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A two-helix motif positions the lysophosphatidic acid acyltransferase active site for catalysis within the membrane bilayer

Nature Structural & Molecular Biology volume 24, pages 666–671 (2017)Cite this article

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Abstract

Phosphatidic acid (PA), the central intermediate in membrane phospholipid synthesis, is generated by two acyltransferases in a pathway conserved in all life forms. The second step in this pathway is catalyzed by 1-acyl-sn-glycerol-3-phosphate acyltransferase, called PlsC in bacteria. Here we present the crystal structure of PlsC from Thermotoga maritima, revealing an unusual hydrophobic/aromatic N-terminal two-helix motif linked to an acyltransferase αβ-domain that contains the catalytic HX4D motif. PlsC dictates the acyl chain composition of the 2-position of phospholipids, and the acyl chain selectivity 'ruler' is an appropriately placed and closed hydrophobic tunnel. We confirmed this by site-directed mutagenesis and membrane composition analysis of Escherichia coli cells that expressed mutant PlsC. Molecular dynamics (MD) simulations showed that the two-helix motif represents a novel substructure that firmly anchors the protein to one leaflet of the membrane. This binding mode allows the PlsC active site to acylate lysophospholipids within the membrane bilayer by using soluble acyl donors.

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Figure 1: Chemical reactions involving LPA acyltransferase (PlsC).
Figure 2: Acyl chain specificity and membrane binding of TmPlsC.
Figure 3: The tertiary structure of TmPlsC.
Figure 4: The active site locale of TmPlsC.
Figure 5: An MD-derived model of TmPlsC bound in the lipid bilayer.

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Acknowledgements

We thank M. Frank for mass spectrometry, and M.-K. Yun for crystallography assistance. Diffraction data were collected at Southeast Regional Collaborative Access Team (SERCAT) beamlines 22-ID and 22-BM at the Advanced Photon Source (APS), Argonne National Laboratory (Argonne, Illinois, USA). Supporting SERCAT institutions may be found at http://www.ser-cat.org/members.html. Use of the APS is supported by the US Department of Energy under Contract No. W-31-109-Eng-38. This work was supported by the NIH (grant GM034496 to C.O.R.), the NCI (Cancer Center core grant CA21765 to St. Jude Children's Research Hospital), and the American Lebanese Syrian Associated Charities (ALSAC).

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Author notes

  1. Rosanna M Robertson

    Present address: US Department of Homeland Security, Science and Technology Directorate, Washington, DC, USA

  2. Stefan Gajewski

    Present address: Nurix, Inc., San Francisco, California, USA

  3. Rosanna M Robertson and Jiangwei Yao: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

    Rosanna M Robertson, Stefan Gajewski, Gyanendra Kumar, Erik W Martin & Stephen W White

  2. Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

    Jiangwei Yao & Charles O Rock

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Contributions

R.M.R., J.Y., C.O.R. and S.W.W. designed the study. R.M.R., S.G. and S.W.W. determined and interpreted the crystal structures. J.Y. and C.O.R. performed and analyzed the biochemical experiments. G.K. performed the modeling and docking studies. E.W.M. performed the MD simulations. All authors contributed to and approved the manuscript.

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Correspondence to Charles O Rock or Stephen W White.

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Integrated supplementary information

Supplementary Figure 1 Complementation of strain SM2-1 with constructs expressing PlsC.

E. coli strain SM2-1 (plsC(Ts)) is defective in PlsC activity. This strain is able to grow at 30 °C, but unable to grow at 42 °C without PlsC complementation. (a) Growth of the temperature-sensitive E. coli strain SM2-1 (plsC(Ts)) transformed with plasmids expressing the empty vector (Control) or PlsC from E. coli (EcPlsC), Chlamydia trachomatis (CtPlsC), or T. maritima (TmPlsC) under the permissive growth condition (30 °C). (b) Growth of the same strain series at the non-permissive temperature (42 °C). (c) Growth of the temperature-sensitive E. coli strain SM2-1 (plsC(Ts)) transformed with plasmids expressing the empty vector (Control) or TmPlsC mutants G25A, G25V, G25L, G25M under the permissive growth condition (30 °C). (d) Growth of the same strain series at the non-permissive temperature (42 °C). Four independent colonies were analyzed for each condition.

Supplementary Figure 2 Acyl chain specificity of PlsC variants.

The temperature-sensitive Escherichia coli strain SM2-1 (plsC(Ts)) was transformed with various PlsC constructs and the PE molecular species were determined in the complemented SM2-1 strains to assess acyl chain selectivity. (a) The 2-position acyl chain composition of strain SM2-1 complemented with E. coli PlsC (EcPlsC), Chlamydia trachomatis PlsC (CtPlsC), Thermotoga maritima PlsC (TmPlsC) and the TmPlsC(G25M) mutant. (b) Molecular species profile of strain SM2-1 complemented with the TmPlsC(G25A) mutant. (c) Molecular species profile of strain SM2-1 complemented with the TmPlsC(G25V) mutant. (d) Molecular species profile of strain SM2-1 complemented with the TmPlsC(G25L) mutant. In (b), (c) and (d), the molecular species containing 16:0 in the 2-position are colored red, while molecular species containing 14:0 in the 2-position are colored blue. In these experiments, the phospholipids from complemented SM2-1 cells were selectively hydrolyzed for the 2-position fatty acid using snake venom phospholipase A2. The hydrolyzed fatty acids were then converted into fatty acid methyl esters and analyzed via gas chromatography. Gas chromatography data are from duplicate biological replicates and the mass spectra shown are representative of duplicate biological replicates.

