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

Introduction

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.

Materials and Methods

The gene fragment encoding the N-terminal 51-288 residues of human HIP14 (we termed it the HIP14ANK) was amplified from ATCC Clone 5266097 and cloned into the NdeI and XhoI restriction sites of a modified pET28b vector containing 6xHis tag followed by SUMO tag, yielding pXC650.

The hexahistidine-SUMO-HIP14ANK fusion protein was expressed in E. coli BL21 (DE3) Gold C⁺ cells (Stratagene) via autoinduction at 37°C ¹⁹. The fusion protein was isolated on a 5 ml nickel-charged HiTrap Chelating HP column (GE Healthcare). The imidazole elution from the nickel-column was cleaved with His-tagged Ulp1 protease ²⁰ overnight while in dialysis against buffer without imidazole at 4°C, leaving two amino acids (HisMet) in front of the HIP14 ankyrin repeat domain. The digested mixture was passed through a nickel-column to remove the His-SUMO tag and the protease. The protein was further purified on a Superdex-75 Prep column (GE HealthCare) and stored in the buffer containing 20 mM Tris pH 8.0, 250 mM NaCl, 5% glycerol, and 5 mM dithiothreitol. The selenomethionine-labeled HIP14ANK protein was produced by a methionine auxotrophic E. coli strain (B834) grown in the PASM-5052 medium in the presence of 92% selenomethionine (125 mg/L) and 8% methionine (10 mg/L) ¹⁹. The growth, induction, and purification were performed following the same protocol as was used for the native protein.

HIP14ANK protein was concentrated to approximately 30 mg/ml. Crystals were obtained by sitting drop with mother liquor containing 100 mM (NH₄)₂SO₄, 25% polyethylene glycol (PEG) 3350, 0.1M Bis-Tris, pH 5.5 at 16°C. Selenomethionine-labeled protein crystals were grown in 200 mM (NH₄)₂SO₄, 30% PEG5KMME, 0.2M MES, pH 5.5. All crystals were cyroprotected by soaking in mother liquor supplemented with 35% xylitol.

Selenium anomalous diffraction data were collected at peak (0.9794 Å) and inflection (0.9796 Å) wavelengths at the SBC-CAT 19-ID beamline at the Advanced Light Source at Argonine National Laboratory on an ADSC Quantum 315 detector. SOLVE ²¹ found 21 selenium sites, which gave a clear, traceable density with many recognizable side chains. Model building with O ²² and refinement with CNS ²³ was then continued using a native data set.

In addition, GST tagged HIP14ANK (pXC648) and GST tagged HP1ß chromodomain were purified and used in the binding of protein domains to peptide SPOT arrays ²⁴.

Results and Discussion

Ankyrin repeats exist in a large number of intracellular and extracellular eukaryotic proteins ^25-27. To investigate whether a subset of ankyrin repeats could function in the recognition of methylated lysines as exemplified by G9a and GLP ¹¹, we searched protein database for sequence homologes of G9a and GLP. We placed particular emphasis on the conservation of aromatic residues corresponding to the cage residues of G9a and GLP, and their appropriate placement on the beta-turn and first alpha helix of neighboring repeats. Table 1 summarizes the protein sequence alignment of ankyrin repeat containing proteins with the potential to bind methyllysine. These proteins have conserved spacing of aromatic, hydrophobic or acidic residues, like in G9a and GLP. The ankyrin repeat domain of HIP14 is one of these candidates.

Table 1.

Sequence alignment of ankyrin repeat domains with the potential to bind methyllysine

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We generated a hexahistidine-SUMO (small ubiquitin-like modifier) tagged construct ²⁸ containing human HIP14 residues 51-288 (pXC650). The SUMO fusion was cleaved off by Ulp1 protease ²⁰. The protein crystallized in the space group P6₁22 and the structure was determined to the resolution of 1.99 Å (Table 2). The crystallographic asymmetric unit contains three molecules, A, B, and C. The three molecules are highly similar, except at the N- and C-ermini (Fig. 1A).

The crystallized HIP14 fragment contains seven ankyrin repeats. Each repeat has a helix-turn-helix-β-turn structure. The helices stack against each other and the β-turns project out at right angles. The sequence conservation between HIP14 and GLP indicates the invariant residues lie between ankyrin repeats 2 and 7 (Fig. 1B and Table 3). Structural superimposition of HIP14 and GLP ankyrin repeats positions the methylated lysine in a surface cage formed by M191, W196, and Y198 of ANK5 and W231 of ANK6 (Fig. 1C). In the GLP structure, an acidic residue interacts with the lone proton of the Nε atom of the di-methylated lysine whose methyl moieties point toward the aromatic residues ¹¹. Surveying structurally characterized cage-like methyl-lysine binding pockets revealed a glutamate or aspartate residue in (or near) the binding pocket having an important role in selecting for lower (mono- or di-) versus higher (tri-) methylation states of lysine ¹⁷. An all-hydrophobic cage in HIP14 suggests that it would bind a tri-methlyated lysine.

Table 3.

Sequence alignment of HIP14 (NP_056151.2) and GLP (Q9H9B1) ankyrin repeats. White-on-black residues (59) are invariant between the two sequences examined , while gray-background positions (22) are conserved (T and S; I, V, L and M; D and E; Q and N; R and K). Cage residues are highlighted in red letters. Grey-letter residues, either in the N-terminus (GLP) or in the C-terminus (HIP14), are not included in the structure. Vertical lines between the blocks indicate conserved hydrophobic residues important for the anti-parallel packing of two helices (in cyan) of each repeat. HIP14 P207 of ANK5 is in a cis-conformation

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The first 17 amino acids of Huntingtin before the polyglutamine track contains a third lysine, K15, followed by a serine, resembling the histone H3 lysine 9 and serine 10. Other substrates of HIP14, SNAP-25, PSD-95, GAD-65, and Synaptotagmin I, all contain sequences resembling histone H3 lysine 4 (H3K4) and H3K9 (supplementary Table 1). Known histone lysine methyltransferases, such as SET7/9 (acting on H3K4) and G9a (acting on H3K9), are able to methylate non-histone targets with consensus sequence as short as two-to-three residues ^24,29-32. Thus, we investigated the potential interaction of the HIP14 ankyrin-repeat domain with modified histone peptides arrays ²⁴. We employed two different peptide arrays including in total >1000 peptides containing >80 known modifications of the H3, H4, H2A and H2B tails in various combinations, but no specific binding to the membranes was detected (not shown). In comparison, as a positive control HP1β shows a strong H3K9 specific signal with the same arrays (Supplementary Figure 1). In addition, there was no detectable binding to the Huntingtin peptides (Supplementary Table 2). We speculate that the methylation of Huntingtin may crosstalk with other modifications, because, in addition to palmitoylation, Huntingtin can be posttranslationally modified either by sumoylation or by ubiquitylation on its N-terminal lysine residues K6 and K9 ³³. The crosstalk between ubiquitylation and methylation of neighboring lysine residues has been observed in regulation of the protein stability of estrogen receptor α ³². It remains to be seen whether Huntingtin is methylated and whether the methylated Huntingtin is recognized by the ankyrin repeats of HIP14.

Supplementary Material