Crystal structure of a membrane-bound o -acyltransferase nature

Membrane-bound O-acyltransferases (MBOATs) are a superfamily of integral transmembrane enzymes that are found in all kingdoms of life 1. 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 remodelling 2, 3. Other MBOATs, including porcupine, hedgehog acyltransferase and ghrelin acyltransferase, catalyse essential lipid modifications of secreted proteins such as Wnt, hedgehog and ghrelin, respectively 4, 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 bacteria 11, 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.

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.

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.

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 K d 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.

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.