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Structure and mechanism of an active lipid-linked oligosaccharide flippase

Nature volume 524, pages 433–438 (2015)Cite this article

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Abstract

The flipping of membrane-embedded lipids containing large, polar head groups is slow and energetically unfavourable, and is therefore catalysed by flippases, the mechanisms of which are unknown. A prominent example of a flipping reaction is the translocation of lipid-linked oligosaccharides that serve as donors in N-linked protein glycosylation. In Campylobacter jejuni, this process is catalysed by the ABC transporter PglK. Here we present a mechanism of PglK-catalysed lipid-linked oligosaccharide flipping based on crystal structures in distinct states, a newly devised in vitro flipping assay, and in vivo studies. PglK can adopt inward- and outward-facing conformations in vitro, but only outward-facing states are required for flipping. While the pyrophosphate-oligosaccharide head group of lipid-linked oligosaccharides enters the translocation cavity and interacts with positively charged side chains, the lipidic polyprenyl tail binds and activates the transporter but remains exposed to the lipid bilayer during the reaction. The proposed mechanism is distinct from the classical alternating-access model applied to other transporters.

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Figure 1: In vitro LLO flipping assay.
Figure 2: Structures of PglK in distinct conformations.
Figure 3: Structural and biochemical characterization of the external helix EH.
Figure 4: Analysis of translocation pathway.
Figure 5: Proposed LLO flipping mechanism including five states (circled numbers).

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors were deposited with the RCSB Protein Data Bank (PDB) under accessions 5C78 (apo-inward-1), 5C76 (apo-inward-2) and 5C73 (outward-occluded).

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Acknowledgements

We thank the staff scientists at the PX beamline of the Swiss Light Source for help with data collection, and M. Napiorkowska and A.Ramirez for assistance with PglB assays. This work was supported by the Swiss National Science Foundation (SNF 31003A–146191 to K.P.L. and Transglyco Sinergia program to M.A., J.-L.R. and K.P.L.). C.P. acknowledges support from the ETH postdoctoral fellowship program.

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Author notes

  1. Sabina Gerber

    Present address: † Present address: GlycoVaxyn AG, Grabenstrasse 3, 8952 Schlieren, Switzerland.,

Authors and Affiliations

  1. Institute of Molecular Biology and Biophysics, ETH Zürich, Zürich, CH-8093, Switzerland

    Camilo Perez, Sabina Gerber, Monika Bucher & Kaspar P. Locher

  2. Department of Chemistry and Biochemistry, University of Berne, Berne, CH-3012, Switzerland

    Jérémy Boilevin, Tamis Darbre & Jean-Louis Reymond

  3. Institute of Microbiology, ETH Zürich, Zürich, CH-8093, Switzerland

    Markus Aebi

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Contributions

C.P. determined the structures of PglK, established the in vitro flipping assay, and performed in vivo flipping studies. S.G. crystallized PglK in the apo-inward-2 state, M.B. assisted in expression and purification of PglK. J.B., T.D. and J.-L.R. synthesized LLO analogues. K.P.L., S.G. and C.P. conceived the project. K.P.L., M.A., and C.P. analysed the data. K.P.L. and C.P. wrote the manuscript.

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Correspondence to Kaspar P. Locher.

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Extended data figures and tables

Extended Data Figure 1 Structures of LLOs and chemical analogues used in this study.

Extended Data Figure 2 LLO synthesis and N-glycosylation in C. jejuni.

Red hexagon denotes di-N-acetylbacillosamine; yellow square denotes N-acetylgalactosamine; and blue circle denotes glucose. The LLO polyprenyl tail is shown in purple. A periplasmic polypeptide chain is shown in blue. PglH (red) is the glycosyltransferace used in the in vitro tLLO flipping assays. Und-PP-ase, undecaprenyl pyrophosphatase.

Extended Data Figure 3 Biochemical characterization of wild-type and mutant PglK.

a, In vitro tLLO flipping rates of PglK wild-type and E510Q mutant. Bars indicate initial velocities. b, In vitro tLLO flipping of PglK mutants E510Q, EHm1 and EHm2. +ADP, assay in the presence of 4 mM ADP; −ATP, assay in the absence of ATP. Liposomes, empty liposomes containing only tLLO. ADP alone does not cause tLLO to disappear from the external liposomes leaflet. c, In vivo LLO flipping of wild-type PglK and E510Q mutant expressed in E. coli SCM6 cells containing the C. jejuni pgl operon30. E.V., empty vector, N.I.C., non-induced cells. Anti-glycan refers to HR6 antibody recognizing the N-glycan of a substrate protein (see Methods), whereas anti-PglK is used to monitor PglK expression level. d, Determination of Km values of ATP hydrolysis and ATP-driven in vitro tLLO flipping. The black curve represents the ATPase rate of PglK at distinct ATP concentrations. The blue curve represents the initial LLO flipping rate in proteoliposomes at distinct ATP concentrations. Cpm, counts per million. e, ATPase activity in the presence (+) or absence (−) of native LLO of wild-type PglK and E510Q mutant in detergent (LMNG) or proteoliposomes. f, ATPase activity of PglK in detergent in the presence of LLO (20 μM), tLLO (20 μM), diverse drugs (50 μM) and lipid A (20 μM). g, Determination of Km values of ATP hydrolysis in the presence (+) or absence (−) of LLO (20 μM). Stimulation results in higher Vmax, while the Km for ATP remains almost unaltered in the presence (Km(+LLO) = 0.54 ± 0.03 mM) or absence (Km(−LLO) = 0.36 ± 0.04 mM) of native LLO. h, Determination of Km value of ATPase stimulation by native LLO in detergent (rates are normalized against the basal activity in absence of LLO). Error bars denote s.d. (n = 3).

