecule inhibitor IMP-1575 revealed conformational changes in the active site which occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the novel mechanism by which HHAT adapts the membrane environment to transfer a long chain fatty acid across the ER membrane from cytosolic acyl-CoA to a luminal protein substrate. This structure of a member of the protein-substrate membrane-bound O-acyltransferase (MBOAT) superfamily provides a blueprint for other protein substrate MBOATs, such as WNT morphogen acyltransferase Porcupine and ghrelin O-acyltransferase GOAT, and a template for future drug discovery.

PyLipID: A Python Package for Analysis of Protein–Lipid Interactions from Molecular Dynamics Simulations

“…In particular, the analysis of residence times has allowed us to separate interactions/sites in annular and specific cholesterol binding sites, the latter showing longer residence times and having enriched interactions with Leu, Gly, and Ala residues. Extending this approach to a couple of anionic phospholipids suggests that long residence time binding sites correlate with those observed experimentally in cryo-EM structures, indicating how PyLipID may be used to aid the assignment and analysis of lipid-like density in newly determined structures …”

“…Extending this approach to a couple of anionic phospholipids suggests that long residence time binding sites correlate with those observed experimentally in cryo-EM structures, indicating how PyLipID may be used to aid the assignment and analysis of lipid-like density in newly determined structures. 75 ■ ASSOCIATED CONTENT * sı Supporting Information…”

A remarkably specific ligand reveals ghrelinO-acyltransferase interacts with extracellular peptides and exhibits unexpected cellular localization for a secretory pathway enzyme

“…Computational modeling of GOAT supports the presence of an internal transmembrane channel that could act as a pore, 29 and similar channels are observed in the crystal structure of the bacterial MBOAT DltB, the cryo-EM structures of Hhat, and structural models of PORCN. 32,[52][53][54][55] We note our imaging studies do not support colocalization of ligand 15 with GOAT in either transfected HEK 293 cells or prostate cancer cells, which could potentially support ligand transport (mechanism 2) rather than enzymeligand internalization (mechanism 1). However, our studies were performed with fixed cells and the short length and small number of amine groups within the peptide ligand may lead to inefficient ligand crosslinking during fixation allowing intracellular dispersion during sample preparation.…”

A combined computational-biochemical approach offers an accelerated path to membrane protein solubilization

“…This model matched the published mouse GOAT topology, and revealed the helical cluster "MBOAT core" and transmembrane channel now considered a hallmark of MBOAT-family enzymes that modify protein substrates. 23,[35][36][37][38][39] Despite the lack of hGOAT solubilization and purification, extensive functional studies have determined the substrate selectivity of GOAT and provided insight into the enzyme's catalytic mechanism. 32,[40][41][42][43][44][45] Building on this foundation, solubilization and purification of hGOAT will facilitate functional and structural studies of this enzyme and its acylation activity.…”