The Cysteine-rich Domain of the DHHC3 Palmitoyltransferase Is Palmitoylated and Contains Tightly Bound Zinc

Abstract

DHHC palmitoyltransferases catalyze the addition of the fatty acid palmitate to proteins on the cytoplasmic leaflet of cell membranes. There are 23 members of the highly diverse mammalian DHHC protein family, all of which contain a conserved catalytic domain called the cysteine-rich domain (CRD). DHHC proteins transfer palmitate via a two-step catalytic mechanism in which the enzyme first modifies itself with palmitate in a process termed autoacylation. The enzyme then transfers palmitate from itself onto substrate proteins. The number and location of palmitoylated cysteines in the autoacylated intermediate is unknown. In this study, we present evidence using mass spectrometry that DHHC3 is palmitoylated at the cysteine in the DHHC motif. Mutation of highly conserved CRD cysteines outside the DHHC motif resulted in activity deficits and a structural perturbation revealed by limited proteolysis. Treatment of DHHC3 with chelating agents in vitro replicated both the specific structural perturbations and activity deficits observed in conserved cysteine mutants, suggesting metal ion-binding in the CRD. Using the fluorescent indicator mag-fura-2, the metal released from DHHC3 was identified as zinc. The stoichiometry of zinc binding was measured as 2 mol of zinc/mol of DHHC3 protein. Taken together, our data demonstrate that coordination of zinc ions by cysteine residues within the CRD is required for the structural integrity of DHHC proteins.Palmitoylation describes the attachment of a 16-carbon fatty acid to cysteine residues in proteins through a reversible thioester bond. Screens of the palmitoyl proteome have identified hundreds of palmitoylated proteins (1-4), including many involved in the pathogenesis of human diseases. Palmitate serves as a membrane anchor for many cytoplasmic proteins and is also present on numerous integral membrane proteins.

ZDHHC3 Tyrosine Phosphorylation Regulates Neural Cell Adhesion Molecule Palmitoylation

“…As shown in Fig. 7D, mutation of the PPFP sequence to APFA did not abolish Src-ZDHHC3 interaction, suggesting that other sites can be involved in enzyme-substrate interaction in the case of Srcmediated ZDHHC3 phosphorylation (23).…”

Fatty acylation of proteins: The long and the short of it

“…First, palmitate is attached to the cysteine in the DHHC motif[69]. This autoacylation reaction results in formation of an acyl-enzyme intermediate, which then transfers palmitate to a cysteine residue within the acceptor protein substrate.…”

“…This autoacylation reaction results in formation of an acyl-enzyme intermediate, which then transfers palmitate to a cysteine residue within the acceptor protein substrate. Structural integrity of DHHC enzymes is maintained by coordination of two zinc ions to conserved cysteines within the cysteine rich domain[69]. Although mutation of the DHHC cysteine residue typically generates an inactive enzyme mutant, there are examples of yeast DHHC proteins that can function at reduced efficiency with alanine or arginine in place of the DHHC cysteine[70].…”

Progress toward Understanding Protein S-acylation: Prospective in Plants

“…Most importantly, PATs also have a highly conserved catalytic Asp-His-His-Cys Cysteine Rich Domain (DHHC-CRD) of ~50 amino acids (Roth et al, 2002). This domain was proposed as Cx2Cx9HCx2Cx4DHHCx5Cx4Nx3F (Mitchell et al, 2006), usually residing on the cytoplasmic face of membranes between transmembrane domains (TMD) 2 and 3 of PATs (Gottlieb et al, 2015). It was reported that mutation of cysteine in DHHC domain inhibits both acyl intermediate formation and acyl chain transfer activity of PATs (Mitchell et al, 2006; Gottlieb et al, 2015).…”

“…This domain was proposed as Cx2Cx9HCx2Cx4DHHCx5Cx4Nx3F (Mitchell et al, 2006), usually residing on the cytoplasmic face of membranes between transmembrane domains (TMD) 2 and 3 of PATs (Gottlieb et al, 2015). It was reported that mutation of cysteine in DHHC domain inhibits both acyl intermediate formation and acyl chain transfer activity of PATs (Mitchell et al, 2006; Gottlieb et al, 2015). Indeed, when cysteine residue in the DHHC motif of AtPAT24, AtPAT10 and AtPAT14 of Arabidopsis was mutated to alanine or serine, all 3 AtPATs lost their PAT activities (Hemsley et al, 2005; Qi et al, 2013; Li et al, 2016).…”