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. 2019 Sep 27;294(39):14166-14174.
doi: 10.1074/jbc.AC119.009749. Epub 2019 Aug 14.

The ghrelin O-acyltransferase structure reveals a catalytic channel for transmembrane hormone acylation

Maria B Campaña  1 , Flaviyan Jerome Irudayanathan  2 , Tasha R Davis  1 , Kayleigh R McGovern-Gooch  1 , Rosemary Loftus  1 , Mohammad Ashkar  1 , Najae Escoffery  1 , Melissa Navarro  1 , Michelle A Sieburg  1 , Shikha Nangia  3   4 , James L Hougland  5   4
Affiliations

The ghrelin O-acyltransferase structure reveals a catalytic channel for transmembrane hormone acylation

Maria B Campaña et al. J Biol Chem. .

Abstract

Integral membrane proteins represent a large and diverse portion of the proteome and are often recalcitrant to purification, impeding studies essential for understanding protein structure and function. By combining co-evolutionary constraints and computational modeling with biochemical validation through site-directed mutagenesis and enzyme activity assays, we demonstrate here a synergistic approach to structurally model purification-resistant topologically complex integral membrane proteins. We report the first structural model of a eukaryotic membrane-bound O-acyltransferase (MBOAT), ghrelin O-acyltransferase (GOAT), which modifies the metabolism-regulating hormone ghrelin. Our structure, generated in the absence of any experimental structural data, revealed an unanticipated strategy for transmembrane protein acylation with catalysis occurring in an internal channel connecting the endoplasmic reticulum lumen and cytoplasm. This finding validated the power of our approach to generate predictive structural models for other experimentally challenging integral membrane proteins. Our results illuminate novel aspects of membrane protein function and represent key steps for advancing structure-guided inhibitor design to target therapeutically important but experimentally intractable membrane proteins.

Keywords: acyltransferase; co-evolutionary constraint; computational modeling; ghrelin O-acyltransferase (GOAT); integral membrane protein; membrane enzyme; membrane protein; membrane-bound O-acyltransferase (MBOAT); post-translational modification (PTM); protein acylation; protein structure; protein structure prediction; structural biology.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Structural model of hGOAT generated by computational methods. A, schematic of ghrelin octanoylation by hGOAT showing the predicted transmembrane topology of hGOAT containing 11 transmembrane helix domains (TM1–11), two intramembrane domains (IM1–2), and loop regions generated using Protter (43). B, octanoylation of a ghrelin-mimetic fluorescent peptide by recombinant hGOAT. C, contact maps for hGOAT showing the probability for a co-evolutionary contact from RaptorX analysis (i) and amino acid contacts in the final optimized hGOAT structure (ii). D, structure of hGOAT in an ER-mimetic lipid membrane, correlated to color-coded membrane topology in A. E, illustration of the internal channel within hGOAT (green) transiting from the ER lumen to the cytoplasm, with the channel determined by the CAVER 3.0 plugin in PyMOL (33). F, structural overlay of hGOAT and DltB showing the absolutely conserved histidine residues (hGOAT His-338 (teal) and DltB His-336 (purple) (Protein Data Bank code 6BUG, chain C) within these acyltransferases.
Figure 2.
Figure 2.
Mutagenesis studies support the location and functional importance of the hGOAT internal channel. A–C, alanine mutations mapped onto the hGOAT structure, with each sphere denoting the α-carbon of the mutated residue. Spheres are colored as follows. Blue, alanine variants with octanoylation activity within 3-fold of WT hGOAT; purple, alanine variants with impaired octanoylation activity (>3-fold loss compared with WT hGOAT); red, inactive alanine variants. A, view from lumenal face; B, view from cytoplasmic face; C, side view in the plane of the ER membrane. D, octanoylation activity of hGOAT alanine variants for non-void-contacting (black, n = 21) and void-contacting mutations (green, n = 21), with dotted lines denoting the average acylation activity for each group; *, p < 0.03.
Figure 3.
Figure 3.
The acyl donor binding site within hGOAT. A, structure of octanoyl-CoA bound within hGOAT from a side view in the plane of the ER membrane. B, view from the cytoplasmic face of hGOAT showing the solvent-exposed portions of the CoA component of octanoyl-CoA. C, cutaway view showing the acyl chain–binding pocket within hGOAT, bent sharply upward from the CoA-binding regions on the cytoplasmic face of hGOAT. D and E, interactions between the octanoyl-CoA acyl donor and hGOAT residues; hGOAT residues shown in purple reduce acylation activity under standard reaction conditions when mutated to alanine, and residues shown in red abolish acylation activity upon alanine mutation. F, acylation activity of WT hGOAT and selected hGOAT alanine variants using octanoyl-, lauryl-, or myristoyl-CoA as the sole acyl donor. Activities are normalized to the most reactive hGOAT variant with each acyl donor; individual data points indicate independent trials, and the dotted line indicates the average of three independent trials. G, acyl donor competition demonstrates altered selectivity to a longer acyl donor for F331A and W351A hGOAT variants, consistent with the predicted interaction of these amino acid side chains with the distal end of the octanoyl acyl chain.
Figure 4.
Figure 4.
Proposed pathway for transmembrane ghrelin octanoylation by GOAT. Ghrelin (GSSFL-ghrelin) and octanoyl-CoA enter the GOAT internal channel from the ER lumenal pore and cytoplasmic acyl donor–binding sites, respectively, followed by acyl transfer to the ghrelin serine side chain hydroxyl. Octanoylated ghrelin dissociates to the ER lumen, resulting in the octanoyl chain transiting through the GOAT interior, and CoA is released back to the cytoplasm. The red and blue rectangles represent perimeter helices, the green rectangle represents intramembrane domains forming the cytoplasmic surface of hGOAT, and dotted lines represent binding interactions between the octanoyl-CoA acyl donor and its binding site within hGOAT.
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