Structure of a proton-dependent lipid transporter involved in lipoteichoic acids biosynthesis

Abstract

Lipoteichoic acids (LTAs) are essential cell-wall components in Gram-positive bacteria, including the human pathogen Staphylococcus aureus, contributing to cell adhesion, cell division and antibiotic resistance. Genetic evidence has suggested that LtaA is the flippase that mediates the translocation of the lipid-linked disaccharide that anchors LTA to the cell membrane, a rate-limiting step in S. aureus LTA biogenesis. Here, we present the structure of LtaA, describe its flipping mechanism and show its functional relevance for S. aureus fitness. We demonstrate that LtaA is a proton-coupled antiporter flippase that contributes to S. aureus survival under physiological acidic conditions. Our results provide foundations for the development of new strategies to counteract S. aureus infections.

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Fig. 1: LtaA-catalyzed anchor-LLD flipping.
Fig. 2: S. aureus LtaA structure.
Fig. 3: Amphiphilic cavity characterization.
Fig. 4: LtaA proton coupling and S. aureusltaA growth under acidic conditions.
Fig. 5: Morphology of S. aureus NCTC8325 WT and ∆ltaA mutant, and LTA abundance.
Fig. 6: LtaA anchor-LLD flipping mechanism.

Data availability

Atomic coordinates have been deposited in the Protein Data Bank under accession code PDB 6S7V. Source data for Figs. 1c, 3b,c,e,f, 4a–c and 5c and Extended Fig. 1 are available with the paper online.

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Acknowledgements

We thank the staff at the PX beamline of the Swiss Light Source, Switzerland. We thank G. Cebrero and N. Bärland for providing a control transporter sample. We thank J. Daraspe and M. Rengifo for contributing to TEM images acquisition. We thank U. Lanner, A. Schmidt and T. Müntener for contributing to HPLC–MS and PRM MS studies. This work was supported by the Swiss National Science Foundation (SNSF) (PP00P3_170607 to C.P and 31003A_172861 to J.W.V.). Further funding came from a JPIAMR grant (40AR40_185533 to J.W.V.) and ERC consolidator grant 771534-PneumoCaTChER (to J.W.V). E.L. was funded by the Biozentrum International PhD Program.

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Contributions

B.Z. performed purification and crystallization of LtaA. C.P. assisted B.Z. during data collection, structure determination and docking analysis. B.Z., E.L. and C.P. established and performed in vitro flipping assays. C.P., B.Z. and E.L. analyzed the structural and in vitro functional data. E.L. performed reaction products characterization. X.L. and E.L. performed experiments in live cells. X.L, E.L, C.P. and J.W.-V. analyzed in vivo data. G.M. and S.H. performed NMR analysis. C.P. conceived the project and wrote the manuscript with input from all authors.

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Correspondence to Camilo Perez.

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Peer review information Katarzyna Marcinkiewicz was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Fluorescence quenching analysis of protein-free liposomes.

Representative traces of quenching of liposomes containing NBD-anchor-LLD or NBD-DAG (n ≥ 3). Asterisk marks addition of dithionite. Source data are available with the paper online. F correspond to the fluorescence intensity measured for each time point. Fmax is the average fluorescence measured during the first 200 seconds.

Extended Data Fig. 2 S. aureus LtaA crystallization.

a, SDS-PAGE of samples from different steps of a LtaA purification experiment. Purified protein after cleavage of the His10-tag was used for crystallization. b, Size exclusion chromatography profile of purified LtaA (Superdex 200 10/300 Increase). Gray arrow indicates column void. c, Representative X-ray diffraction images of a LtaA crystal before in situ annealing (left) and after in situ annealing (right). The difference in unit cell dimensions before and after in situ annealing demonstrate shrinking of the unit cell. d, Stereo view (wall-eyed) of the 2Fo-Fc electron density map of the 3.3Å structure of LtaA at 1.0σ level.

Extended Data Fig. 3 2Fo-Fc electron density map.

Individual transmembrane segments of the 3.3Å structure of LtaA at 1.0σ level are shown.

Extended Data Fig. 4 Validation of side-chain register of LtaA model.

a and b, Anomalous electron density map define selenomethionine (SeMet) sites. Contour levels is 4.0σ. Anomalous density was observed for 16 out of 19 SeMet residues in LtaA.

