Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Crystal structure of a membrane-bound O-acyltransferase


Membrane-bound O-acyltransferases (MBOATs) are a superfamily of integral transmembrane enzymes that are found in all kingdoms of life1. In bacteria, MBOATs modify protective cell-surface polymers. In vertebrates, some MBOAT enzymes—such as acyl-coenzyme A:cholesterol acyltransferase and diacylglycerol acyltransferase 1—are responsible for lipid biosynthesis or phospholipid remodelling2,3. Other MBOATs, including porcupine, hedgehog acyltransferase and ghrelin acyltransferase, catalyse essential lipid modifications of secreted proteins such as Wnt, hedgehog and ghrelin, respectively4,5,6,7,8,9,10. Although many MBOAT proteins are important drug targets, little is known about their molecular architecture and functional mechanisms. Here we present crystal structures of DltB, an MBOAT responsible for the d-alanylation of cell-wall teichoic acid in Gram-positive bacteria11,12,13,14,15,16, both alone and in complex with the d-alanyl donor protein DltC. DltB contains a ring of 11 peripheral transmembrane helices, which shield a highly conserved extracellular structural funnel extending into the middle of the lipid bilayer. The conserved catalytic histidine residue is located at the bottom of this funnel and is connected to the intracellular DltC through a narrow tunnel. Mutation of either the catalytic histidine or the DltC-binding site of DltB abolishes the d-alanylation of lipoteichoic acid and sensitizes the Gram-positive bacterium Bacillus subtilis to cell-wall stress, which suggests cross-membrane catalysis involving the tunnel. Structure-guided sequence comparison among DltB and vertebrate MBOATs reveals a conserved structural core and suggests that MBOATs from different organisms have similar catalytic mechanisms. Our structures provide a template for understanding structure–function relationships in MBOATs and for developing therapeutic MBOAT inhibitors.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Overall structure of DltB and its conserved extracellular funnel.
Fig. 2: Structural basis of the DltB–DltC-Ppant interaction.
Fig. 3: Structure of the DltB tunnel and the DltB–DltC-Ppant binding mode provide insight into the molecular mechanism of DltB.
Fig. 4: Conserved regions among bacterial DltB and vertebrate PORCN and GOAT proteins.

Data availability

Atomic structures have been deposited in the Protein Data Bank (PDB) with accession codes 6BUG (crystal form I), 6BUH (crystal form II) and 6BUI (crystal form III). All other data that support the findings of this study are available from the corresponding author upon reasonable request.


  1. Hofmann, K. A superfamily of membrane-bound O-acyltransferases with implications for Wnt signaling. Trends Biochem. Sci. 25, 111–112 (2000).

    Article  CAS  Google Scholar 

  2. Chang, T. Y., Chang, C. C., Ohgami, N. & Yamauchi, Y. Cholesterol sensing, trafficking, and esterification. Annu. Rev. Cell Dev. Biol. 22, 129–157 (2006).

    Article  CAS  Google Scholar 

  3. Liu, Q., Siloto, R. M., Lehner, R., Stone, S. J. & Weselake, R. J. Acyl-CoA:diacylglycerol acyltransferase: molecular biology, biochemistry and biotechnology. Prog. Lipid Res. 51, 350–377 (2012).

    Article  CAS  Google Scholar 

  4. Chang, S. C. & Magee, A. I. Acyltransferases for secreted signalling proteins (review). Mol. Membr. Biol. 26, 104–113 (2009).

    Article  CAS  Google Scholar 

  5. Masumoto, N. et al. Membrane bound O-acyltransferases and their inhibitors. Biochem. Soc. Trans. 43, 246–252 (2015).

    Article  CAS  Google Scholar 

  6. Resh, M. D. Fatty acylation of proteins: the long and the short of it. Prog. Lipid Res. 63, 120–131 (2016).

    Article  CAS  Google Scholar 

  7. Tuladhar, R. & Lum, L. Fatty acyl donor selectivity in membrane bound O-acyltransferases and communal cell fate decision-making. Biochem. Soc. Trans. 43, 235–239 (2015).

    Article  CAS  Google Scholar 

  8. Resh, M. D. Palmitoylation of proteins in cancer. Biochem. Soc. Trans. 45, 409–416 (2017).

    Article  CAS  Google Scholar 

  9. Lanyon-Hogg, T., Faronato, M., Serwa, R. A. & Tate, E. W. Dynamic protein acylation: new substrates, mechanisms, and drug targets. Trends Biochem. Sci. 42, 566–581 (2017).

