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Crystal structure of a phosphorylation-coupled vitamin C transporter

Nature Structural & Molecular Biology volume 22, pages 238–241 (2015)Cite this article

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Abstract

Bacteria use vitamin C (L-ascorbic acid) as a carbon source under anaerobic conditions. The phosphoenolpyruvate-dependent phosphotransferase system (PTS), comprising a transporter (UlaA), a IIB-like enzyme (UlaB) and a IIA-like enzyme (UlaC), is required for the anaerobic uptake of vitamin C and its phosphorylation to L-ascorbate 6-phosphate. Here, we present the crystal structures of vitamin C–bound UlaA from Escherichia coli in two conformations at 1.65-Å and 2.35-Å resolution. UlaA forms a homodimer and exhibits a new fold. Each UlaA protomer consists of 11 transmembrane segments arranged into a ‘V-motif’ domain and a ‘core’ domain. The V motifs form the interface between the two protomers, and the core-domain residues coordinate vitamin C. The alternating access of the substrate from the opposite side of the cell membrane may be achieved through rigid-body rotation of the core relative to the V motif.

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Figure 1: Overall structure of UlaA and vitamin C coordination.
Figure 2: Accessibility of the ligand-binding site in the outward-open and occluded states.
Figure 3: Speculative model of the UlaA transport activity.

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Acknowledgements

We thank Shanghai Synchrotron Radiation Source for access to beamline BL17U, SPring-8 for access to beamline BL41XU and the Brookhaven National Synchrotron Light Source for access to beamline X29A. We thank N. Yan and E. Coutavas for discussion and comments on the manuscript and D. King (University of California, Berkeley) for MS analysis. This work was supported by funds from the Ministry of Science and Technology of China (grant nos. 2011CB911102 and 2015CB910104), Tsinghua University 985 Phase II funds, National Natural Science Foundation of China (31321062) and Beijing Municipal Commissions of Education and Science and Technology to J.W. We acknowledge the China National Center for Protein Sciences, Beijing, for providing the facility support. X.L. is supported as the Gordon and Betty Moore Foundation Fellow of the Life Sciences Research Foundation.

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Author notes

  1. Ping Luo and Xinzhe Yu: These authors contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Life Sciences, Tsinghua University, Beijing, China

    Ping Luo, Weiguang Wang & Jiawei Wang

  2. Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China

    Ping Luo, Xinzhe Yu, Weiguang Wang, Shilong Fan & Jiawei Wang

  3. Ministry of Education Key Laboratory of Protein Science, School of Life Sciences, Tsinghua University, Beijing, China

    Xinzhe Yu

  4. Laboratory of Cell Biology, Rockefeller University, New York, New York, USA

    Xiaochun Li

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Contributions

P.L., X.Y., X.L. and J.W. designed all experiments. P.L., X.Y., W.W., S.F. and X.L. performed the experiments. All authors analyzed the data. P.L., X.Y., X.L. and J.W. contributed to manuscript preparation. J.W. wrote the manuscript.

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Correspondence to Xiaochun Li or Jiawei Wang.

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Integrated supplementary information

Supplementary Figure 1 Sequence alignment of E. coli UlaA with homologs from other species and organisms and E. coli UlaA topology diagram.

(a) Secondary structural elements of UlaA are indicated above the sequence alignment. Conserved amino acids are colored red and yellow in decreasing degrees of conservation, and those only conserved in TM11 of various UlaAs, not including VpeC, are colored green. UlaA is spatially organized into “V motif 1” (cyan), “Core 1” (yellow), “V motif 2” (Magenta), “Core 2” (orange), and “TM11” (gray) subdomains. The listed UlaA homologs include UlaA from Escherichia coli (GI: 606808039), Corynebacterium pseudotuberculosis (GI: 503005968), Streptomyces gancidicus (GI: 493093377), Bacillus licheniformis (GI: 489271278), Vibrio cholerae (GI: 669371971), Pasteurella multocida (GI: 504480874), Mycoplasma pulmonis (GI: 499227462), and VpeC from Escherichia coli (GI: 446267492). In the bacteria, e.g. Vibrio cholera, Pasteurella multocida, and Mycoplasma pulmonis, UlaB domains are fused C-terminal to the UlaA domains. The sequences were aligned with ClustalW (Thompson, J.D., Gibson, T. & Higgins, D.G. Multiple sequence alignment using ClustalW and ClustalX. Current protocols in bioinformatics, 2.3. 1-2.3. 22 (2002)). (b) The helices of UlaA are denoted as cylinders and β-sheet as arrows. The diagram is oriented with the extracellular side on top. The helix coloring scheme is consistent with that used in Fig. 1a. The black lines show the approximate location of the membrane, and the N- and C-termini are labelled. The structural repeats are highlighted by the trapezoids, which emphasize their relative orientation. The TM segments comprising the transporter cores are denoted as “Core 1” and “Core 2”, while those comprising the V motif are denoted as “V motif 1” and “V motif 2”, respectively, as in Fig. 1c.

Supplementary Figure 2 Representative electron density map of UlaA.

The stereo view of the 2Fo-Fc electron density of UlaA in C2 crystal form is shown. The electron density is contoured at 1.5 σ.

Supplementary Figure 3 Electrostatic-potential surface representations of UlaA dimer and superimposition of the two ‘inverted’ structural repeats within UlaA.

(a) The structure of the UlaA dimer is shown in cartoon and electrostatic potential surface representations as viewed within the plane of the membrane, represented as a grey rectangle. UlaA is spatially organized into “V motif 1” (cyan), “Core1” (yellow), “V motif 2” (Magenta), “Core 2” (orange), and “TM11” (gray) subdomains. Vitamin C is shown in ball-and-stick representation. (b) Viewed from the intracellular side of the membrane. (c) Superimposition of “Core 1” and “Core 2” subdomains. (d) Superimposition of “V motif 1” and “V motif 2” subdomains.

Supplementary Figure 4 L-ascorbate coordination by UlaA.

(a) Vitamin C is located in a concave pocket. The “Core 1” and “Core 2” subdomains of UlaA are shown in surface electrostatic potential. (b) The 2Fo-Fc electron density for vitamin C after refinement is contoured at 1.5 σ.

Supplementary Figure 5 Measurement of dissociation constants between UlaA variants and vitamin C by isothermal titration calorimetry (ITC).

Raw data are shown in the upper panels and the integrated heats per injection are plotted in the lower panel. The solid lines through the data are fits to the single site model. (a) Wild-type data. (e) The ligand (6 mM vitamin C) is titrated into buffer as a control. ITC data for UlaA variants with hydrogen-bond contributions are shown in (b-d) and (f-i). Data with van der Waals contributions are shown in (j-l). All the ITC experiments were performed independently three times.

Supplementary Figure 6 Structural changes between two crystal forms.

(a) Structural differences between the outward-open state and occluded state, shown from the periplasm, highlighting the overall rigid-body rotation of the core domain relative to the V motif domain. The disorder region in P21B is indicated by black ellipse. (b) Distances of Cα atoms between the core domains of C2A' and P21B molecules. The color scheme used is the same as that in Fig. 1c.

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Luo, P., Yu, X., Wang, W. et al. Crystal structure of a phosphorylation-coupled vitamin C transporter. Nat Struct Mol Biol 22, 238–241 (2015). https://doi.org/10.1038/nsmb.2975

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