DHHC20 palmitoyl-transferase reshapes the membrane to foster catalysis

Abstract

Cysteine palmitoylation, a form of S-acylation, is a key post-translational modification in cellular signaling. This type of reversible lipidation is catalyzed by a family of integral membrane proteins known as DHHC acyltransferases. The first step in the S-acylation process is the recognition of free acyl-CoA from the lipid bilayer. The DHHC enzyme then becomes autoacylated, at a site defined by a conserved Asp-His-His-Cys motif. This reaction entails ionization of the catalytic Cys. Intriguingly, in known DHHC structures this catalytic Cys appears to be exposed to the hydrophobic interior of the lipid membrane, which would be highly unfavorable for a negatively charged nucleophile, thus hindering auto-acylation. Here, we use biochemical and computational methods to reconcile these seemingly contradicting facts. First, we experimentally demonstrate that human DHHC20 is active when reconstituted in POPC nanodiscs. Microsecond-long all-atom molecular dynamics simulations are then calculated for hDHHC20 and for different acyl-CoA forms, also in POPC. Strikingly, we observe that hDHHC20 induces a drastic deformation in the membrane, particularly on the cytoplasmic side where autoacylation occurs. As a result, the catalytic Cys becomes hydrated and optimally positioned to encounter the cleavage site in acyl-CoA. In summary, we hypothesize that DHHC enzymes locally reshape the membrane to foster a morphology that is specifically adapted for acyl-CoA recognition and auto-acylation.3 Significance StatementPalmitoylation, the most common form of S-acylation and the only reversible type of protein lipidation, is ubiquitous in eukaryotic cells. In humans, for example, it has been estimated that as much as ~10% of the proteome becomes palmitoylated, i.e. thousands of proteins. Accordingly, protein palmitoylation touches every important aspect of human physiology, both in health and disease. Despite its biological and biomedical importance, little is known about the molecular mechanism of the enzymes that catalyze this post-translational modification, known as DHHC acyltransferases. Here, we leverage the recently-determined atomic-resolution structure of human DHHC20 to gain novel insights into the mechanism of this class of enzymes, using both experimental and computational approaches.

Palmitoylation of the K ATP channel Kir6.2 subunit promotes channel opening by regulating PIP 2 sensitivity

“…Although the sequences surrounding the target cysteine are involved in recognition by specific DHHCs (20), a recent cysteine-scanning mutagenesis study demonstrated that S-palmitoylation can occur at any cysteine residue near the inner membrane interface, without an apparent need for an amino acid "consensus" sequence (21). The same study demonstrated that palmitoylation can occur up to 8 Å (e.g., approximately five to six residues/1.5 turn of an α-helix) into the membrane (21), most likely due to the ability of DHHC enzymes to locally reshape the membrane (22). Several of the ion channel palmitoylation sites identified to date are in fact near a membrane interface (7).…”

Structure and Mechanism of Hedgehog Acyl Transferase

“…The CoA phosphate groups are coordinated by a metal ion, built as a magnesium ion based on coordination geometry (Harding, 2001), and several hydrogen bonds mediated by Arg336 and Ser357. The Palm-CoA moiety is shifted along the membrane normal in the HHAT binding site compared to its average simulated position in a membrane bilayer (Stix et al, 2020). This locates the Palm-CoA thioester in the core of the HHAT cavity close to the proposed reaction center residues His379 and Asp339 (Fig.…”

“…The CHARMM-36 forcefield (Huang and MacKerell, 2013) was used to describe all components. Palm-CoA parameters were derived from Stix et al (Stix et al, 2020). Standard CHARMM-36 parameters were used for the Mg ion bound to Palm-CoA (which remained bound without the need for additional restraints) and for the heme/Fe complex.…”

Nanodiscs: A toolkit for membrane protein science

“…Examples include the dimeric heme cyclooxygenase (COX) 183,184 the flavoprotein human monoamine oxidase A 185 and methane monooxygenase 186 . DHHC acyltransferase (containing four trans‐membrane helices) was incorporated into the larger MSPE3D1‐POPC Nanodiscs for functional studies to show that significant membrane deformation is enabling hydration of catalytic cysteine at the active site inside the membrane 187 …”