    Article  CAS  Google Scholar 

  10. Madan, B. & Virshup, D. M. Targeting Wnts at the source—new mechanisms, new biomarkers, new drugs. Mol. Cancer Ther. 14, 1087–1094 (2015).

    Article  CAS  Google Scholar 

  11. Perego, M. et al. Incorporation of d-alanine into lipoteichoic acid and wall teichoic acid in Bacillus subtilis. Identification of genes and regulation. J. Biol. Chem. 270, 15598–15606 (1995).

    Article  CAS  Google Scholar 

  12. Neuhaus, F. C., Heaton, M. P., Debabov, D. V. & Zhang, Q. The dlt operon in the biosynthesis of d-alanyl-lipoteichoic acid in Lactobacillus casei. Microb. Drug Resist. 2, 77–84 (1996).

    Article  CAS  Google Scholar 

  13. Reichmann, N. T., Cassona, C. P. & Gründling, A. Revised mechanism of d-alanine incorporation into cell wall polymers in Gram-positive bacteria. Microbiology 159, 1868–1877 (2013).

    Article  CAS  Google Scholar 

  14. Pasquina, L. et al. A synthetic lethal approach for compound and target identification in Staphylococcus aureus. Nat. Chem. Biol. 12, 40–45 (2016).

    Article  CAS  Google Scholar 

  15. Neuhaus, F. C. & Baddiley, J. A continuum of anionic charge: structures and functions of d-alanyl-teichoic acids in Gram-positive bacteria. Microbiol. Mol. Biol. Rev. 67, 686–723 (2003).

    Article  CAS  Google Scholar 

  16. Peschel, A. et al. Inactivation of the dlt operon in Staphylococcus aureus confers sensitivity to defensins, protegrins, and other antimicrobial peptides. J. Biol. Chem. 274, 8405–8410 (1999).

    Article  CAS  Google Scholar 

  17. Rios-Esteves, J., Haugen, B. & Resh, M. D. Identification of key residues and regions important for porcupine-mediated Wnt acylation. J. Biol. Chem. 289, 17009–17019 (2014).

    Article  CAS  Google Scholar 

  18. Yang, J., Brown, M. S., Liang, G., Grishin, N. V. & Goldstein, J. L. Identification of the acyltransferase that octanoylates ghrelin, an appetite-stimulating peptide hormone. Cell 132, 387–396 (2008).

    Article  CAS  Google Scholar 

  19. Buglino, J. A. & Resh, M. D. Identification of conserved regions and residues within hedgehog acyltransferase critical for palmitoylation of sonic hedgehog. PLoS ONE 5, e11195 (2010).

    Article  ADS  Google Scholar 

  20. Das, A., Davis, M. A. & Rudel, L. L. Identification of putative active site residues of ACAT enzymes. J. Lipid Res. 49, 1770–1781 (2008).

    Article  CAS  Google Scholar 

  21. Lin, S., Lu, X., Chang, C. C. & Chang, T. Y. Human acyl-coenzyme A:cholesterol acyltransferase expressed in chinese hamster ovary cells: membrane topology and active site location. Mol. Biol. Cell 14, 2447–2460 (2003).

    Article  CAS  Google Scholar 

  22. McFie, P. J., Stone, S. L., Banman, S. L. & Stone, S. J. Topological orientation of acyl-CoA:diacylglycerol acyltransferase-1 (DGAT1) and identification of a putative active site histidine and the role of the N terminus in dimer/tetramer formation. J. Biol. Chem. 285, 37377–37387 (2010).

    Article  CAS  Google Scholar 

  23. Viana, D. et al. A single natural nucleotide mutation alters bacterial pathogen host tropism. Nat. Genet. 47, 361–366 (2015).

    Article  CAS  Google Scholar 

  24. Jogl, G., Hsiao, Y. S. & Tong, L. Structure and function of carnitine acyltransferases. Ann. NY Acad. Sci. 1033, 17–29 (2004).

    Article  ADS  CAS  Google Scholar 

  25. Taylor, M. S. et al. Architectural organization of the metabolic regulatory enzyme ghrelin O-acyltransferase. J. Biol. Chem. 288, 32211–32228 (2013).

    Article  CAS  Google Scholar 

  26. Matevossian, A. & Resh, M. D. Membrane topology of hedgehog acyltransferase. J. Biol. Chem. 290, 2235–2243 (2015).

    Article  CAS  Google Scholar 

  27. Konitsiotis, A. D. et al. Topological analysis of hedgehog acyltransferase, a multipalmitoylated transmembrane protein. J. Biol. Chem. 290, 3293–3307 (2015).