Extended Data Fig. 5 LtaA structure analysis.

a, Overall structure of LtaA. The N-terminal domain is shown in light-orange, C-terminal domain is shown in light-blue, the cytoplasmic helical loop connecting the N-terminal and C-terminal domains is shown in gray. b, Vacuum electrostatic surface representation of LtaA showing side views of the protein. c, Top view of LtaA showing residues participating in the motif-G sequence (G345(X)8G(X)3GP(X)2GG363) in TM11 and motif-G-like sequence in TM5.d, Cytoplasmic view of LtaA showing TMs and loops blocking the access to the central cavity.

Extended Data Fig. 6 Sequence conservation analysis.

A multiple sequence alignment of 76 LtaA homologues found in related Staphylococcus species or other Gram-positive bacteria was generated. Top view of LtaA, residues in N-terminal and C-terminal cavity are colored by sequence conservation (ConSurf server).

Extended Data Fig. 7 Docking analysis and structures of compounds used in this study.

a, Models of lipid-linked-disaccharide docked into the amphiphilic cavity of LtaA. Lipid-linked-disaccharide is shown in black and red sticks. Green surface shows the amphiphilic central cavity of LtaA. b, Structures of disaccharides and Anchor-LLD (β-D-Glc-(1→6)-β-D-Glc-(1→3)-diacylglycerol).

Extended Data Fig. 8 Liquid chromatography mass spectrometry (LC-MS) analysis of relative abundance of LtaA and variants in S. aureus membranes.

Chromatographic separation of peptides was carried out using an EASY nano-LC 1000 system. Mass spectrometry analysis was performed on a Q-Exactive mass spectrometer equipped with a nanoelectrospray ion source. Three peptides ions of LtaA, LTNYNTRPVK (2+ and 3+ ion) and MQDSSLNNYANHK (2+) could be confidently identified and were used for label-free parallel reaction monitoring (PRM) quantification. The integrated peak areas of the 3 peptide ions quantified by PRM were summed and employed for LtaA quantification. The histogram shows relative abundances of LtaA and variants from independent experiments (n = 3).

Extended Data Fig. 9 Phenotypes of S. aureus WT, ∆ltaA, and LtaA mutants.

a, Over-expression of LtaA mutants. S. aureus strain NCTC8325 growth on C+Y agar plates in the presence of 0.1 mM IPTG incubated at 37 °C and 5% CO2. b, Over-expression of LtaA mutants. S. aureus strain NCTC8325 growth on C+Y agar plates in the presence of 0.1 mM IPTG incubated at 37 °C under different pH conditions. LtaA WT represents ∆ltaA mutant complemented with wild type ltaA on pLOW vector; Ctrl vector indicates ∆ltaA mutant complemented with pLOW carrying a functionally unrelated gene as vehicle control; the other labels represent ∆ltaA mutant complemented with ltaA with corresponding point mutations. c, Transmission electron microscopy (TEM) images at low magnification showing the morphology of S. aureus NCTC8325 WT and ∆ltaA mutant.

Extended Data Fig. 10 LtaA-catalyzed lipid-linked-disaccharide flipping and proton gradients.

a, Representative traces of flipping assays with a control transporter (bacterial choline transporter) in the presence of different proton gradients, in and out denote pH of buffer inside and outside of liposomes, respectively (n ≥ 3). Asterisk marks addition of dithionite. F correspond to the fluorescence intensity measured for each time point. Fmax is the average fluorescence measured during the first 200 seconds. b, Scheme of LtaA-catalyzed lipid-linked-disaccharide flipping under an outward proton gradient (top), no gradient (center), and an inward proton gradient (bottom). Under application of a pH gradient (H+), LtaA (yellow boxes) translocates NBD-anchor-LLD (red spheres) contrary to the proton gradient. Addition of dithionite (dit.) then reduces exposed and exchanged NBD-anchor-LLD (black spheres). The extent of quenching is in accordance to the direction of the pH gradient. Full fluorescence quenching will be achieved after prolonged incubation (dashed arrows).

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Zhang, B., Liu, X., Lambert, E. et al. Structure of a proton-dependent lipid transporter involved in lipoteichoic acids biosynthesis. Nat Struct Mol Biol 27, 561–569 (2020). https://doi.org/10.1038/s41594-020-0425-5

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