Extended Data Figure 4 Electron density maps.

a, b, Stereo views (cross-eyed) of the 2Fo − Fc electron density maps for the complete PglK dimer of the structures at 2.9 and 3.9 Å, respectively. c, Stereo view of the non-crystallographic symmetry (NCS)-averaged electron density map for the complete PglK dimer of the structure at 5.9 Å and close-up of the NBDs showing the Fo − Fc electron density map for the bound ADP molecules. 2Fo − Fc and NCS maps are shown at 1.0σ level. Fo − Fc maps are shown at 3.0σ level.

Extended Data Figure 5 Validation of side-chain register of outward-occluded PglK model.

a–c, Anomalous electron density maps define selenomethionine (a), cysteine-bound mercury (b) and PtCl4 sites (c). Contour levels are between 4.0 to 5.0σ. In a, anomalous density was observed for 9 out of 10 selenomethionines of PglK.

Extended Data Figure 6 Structural features of PglK.

a, Ribbon diagram of one PglK subunit depicting the secondary structure arrangement, based on the Sav1866 nomenclature36. b, Conformational changes of TM4 and TM5 visualized after superposition of subunits of apo-inward-1 (light orange) and apo-inward-2 (dark orange) structures. c, Structures of the antibacterial peptide ABC exporter McjD in occluded state (PDB code 4PLO), PglK in outward-occluded state, and the ABC exporter Sav1866 (PDB code 2HYD) in outward-open state. Transmembrane helices TM1 and TM3 (purple) define the extent of the external opening. Subunits in each dimer are shown in orange and grey. d, Side and cytoplasmic view cut-off of PglK apo-inward-1 structure and vacuum electrostatic surface representation showing the internal cavity.

Extended Data Figure 7 PglK crystal packing.

a–c, The main crystal contacts of apo-inward-1 (2.9 Å resolution) (a), apo-inward-2 (3.9 Å resolution) (b), and outward-occluded (5.9 Å resolution) (c) states.

Extended Data Figure 8 Putative interactions of native and synthetic LLO analogues with PglK in apo-inward and outward-occluded states.

a, b, Native LLO (a) and synthetic LLO analogues (b). Red hexagon denotes di-N-acetylbacillosamine; yellow square denotes N-acetylgalactosamine; blue circle denotes glucose; and blue square denotes N-acetylglucosamine. The LLO polyprenyl tail is shown in purple.

Extended Data Figure 9 Product analysis and control experiments of PglK-catalysed in vitro flipping assay.

a, Product analysis of tLLO glycosylation catalysed by PglH. The reaction products were analysed by in vitro glycosylation of a fluorescently labelled substrate peptide (DQNAT sequon) catalysed by PglB as reported earlier30,31. Depending on the presence and size of the N-glycan, peptides show a different mobility after Tricine–SDS–PAGE. Bands were visualized using a fluorescence gel scan (488 nm excitation and 526 nm emission). Lane 1, product of the deglycosylation reaction of hexasaccharide LLO catalysed by α-N-acetylgalactosaminidase, which removes terminal GalNAc molecules. This demonstrates the purity of the LLO used with respect to GalNAc. Lane 2, tLLO used in biochemical assays. Lane 3, products of tLLO glycosylation when the tLLO:GalNAc molar ratio is 2:1. Lane 4, products of tLLO glycosylation when the tLLO:GalNAc molar ratio is 1:1. Lane 5, products of tLLO glycosylation when the tLLO:GalNAc molar ratio is 1:10. b, PglK proteoliposomes incubated in the presence of 4 mM ADP. The level of tLLO in the external leaflet after t = 40 min remains unchanged (104.2 ± 10.8%) relative to the amount at t = 0. ADP alone does not cause a decrease in the concentration of tLLO in the external liposomes leaflet, ruling out a potential sequestration of tLLO in the outward-occludded state of PglK. c, Determination of tLLO orientation in proteoliposomes. Disrupted liposomes (0.3% Triton X-100) and non-disrupted proteoliposomes were incubated with PglH in the presence of excess GalNAc. The amount of glycopeptide was determined from band intensities of fluorescence gel scans. 48.2 ± 7.5% of tLLO is located in the outer leaflet of the bilayer. d, Determination of PglK orientation in proteoliposomes. The fully functional mutant PglK(C269L/C352S/C386S/C549L/S544C) was reconstituted in proteoliposomes and labelled with negatively charged Alexa Fluor 488 C5 maleimide. The fluorescence of non-disrupted and disrupted proteoliposomes was compared. 51.8 ± 2.8% of the PglK molecules are oriented with NBDs facing outwards. Red hexagon denotes di-N-acetylbacillosamine; yellow square denotes N-acetylgalactosamine.

Extended Data Table 1 X-ray data collection and refinement statistics
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Perez, C., Gerber, S., Boilevin, J. et al. Structure and mechanism of an active lipid-linked oligosaccharide flippase. Nature 524, 433–438 (2015). https://doi.org/10.1038/nature14953

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