The ankyrin repeat domain of Huntingtin interacting protein 14 contains a surface aromatic cage, a potential site for methyl‐lysine binding

Abstract

Huntingtin interacting protein 14 (HIP14), a membrane-bound palmitoyl transferase, palmitoylates a number of neuronal proteins (including Huntingtin) and affects the trafficking, stability, aggregation, and/or functional activity of substrate proteins. HIP14 contains an N-terminal ankyrin repeat domain that may function in its substrate recognition. Sequence analysis suggests that the HIP14 ankyrin repeats share approximately 50% identity with the ankyrin repeats of G9a and G9a-like protein (GLP) histone lysine methyltransferases. The crystal structure of the HIP14 ankyrin repeats reveals a surface aromatic cage, formed by two tryptophans, one tyrosine, and one methionine. The all-hydrophobic cage resembles the tri-methylated lysine binding pocket of the plant homeodomain (PHD) of human BPTF (bromodomain and PHD domain transcription factor) 1. HIP14, a Huntingtin interacting protein 2, is a 633-residue protein, including 7-8 ankyrin repeats in the N-terminal region followed by five predicted transmembrane helices. The protein contains a signature DHHC palmitoyl transferases motif located close to the predicted fourth transmembrane helix 3. Importantly, the ankyrin repeats (a protein-protein interaction domain that may function in substrate recognition) and the DHHC sequence (the hypothetical active site) are both predicted to reside on the cytoplasmic face of the lipid bilayer 4, presumably allowing the substrate recognition and the active site to interact with the same substrate. Posttranslational palmitoylation involves the attachment of the saturated C16 fatty acid palmitate to specific cysteines via a thioester linkage 5-7. HIP14 palmitoylates Huntingtin at cysteine 214 8. Other substrates of HIP14-mediated palmitoylation include SNAP-25 (synaptosome associated protein 25 kDa), PSD-95 (postsynaptic density 95 kDa), GAD-65 (glutamate decarboxylase 65 kDa), and Synaptotagmin I 9. Ankyrin repeats are known to mediate protein-protein interactions 10. Recently we showed that the ankyrin repeat domains of G9a and GLP, two euchromatin associated histone lysine methyltranferases, bind N-terminal histone H3 peptides containing mono- or di-methylated lysine 9 (H3K9me1, me2) via a partial aromatic cage with three tryptophans and one acidic residue 11. Besides the ankyrin repeats of G9a and GLP, other protein domains including the Chromodomain 12, plant homeodomain 1,13-15, and Tudor domain 16, also recognize methylated lysines (Review 17). The common mode of methyl-lysine interactions is via a surface aromatic cage consisting of 2-4 aromatic residues. These aromatic cages are highly selective for methyllysine. In a study of Chromodomain-methyllysine interaction, binding was driven primarily by cation-π interactions (i.e, a methyllysine carries a positive charge), and secondarily by the packing of methyl group(s) against the aromatic ring(s) of the cage, with the hydrophobic effect contributing the least to binding 18. This eliminates the possibility of such cages generically binding hydrophobic residues, and suggests that other methyllysine binding ankyrin repeats could be identified by the presence of a surface aromatic cage.