    Article  CAS  Google Scholar 

  28. Barnett, B. P. et al. Glucose and weight control in mice with a designed ghrelin O-acyltransferase inhibitor. Science 330, 1689–1692 (2010).

    Article  ADS  CAS  Google Scholar 

  29. Ho, S. Y. & Keller, T. H. The use of porcupine inhibitors to target Wnt-driven cancers. Bioorg. Med. Chem. Lett. 25, 5472–5476 (2015).

    Article  CAS  Google Scholar 

  30. Chen, B. et al. Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer. Nat. Chem. Biol. 5, 100–107 (2009).

    Article  ADS  CAS  Google Scholar 

  31. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  32. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  33. Zimmermann, S. et al. High-resolution structures of the d-alanyl carrier protein (Dcp) DltC from Bacillus subtilis reveal equivalent conformations of apo- and holo-forms. FEBS Lett. 589, 2283–2289 (2015).

    Article  CAS  Google Scholar 

  34. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  35. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).

    Article  Google Scholar 

  36. Delano, W. L. & Brünger, A. T. Helix packing in proteins: prediction and energetic analysis of dimeric, trimeric, and tetrameric GCN4 coiled coil structures. Proteins 20, 105–123 (1994).

    Article  CAS  Google Scholar 

  37. Ashkenazy, H. et al. ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules. Nucleic Acids Res. 44, W344–W350 (2016).

    Article  CAS  Google Scholar 

  38. Taly, J. F. et al. Using the T-Coffee package to build multiple sequence alignments of protein, RNA, DNA sequences and 3D structures. Nat. Protoc. 6, 1669–1682 (2011).

    Article  CAS  Google Scholar 

  39. Guariglia-Oropeza, V. & Helmann, J. D. Bacillus subtilis σV confers lysozyme resistance by activation of two cell wall modification pathways, peptidoglycan O-acetylation and d-alanylation of teichoic acids. J. Bacteriol. 193, 6223–6232 (2011).

    Article  CAS  Google Scholar 

Download references


We are grateful to the staff at Advanced Light Source beamlines 5.0.1, 8.2.1 and 8.2.2 for assistance with synchrotron data collection. We thank N. Zheng and P. Hsu for comments on this manuscript, T. Hinds for discussion and advice on assays, S. Ovchinnikov for computational modelling, L. Kruse for use of the radioactive gel scanner and M. Ragheb for construction of the dlt operon deletion strain. This work was supported by National Institutes of Health grant R01 GM127316 to W.X. and a Jane Coffin Childs postdoctoral fellowship to D.M. This work was also supported by Chinese Academy of Sciences grant XDB08010303 to Z.R. and W.X., the National Institute of Health grant DP2GM110773 to H.M. and the Bacterial Pathogenesis Training Grant 5T32AI055396-13 to K.S.L.

Reviewer information

Nature thanks E. Tate and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Authors and Affiliations



D.M. carried out protein purification, crystallization, and related binding and enzymatic analysis. D.M. and Z.W. collected diffraction data and determined the crystal structure. Z.W. performed structural refinement. C.N.M. constructed the B. subtilis strains, and C.N.M., K.S.L. and H.M. performed the cell survival assays. P.L. contributed to molecular cloning and sample preparation. X.L. and Z.R. contributed to screening of other MBOAT proteins. D.M., Z.W. and W.X. analysed structural data and wrote the paper. All authors participated in manuscript revision and analysis of biochemical data.

Corresponding author

Correspondence to Wenqing Xu.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 MBOAT-catalysed reactions and chemical structures of MBOAT substrates.

a, General reaction catalysed by MBOATs. b, Structure of CoA and acyl-CoA. The red rectangle highlights the Ppant prosthetic group within the CoA structure. For known acyl-group donors of MBOATs, the acyl groups are covalently linked with a sulfhydryl group (for example, that of Ppant in acyl-CoA or DltC-Ppant). c, Comparison of acyl-group donors and acceptors of PORCN, GOAT, DGAT1, ACAT and DltB. In the acyl-group donor column, the red dashed lines indicate the bonds that are broken during acyl-transfer reactions. In the acyl-group acceptor column, the hydroxyl groups that accept acyl groups are highlighted in red. ACAT1, ACAT2 and DGAT1 use saturated and unsaturated long-chain acyl-CoA. d, The reaction catalysed by DltB. DltB catalyses d-alanylation of both wall teichoic acid and LTA. Because the d-alanylation of wall teichoic acid is at least partially dependent on LTA d-alanylation, here we discuss only the d-alanylation of LTA. DltB transfers d-alanyl groups onto hydroxyl groups of the polyglycerolphosphate chain of the LTA molecule. For simplicity, only the type I LTA structure is shown here. The fatty-acid chains are responsible for the anchoring of LTA to the membrane of Gram-positive bacteria.

