Substrate and product complexes reveal mechanisms of Hedgehog acylation by HHAT

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.

Structure, mechanism, and inhibition of Hedgehog acyltransferase

“…The strong heme density on Cys324 is clearly distinct from the lipid-like attachments we observe for multiple cysteines in the HHAT structure ( Figure S5 ), with partial density consistent with reversible long-chain S -acylation. Our discovery agrees with a recent structure of HHAT in complex with antibody fragments that also revealed a heme group in a similar conformation ( Jiang et al., 2021 ) ( Figures S6 A and S6B).…”

“…This locates the Palm-CoA thioester in the core of the HHAT cavity close to the proposed reaction center residues His379 and Asp339 ( Figure 2 E) that have previously been shown to be important for the catalysis of palmitoyl transfer to SHH ( Buglino and Resh, 2010 ; Hofmann, 2000 ). We observe a similar overall conformation of the co-factor and binding pocket when compared to the structure of HHAT bound to Palm-CoA ( Jiang et al., 2021 ) ( Figure S6 C). However, some small conformational changes in the binding mode near the catalytic center are observed.…”

“…It has recently been postulated that Palm-CoA enters HHAT through an “archway” formed between α10 and α11 ( Jiang et al., 2021 ). The authors observed extra cryo-EM density in this region, which they attribute to Palm-CoA (as deposited at the Electron Microscopy Data Bank under EMD-23836 and EMD-23837) ( Figure S6 E, left panel).…”

“…Topological studies suggested a similar domain for HHAT ( Konitsiotis et al., 2015 ; Matevossian and Resh, 2015b ). A recent structure of HHAT ( Jiang et al., 2021 ) demonstrates that this is indeed the case, and provides intriguing insights into the likely mechanism of this complex protein-acylating enzyme. Here, we present a high-resolution cryogenic electron microscopy (cryo-EM) structure of HHAT in complex with the non-hydrolyzable Palm-CoA analog palmityl-CoA (nhPalm-CoA) and an inhibitory megabody.…”

Open Biology: overview for special issue on dynamics of protein fatty acylation

“…However, detailed insight into the molecular mechanism of individual fatty acylation reactions remains challenging and is an ongoing endeavour. One of the most exciting advances in the field of fatty acylation has been the elucidation of three-dimensional structures of zDHHC and MBOAT enzymes, some of which were reported as this special issue was being assembled [12,[20][21][22][23]. This information will not only shed light on the catalytic mechanisms employed by fatty acyltransferases but will also aid in the design and optimization of selective small molecule inhibitors to be used for therapeutic targeting in disease.…”