Fatty acyl recognition and transfer by an integral membrane S-acyltransferase

Fattening up proteins Many eukaryotic proteins are modified by the attachment of lipids, and these modifications can alter how proteins interact with cellular membranes. Rana et al. present x-ray crystal structures of an integral membrane enzyme that appends a fatty acyl chain onto a cysteine residue of target proteins. The enzyme active site is situated at the membrane surface, thus explaining the enzyme's preference for substrates that are already membrane-associated. The structure of a fatty acid-like inhibitor bound within a hydrophobic cavity elucidates the mechanism for the enzyme's acyl chain specificity. Science, this issue p. eaao6326 A lipid-binding cavity determines acyl chain length for a membrane-bound acyltransferase. INTRODUCTION Hundreds of cellular proteins are modified by posttranslational S-acylation of cysteines, commonly known as protein palmitoylation. Unlike other lipid attachments, which are thought to be permanent, palmitoylation can be reversed by cellular thioesterases, enabling dynamic modulation of the local hydrophobicity of substrate proteins. In humans, palmitoylation is catalyzed by 23 members of the DHHC family of integral membrane enzymes, which contain a signature Asp-His-His-Cys (DHHC) motif. DHHC enzymes use acyl–coenzyme A (CoA) (predominantly palmitoyl-CoA) to generate an acyl-enzyme intermediate from which the acyl chain is subsequently transferred to a substrate. With a recent systems-level analysis suggesting that more than 10% of the proteome is palmitoylated, the complexity of protein palmitoylation approaches that of protein phosphorylation and ubiquitylation. Nonetheless, fundamental aspects of DHHC enzymes, including their mechanism of catalysis and acyl-CoA binding and recognition, have been challenging to analyze without detailed structural information. RATIONALE To obtain insights into the structural mechanism of DHHC enzymes, we solved the crystal structures of two DHHC family members: human DHHC20 and a catalytically inactive mutant of zebrafish DHHC15. We also purified and solved the structure of human DHHC20 conjugated to an irreversible inhibitor that mimics the acylated enzyme intermediate. We carried out structure-guided mutagenesis experiments to test residues important for enzyme function and to engineer mutant enzymes with altered acyl-CoA selectivity. RESULTS The four transmembrane helices of hDHHC20 and zfDHHS15 form a tepee-like structure with the active site, contained in the highly conserved cytosolic DHHC cysteine-rich domain, at the membrane-cytosol interface. The cysteine-rich domain binds two zinc (Zn2+) ions that impart structural stability, but do not actively coordinate the nucleophilic cysteine. The C-terminal domain has an unanticipated amphipathic helix and a hydrophobic loop that together form a supporting structure for the transmembrane domain. The transmembrane domain forms a cavity where the acyl chain of acyl-CoA is inserted. Cavity-lining residues are determinants of fatty acyl recognition and chain-length selectivity. Our structures enabled us to engineer mutants of human DHHC20 with altered acyl chain–length selectivities. CONCLUSION By elucidating the location of the active site at the membrane-aqueous interface, our structures readily explain why candidate cysteines for palmitoylation are proximal to the membrane. The active site has a catalytic triad-like arrangement of aspartic acid and histidine residues that activate the nucleophilic cysteine. The C-terminal domain has conserved motifs that form critical interactions with the active site and the rest of the protein. The structures reported here set the stage for the development of structure-based small molecule probes and tools such as orthogonal DHHC enzyme–fatty acyl–CoA pairs that will likely help investigate the enzyme-substrate network of this biologically and biomedically important family of enzymes. Molecular view of DHHC palmitoyltransferases. Human DHHC20 palmitoyltransferase (yellow) shown localized in the Golgi body membrane (green stacks). The Zn2+ ions are shown as gray spheres and the acyl chain of the palmitoyl group in white sticks. A hypothetical substrate (blue) approaches the palmitoyltransferase from the left and, after palmitoylation, is localized to the Golgi body membrane through anchoring of the palmitoyl group, now transferred to the substrate. Image credit: J. Swan, National Institutes of Health DHHC (Asp-His-His-Cys) palmitoyltransferases are eukaryotic integral membrane enzymes that catalyze protein palmitoylation, which is important in a range of physiological processes, including small guanosine triphosphatase (GTPase) signaling, cell adhesion, and neuronal receptor scaffolding. We present crystal structures of two DHHC palmitoyltransferases and a covalent intermediate mimic. The active site resides at the membrane-cytosol interface, which allows the enzyme to catalyze thioester-exchange chemistry by using fatty acyl–coenzyme A and explains why membrane-proximal cysteines are candidates for palmitoylation. The acyl chain binds in a cavity formed by the transmembrane domain. We propose a mechanism for acyl chain–length selectivity in DHHC enzymes on the basis of cavity mutants with preferences for shorter and longer acyl chains.