title: Zinc co-ordination by the DHHC cysteine-rich domain of the palmitoyltransferase Swf1.
abstract: S-acylation, commonly known as palmitoylation, is a widespread post-translational modification of proteins that consists of the thioesterification of one or more cysteine residues with fatty acids. This modification is catalysed by a family of PATs (palmitoyltransferases), characterized by the presence of a 50-residue long DHHC-CRD (Asp-His-His-Cys cysteine-rich domain). To gain knowledge on the structure-function relationships of these proteins, we carried out a random-mutagenesis assay designed to uncover essential amino acids in Swf1, the yeast PAT responsible for the palmitoylation of SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins. We identified 21 novel loss-of-function mutations, which are mostly localized within the DHHC-CRD. Modelling of the tertiary structure of the Swf1 DHHC domain suggests that it could fold as a zinc-finger domain, co-ordinating two zinc atoms in a CCHC arrangement. All residues predicted to be involved in the co-ordination of zinc were found to be essential for Swf1 function in the screen. Moreover, these mutations result in unstable proteins, in agreement with a structural role for these zinc fingers. The conservation of amino acids predicted to form each zinc-binding pocket suggests a shared function, as the selective pressure to maintain them is lost upon mutation of one of them. A Swf1 orthologue that lacks one of the zinc-binding pockets is able to complement a yeast swf1∆ strain, possibly because a similar fold can be stabilized by hydrogen bonds instead of zinc co-ordination. Finally, we show directly that recombinant Swf1 DHHC-CRD is able to bind zinc. Sequence analyses of DHHC domains allowed us to present models of the zinc-binding properties for all PATs.
authors.affiliation: Centro de Investigaciones en Química Biológica de Córdoba, CIQUIBIC (UNC-CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, X5000HUA Córdoba, Argentina.
authors.forename: Ayelén
authors.initials: A
authors.lastname: González Montoro
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Quiroga
authors.forename: Javier
authors.initials: J
authors.lastname: Valdez Taubas
mesh_terms: D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D001665:Binding Sites; D056831:Coordination Complexes / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055786:Gene Knockout Techniques; D005816:Genetic Complementation Test; D006801:Humans; D054878:Lipoylation; D010802:Phylogeny; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae; D020539:Sequence Analysis, Protein; D040681:Structural Homology, Protein; D025062:Yarrowia / Q000201:enzymology*; D015032:Zinc / Q000737:chemistry*
chemical_list: D056831:Coordination Complexes; D005656:Fungal Proteins; D000217:Acyltransferases; D015032:Zinc