Zinc coordination by the DHHC cysteine-rich domain of the palmitoyltransferase Swf1

AYELEN GONZALEZ MONTORO; RODRIGO QUIROGA; JAVIER VALDEZ TAUBAS

BIOCHEMICAL JOURNAL

PORTLAND PRESS LTD

Lugar: Londres; Año: 2013 p. 427 - 435

0264-6021

S-acylation, commonly known as palmitoylation, is a widespread post-translational modification of proteins that consists in the tioestherification of one or more cysteine residues with fatty acids. This modification is catalyzed by a family of palmitoyltransferases (PATs), characterized by the presence of a 50-residue long Cysteine-Rich Domain (DHHC-CRD). 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 proteins. We identified 21 novel loss-of-function mutations, which are mostly localized within the DHHC-CRD. Modeling of the tertiary structure of the Swf1 DHHC domain suggests that it could fold as a zinc-finger domain, coordinating two zinc atoms in a CCHC arrangement. All residues predicted to be involved in the coordination 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 coordination. 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.