Structure and Mechanism of DHHC Protein Acyltransferases

Principal Investigator: Maurine Linder

Co-PI: Toshimitsu Kawate

Department of Molecular Medicine
Sponsor: NIH-National Institute of Neurological Disorder and Stroke (NINDS)
Grant Number: 5R21NS101390-02
Title: Structure and Mechanism of DHHC Protein Acyltransferases
Project Amount: $197,035
Project Period: September 2018 to August 2019

DESCRIPTION (provided by applicant): 

Reversible protein S-­palmitoylation is an abundant posttranslational modification in the nervous system and is a key regulatory mechanism in numerous neuronal signaling pathways. Substrates for protein S-­palmitoylation include neurotransmitter receptors and transporters, signal transducing GTPases, and synaptic scaffolds. Members of the DHHC family of protein acyltransferases mediate the addition of palmitate or other long-­chain fatty acids to proteins and have been linked to a number of neurological disorders including intellectual disability and Huntington’s Disease. The importance of S-­palmitoylation as a regulatory modification in neuronal function and the alterations in S-­palmitoylation associated with human disease warrant a mechanistic understanding of the enzymes that mediate this process. Accordingly, our long-­term goals are to elucidate the molecular mechanisms underlying DHHC enzyme activity and its regulation by determining the first atomic resolution structure of a DHHC protein by x-­ray crystallography. The goals of this proposal are to understand how self-­association of DHHC proteins regulates their enzyme activity and to crystallize a DHHC protein. Using a bioluminescence resonance energy transfer assay in cells and in vitro enzyme assays with purified proteins, we determined that DHHC2 and DHHC3 proteins self-­associate and that the extent of self-­association tightly correlated with its enzyme activity. Furthermore, our pre-­crystallization screening revealed that monodisperse and stable DHHC protein constructs exist predominantly as dimers. Based on these findings, we propose that DHHC PATs exist in a monomer-­dimer equilibrium in cell membranes, where the monomeric form is active and the associated form is inactive. Testing this hypothesis is key to understanding how enzyme activity is regulated and critical for identifying conditions that will stabilize the protein for crystallization.