Extended Data Fig. 2 Purification of DltB, DltC-Ppant and DltB mutants.

a, SEC profile of DltB. DltB can be purified to homogeneity in most detergents and is well-behaved during SEC. b, SDS–PAGE and SEC profile of DltC. c, Mass spectrometry analysis of DltC species. This indicates that purified DltC has a molecular mass of 9,590 Da, which is equal to the calculated molecular mass of Ppant-modified DltC, referred to as DltC-Ppant. d, SEC profile of wild-type and mutant DltB proteins. DltB mutants including V305D/I306D, S293A, H289A and H336A are properly folded, as they migrate predominantly as a monomeric peak, similar to wild-type DltB.

Extended Data Fig. 3 Electron density map of DltB.

a, Stereo experimental electron density map, using phases derived from an Au-SAD phasing (Extended Data Table 1). This 2FoFc map is contoured at 1.0σ. DltB backbone tracing is shown in red. b, The final 2FoFc electron density map of the crystal form II (Extended Data Table 1). This map is contoured at 1.0σ, shown in stereo and in an orientation approximately looking down the funnel. The catalytic His336 as well as His289 (another conserved residue (either His or Asn) among MBOAT proteins) are labelled. Both His336 and His289 are located at the bottom of the extracellular funnel, and sandwich the top opening of the transmembrane tunnel.

Extended Data Fig. 4 Stereo view of DltB structure, and an extracellular ‘ring’ of DltB residues associated with a switch of pathogen host.

a, The ‘front’ side view of DltB (stereo view is provided). b, The ‘top’ view of DltB, looking from the extracellular space (stereo view is provided). The His336 side chain is shown as sticks. The extracellular funnel is clear at this angle. c, Cartoon illustration of the N- and C-ridges of DltB in two orthogonal views. d, Locations of pathogen-host-sensitive sites in S. aureus DltB (I2, V61, T113, H121, I227, Q231, Y247, Y250, Y346, G401 and K402) are labelled with red balls in corresponding residues of the S. thermophilus DltB structure. It is clear that all 11 sites are located at the apex of the extracellular ridge of DltB. S. aureus DltB T113 is not conserved and does not have a corresponding residue in other DltBs (see Extended Data Fig. 5): here, the position of its closest residue is labelled. The intracellular DltC is shown in magenta. The DltB structure in these two panels are related with a 45° rotation.

Extended Data Fig. 5 DltB sequence alignment.

DltB sequences of representatives from 10 different genera of Gram-positive bacteria were chosen for sequence alignment using the T-Coffee server. Secondary structural elements of DltB are indicated above the alignment. Residues that form the funnel are identified by purple squares, and residues that form the tunnel are identified with dark red dots. DltB residues involved in direct interaction with DltC are indicated with orange inverted triangles. Residues corresponding to the three sites for which single-point mutations desensitize S. aureus to inhibition by m-AMSA are indicated with blue triangles. Residues of S. aureus DltB, the mutation of which alter the host preference from being human-specific to being capable of infecting rabbits, are indicated with green diamonds. A red star highlights the histidine residue that is completely conserved among MBOATs. ST, S. thermophilus; BS, B. subtilis; LC, L. casei; SA, S. aureus; Lm, Listeria monocytogenes; EF, Enterococcus faecalis; CD, Clostridioides difficile; LM, Leuconostoc mesenteroides; LS, Lysinibacillus sphaericus; BT, Brochothrix thermosphacta.

Extended Data Fig. 6 GST pull-down and Octet assays for analysis of the interaction between DltB and DltC-Ppant.

a, Results of using wild-type GST–DltC to pull-down either wild-type or mutant DltB, with GST to pull-down wild-type DltB as a negative control. Lanes 1–5 show inputs in this experiment. Pull-down results demonstrate that DltB and DltC can form a stable complex at an almost 1:1 molar ratio. DltB(V305D) loses most of its capacity to bind to wild-type GST–DltC, whereas the binding between DltB and DltC was completely abolished with the double mutant DltB(V305D/I306D). b, Results of using wild-type or mutant GST–DltC to pull-down wild-type DltB. Lanes 1–5 show inputs in this experiment. The mutant GST–DltC(V39D) runs slightly slower than wild-type GST–DltC and GST–DltC(V39R) on SDS–PAGE. Both GST–DltC(V39D) and GST–DltC(V39R) lost most of their capacity to bind with wild-type DltB. Pull-down experiments were performed at least twice technically, with the same results. c. Binding-affinity measurements for DltB and DltC using the Octet technique. Wild-type GST–DltC-Ppant and GST–DltC(S35A) show similar binding affinities with wild-type DltB. Data are shown in blue, with the corresponding fits in red. The DltB concentration gradient used here is: 0.03 µM, 0.1 µM, 0.3 µM, 1 µM, 3 µM, 10 µM. Octet assays were performed twice technically. d, Summary of Octet binding assay. Wild-type DltC and GST–DltC(S35A) show similar binding affinities to wild-type DltB. Mean Kd values and s.d. are shown for each assay. Mutation of residues on the binding surface of either DltB or DltC can reduce or abolish their binding.