Supplementary Figure 3 Biochemical characterization of TmPlsC.

(a) TmPlsC was overexpressed and purified by affinity chromatography. Activity versus enzyme concentration was determined for the purified TmPlsC using saturating concentrations of 1-hexadecanoyl-sn-glycero-3-phosphate (16:0-LPA) and [14C]16:0-ACP. Inset, SDS-PAGE shows the purity of TmPlsC. Lane 1, standards; lane 2, TmPlsC. (b) TmPlsC catalyzed 1-acyl-sn-glycerol-3-phosphate acyltransferase activity was compared using the [14C]16:0-ACP or [14C]16:0-CoA as the acyl donor and 16:0-LPA. The TmPlsC was able to use both acyl-donors, with higher activity using [14C]16:0-ACP. (c) The specific activity of the purified wild type TmPlsC, TmPlsC-H1 (missing the N-terminal helix α1), and TmPlsC-H12 (missing both N-terminal helices α1 and α2) proteins. The TmPlsC-H1 protein had a greater than 100 fold decrease in activity compared to the wild type, while the TmPlsC-H12 protein was inactive. The data were derived from duplicate experiments. See Supplementary Data Set 1 for additional information.

Supplementary Figure 4 TmPlsC electron density.

(a) The active site locale. (b) The N-terminal two-helix motif. (c) Putative acyl chain within the specificity tunnel. The electron densities are from 2Fo-Fc maps contoured at 1.4 σ in (a) and 1 σ in (b) and (c).

Supplementary Figure 5 The primary, secondary and tertiary structure of TmPlsC.

(a) The amino acid sequence highlighting the N-terminal two-helix motif (orange), and the ab catalytic domain (light blue α-helices and light green β-strands). Yellow, active site residues; magenta, exposed aromatic residues proposed to interact with the lipid bilayer; blue, basic residues proposed to interact with the membrane phospholipids; pink, residues that line the hydrophobic selectivity tunnel. The four conserved signature AGPAT family motifs are boxed and labeled. The figure was prepared using ENDscript 2 (Robert & Gouet, Nucleic Acids Res 42, W320-4 (2014). (b) AGPAT family motifs in the ab domain. Motifs 1 (light green), 3 (magenta) and 4 (teal) are at the active site, and motif 2 (purple) is in a flexible loop.

Supplementary Figure 6 Structural conservation of the ab-acyltransferase catalytic domain.

(a) PlsC. (b) G3P acyltransferase (GPAT). (c) Mycobacterial PatA. (d) Overlay of the three structures. In each figure, the acyltransferase domain is colored light blue (α-helices) and light green (β-strands and loops), and the different additional α-helical subdomains are colored orange (PlsC), brown (GPAT) and pink (PatA). The catalytic histidine (yellow carbon sticks) shows the common location of the active site.

Supplementary Figure 7 The packing of the N-terminal two-helix motif in the TmPlsC crystal.

In the I23 crystal lattice, the two TmPlsC molecules in the asymmetric unit pack around the three-fold axis via their two-helix motifs to create a double ring trimeric assembly. (a) (Side view) and (b) (Top view). Each trimer in the assembly encloses electron density (blue) that is consistent with bound phospholipid molecules that are aligned around the axis in a fashion that approximates how TmPlsC engages one leaflet of the membrane. The three asymmetric units are colored green, magenta and brown. The central electron density is best resolved in assembly 1, and (c) and (d) show the side and top views and the nine refined phospholipid molecules, three for each TmPlsC molecule. (e) Close up of the electron densities shown in (c) and (d). (f) The electron density for the best resolved phospholipid shown in (e). The electron densities are from 2Fo-Fc maps contoured at 1 σ.

Supplementary Figure 8 Diffusion of TmPlsC in the MD simulation box.

The root mean squared deviation of the protein center of mass from the starting position (after equilibration) was calculated in the xy plane (blue circles) showing diffusion in the membrane. The root mean squared vertical displacement of the protein center of mass versus the starting position (red circles) was calculated and then normalized for displacement of the entire membrane in the simulation box. This plot shows that the protein converged on a defined depth in the membrane.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8. (PDF 1088 kb)

Supplementary Data set 1

Supplementary Data Set 1. (PDF 271 kb)

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Robertson, R., Yao, J., Gajewski, S. et al. A two-helix motif positions the lysophosphatidic acid acyltransferase active site for catalysis within the membrane bilayer. Nat Struct Mol Biol 24, 666–671 (2017). https://doi.org/10.1038/nsmb.3436

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