Bivalent recognition of fatty acyl-CoA by a human integral membrane palmitoyltransferase

Abstract

S-acylation, also known as palmitoylation, is the most abundant form of protein lipidation in humans. This reversible posttranslational modification, which targets thousands of proteins, is catalyzed by 23 members of the DHHC family of integral membrane enzymes. DHHC enzymes use fatty acyl-CoA as the ubiquitous fatty acyl donor and become autoacylated at a catalytic cysteine; this intermediate subsequently transfers the fatty acyl group to a cysteine in the target protein. Protein S-acylation intersects with almost all areas of human physiology, and several DHHC enzymes are considered as possible therapeutic targets against diseases such as cancer. These efforts would greatly benefit from a detailed understanding of the molecular basis for this crucial enzymatic reaction. Here, we combine X-ray crystallography with all-atom molecular dynamics simulations to elucidate the structure of the precatalytic complex of human DHHC20 in complex with palmitoyl CoA. The resulting structure reveals that the fatty acyl chain inserts into a hydrophobic pocket within the transmembrane spanning region of the protein, whereas the CoA headgroup is recognized by the cytosolic domain through polar and ionic interactions. Biochemical experiments corroborate the predictions from our structural model. We show, using both computational and experimental analyses, that palmitoyl CoA acts as a bivalent ligand where the interaction of the DHHC enzyme with both the fatty acyl chain and the CoA headgroup is important for catalytic chemistry to proceed. This bivalency explains how, in the presence of high concentrations of free CoA under physiological conditions, DHHC enzymes can efficiently use palmitoyl CoA as a substrate for autoacylation.

A High-Throughput Fluorescent Turn-On Assay for Inhibitors of DHHC Family Proteins

“…Therefore, we reasoned that removal of the methyl amide from DPP-5 would both minimize steric clashes and result in a better acyl-CoA mimic ( PTP-1 ) (see Figure A). Docking against zDHHC20 revealed a tightly bound ligand–receptor complex, but also that the probe did not engage a highly conserved key residue of the basic patch, Lys135 , (Figure S2 in the Supporting Information). In silico screening to maximize this ADP-binding pocket interaction demonstrated that replacement of succinic acid with glutaric acid ( PTP-2 ) resulted in further optimized interactions (Figure A).…”

Structural Exploration on Palmitoyltransferase DHHC3 from Homo sapiens

“…On the other hand, whether DHHC3 is the specific palmitoyltransferase for LAMTOR1 deserves further investigations. Recently, Lee et al indicated that the addition of palmitoyl-CoA to an inactive mutant of DHHC20 (hDHHS20) can be successfully crystallized [ 24 ]. This crystal structure revealed that palmitoyl-CoA locates at the interface between two hDHHS20 monomers and partially inserts into the acyl chain binding cavity, which gives the clue to improve crystallization conditions to obtain the DHHC3 structure at atomic resolution.…”

“…The crystal structure of a complex formed by hDHHC20 with its inhibitor 2-bromopalmitate (2-BP) to mimic the acyl intermediate was further solved, showing that the acyl chain is inserted into an enzyme cavity composed of the four TM helices within the membrane bilayer, in which TM3 contributes the most in contact with the acyl chain [ 23 ]. They further reported a structure of a catalytically inactive mutant hDHHS20 in a complex with palmitoyl-CoA and elucidated that the polar and ionic interactions between the CoA headgroup and the cytosolic domain are also important for the substrate binding [ 24 ].…”

Charting the Chemical Space of Acrylamide-Based Inhibitors of zDHHC20

“…We aimed to mimic the native substrate of zDHHC20, acyl CoA, which interacts not only with the lipid-binding pocket but also with CoA-binding components, including an ADP-binding pocket. In fact, it has been shown that bivalent recognition is critical for DHHC–substrate binding . The CoA can be seen as three elements: a cysteamine, a pantothenic acid linker, and a 3′-phosphoadenosine diphosphate.…”

“…In designing the next generation of DHHC inhibitors, improving selectivity will likely mean reducing the lipophilicity and/or targeting additional binding motifs, such as the CoA binding pocket. In the present study, we report an expanded structure–activity relationship (SAR) study of CMA and its derivatives against zDHHC20 (Figure ), the most structurally characterized family member, − and the homologous zDHHC2, with an eye toward understanding the limits and potential of the acrylamide scaffold.…”