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. 2021 Jun 11;372(6547):1215-1219.
doi: 10.1126/science.abg4998.

Substrate and product complexes reveal mechanisms of Hedgehog acylation by HHAT

Yiyang Jiang  1 , Thomas L Benz  1 , Stephen B Long  2
Affiliations

Substrate and product complexes reveal mechanisms of Hedgehog acylation by HHAT

Yiyang Jiang et al. Science. .

Abstract

Hedgehog proteins govern crucial developmental steps in animals and drive certain human cancers. Before they can function as signaling molecules, Hedgehog precursor proteins must undergo amino-terminal palmitoylation by Hedgehog acyltransferase (HHAT). We present cryo-electron microscopy structures of human HHAT in complex with its palmitoyl-coenzyme A substrate and of a product complex with a palmitoylated Hedgehog peptide at resolutions of 2.7 and 3.2 angstroms, respectively. The structures reveal how HHAT overcomes the challenges of bringing together substrates that have different physiochemical properties from opposite sides of the endoplasmic reticulum membrane within a membrane-embedded active site for catalysis. These principles are relevant to related enzymes that catalyze the acylation of Wnt and of the appetite-stimulating hormone ghrelin. The structural and mechanistic insights may advance the development of inhibitors for cancer.

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Conflict of interest statement

Competing interests: The authors declare no competing financial interests.

Figures

Fig. 1.
Fig. 1.. Overall structure of HHAT.
(A, B) Cryo-EM reconstruction (A) and cartoon representation (B) of HHAT in complex with palmitoyl-CoA and two Fab antibody fragments, viewed from the membrane. Variable domains of antibodies are shown. (C) Architecture of HHAT; helices drawn as cylinders.
Fig. 2.
Fig. 2.. Palmitoyl-CoA substrate complex.
(A) Binding of two palmitoyl-CoA molecules (sticks with green carbon atoms): one in the active site and one in the archway. Regions of HHAT that contact these ligands are pink; the archway is tan. (B) Detailed view of palmitoyl-CoA substrate in the active site. Its density (green surface) and interacting amino acids (pink sticks) are shown. (C) Palmitoyl-CoA in the archway, shown from the membrane perspective. (D) Slice of the molecular surface of HHAT highlighting the active site cavities and bound ligands. An arrow indicates the carbonyl carbon of palmitoyl-CoA in its substrate binding site.
Fig. 3.
Fig. 3.. Heme prosthetic group.
(A) Density for the heme. (B) Interactions with HHAT. Amino acids contacting the heme are drawn as sticks. Dashed lines indicate hydrogen bonds and interactions with Fe(III).
Fig. 4.
Fig. 4.. Palmitoylated Hedgehog peptide product complex.
(A) Cryo-EM reconstruction, in orthogonal views. (B) Overall structure, highlighting the locations of ligands. Regions in contact with the product are colored blue. (C-D) Close up views of the product, showing cryo-EM density (C) and contacting HHAT residues. (E) A curved arrow indicates repositioning of Trp335 from the substrate complex (light blue) to the product complex (pink). Positioning of the CoA byproduct relative to the substrate is indicated. (F) Slice of the molecular surface showing the product in the reaction chamber and the CoA byproduct in the cytosolic cavity. Dots indicate the disordered C-terminal region of the Hedgehog peptide. (G) Relative enzymatic activity of indicated mutants.
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