Extended Data Fig. 7 Structural details of the DltB–DltC interface and the DltB tunnel.

a, Superposition of crystal structures of DltB and the DltB–DltC complex. There is no significant conformational change in DltB upon the binding of DltC-Ppant. b, Cylinder illustration of the DltB–DltC-Ppant complex, viewed from the bottom of the DltB tunnel. DltB is coloured in rainbow, with DltC in purple. c, Conservation of the DltB tunnel region. Residues involved in tunnel formation are also highly conserved among DltB proteins from different species (Extended Data Fig. 5). d, Stereo view of the DltB tunnel and residues forming this tunnel. The tunnel is formed by three helices from the C-ridge (H13, H14 and H15) and the short H12 helix. Residues involved in tunnel formation in our structures are: Lys282, Trp285, Asn286, Ser293, Phe294, Phe296, Arg297, Phe301, Met302, Tyr325, Asn328, Met329, Met332, Leu353, and His336 (which is also involved in the formation of extracellular funnel).

Extended Data Fig. 8 Survival and LTA d-alanylation assays for wild-type and mutant DltB.

a, Lysozyme susceptibility survival assay. For DltB residues used in both LTA d-alanylation and survival assays, corresponding DltB residue numbers in two species are listed. The endogenous dlt operon was deleted in the B. subtilis strain and complemented with an ectopic copy of the wild-type dlt operon without tag on DltB. Representative images of serial dilutions of cells plated on LB agar (left) and LB agar supplemented with 30 µg ml−1 of lysozyme (right). The genotype of the dltB gene is indicated above the corresponding column of serial dilutions. Dilutions of cells are indicated on the y axis. Mutation of the critical histidine (His328) and residues of DltB involved in binding with DltC(V297/F298) increase the susceptibility to lysozyme of B. subtilis. b, Per cent survival of B. bacillus variants towards lysozyme treatment. This was calculated by dividing the colony-forming units (CFUs) from lysozyme plates by the CFUs from LB-only plates. Data are mean ± s.d. of three biological replicates. The genotype of dltB is indicated at the bottom. B. subtilis strains containing untagged DltB show a similar lysozyme susceptibility pattern to those containing Flag-tagged DltB. c, LTA d-alanylation assay. In experiment 1, the assay time was 120 min after 14C-d-alanine was added, whereas for experiments 2 and 3, the assay time was 30 min. Experiments 2 and 3 are two parallel assays for LTA d-alanylation detection. AMSA represents m-AMSA, a DltB inhibitor.

Extended Data Fig. 9 Comparison and rationalization of topological data.

a, Comparison of HHAT topology data with the DltB structure. b, Comparison of GOAT topology data with the DltB structure. In both panels, secondary structures above DltB sequences are generated from our DltB crystal structure. Reported topology assignments of HHAT and GOAT were achieved using human proteins. Here we highlighted the predicted HHAT or GOAT transmembrane helices for each protein with yellow background within sequences. Residues for human HHAT and GOAT that were experimentally verified to be located on the cytoplasmic side are coloured in red, and residues which are on the lumenal side are coloured in green. Helices and/or loops that are predicted to be associated with the membrane surface or buried halfway within the membrane on the cytoplasmic side are indicated with red and magenta rectangles, respectively. It is clear that the regions corresponding to DltB H7–H14 are topologically more conserved than those forming the DltB N- and C-ridges.

Extended Data Table 1 Data collection, phasing and refinement statistics

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Ma, D., Wang, Z., Merrikh, C.N. et al. Crystal structure of a membrane-bound O-acyltransferase. Nature 562, 286–290 (2018).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


  • Membrane-bound O-acyltransferase (MBOAT)
  • Hedgehog Acyltransferase (HHAT)
  • Funnel Structure
  • Ghrelin O-acyltransferase (GOAT)
  • Acyl-coenzyme A:cholesterol Acyltransferase (ACAT)

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing