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Structural basis of Na+-independent and cooperative substrate/product antiport in CaiT

Nature volume 467, pages 233–236 (2010)Cite this article

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Abstract

Transport of solutes across biological membranes is performed by specialized secondary transport proteins in the lipid bilayer1, and is essential for life. Here we report the structures of the sodium-independent carnitine/butyrobetaine antiporter CaiT from Proteus mirabilis (PmCaiT) at 2.3-Å and from Escherichia coli (EcCaiT) at 3.5-Å resolution. CaiT belongs to the family of betaine/carnitine/choline transporters (BCCT), which are mostly Na+ or H+ dependent, whereas EcCaiT is Na+ and H+ independent2. The three-dimensional architecture of CaiT resembles that of the Na+-dependent transporters LeuT3 and BetP4, but in CaiT a methionine sulphur takes the place of the Na+ ion to coordinate the substrate in the central transport site, accounting for Na+-independent transport. Both CaiT structures show the fully open, inward-facing conformation, and thus complete the set of functional states that describe the alternating access mechanism5. EcCaiT contains two bound butyrobetaine substrate molecules, one in the central transport site, the other in an extracellular binding pocket. In the structure of PmCaiT, a tryptophan side chain occupies the transport site, and access to the extracellular site is blocked. Binding of both substrates to CaiT reconstituted into proteoliposomes is cooperative, with Hill coefficients up to 1.7, indicating that the extracellular site is regulatory. We propose a mechanism whereby the occupied regulatory site increases the binding affinity of the transport site and initiates substrate translocation.

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Figure 1: CaiT structure and bound substrates.
Figure 2: Hydrogen bond networks in PmCaiT.
Figure 3: Substrate transport and binding in CaiT.
Figure 4: Proposed mechanism of cooperative transport.

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Accession codes

Data deposits

Coordinates and structure factors for P. mirabilis CaiT and E. coli CaiT with bound substrate are deposited in the Protein Data Bank under accession numbers 2WSW and 2WSX, respectively..

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Acknowledgements

We thank Ö. Yildiz, T. Barros and R. Wouts for help with computing; S. Ressl for providing the BetP model; K. R Vinothkumar for growing the first crystals of CaiT, J. Hakulinen; F. Joos for help with transport measurements; H. Jung and K. Fendler for discussions; and C. Ziegler and L. Forrest for reading the manuscript. The γ-butyrobetaine used for binding studies was a gift from Lonza (Switzerland). We also thank A. Pauluhn and the X10SA beamline staff at the Swiss Light Source, and the beamline staff at the European Synchrotron Radiation Facility.

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Author notes

  1. Anke C. Terwisscha van Scheltinga

    Present address: Present address: Laboratory of Biophysical Chemistry, Nijenborgh 4, 9747 AG Groningen, The Netherlands.,

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  1. Department of Structural Biology, Max Planck Institute of Biophysics, Max-von-Laue Strasse 3, 60438 Frankfurt am Main, Germany,

    Sabrina Schulze, Stefan Köster, Ulrike Geldmacher, Anke C. Terwisscha van Scheltinga & Werner Kühlbrandt

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Contributions

Experiments were performed by S.S. and U.G.; cloning and mutagenesis was performed by S.K. Crystals were grown by S.S., diffraction data were collected and processed by S.S. and A.C.T.v.S., and the structures were analysed by S.S., A.C.T.v.S. and W.K. S.S. and W.K. wrote the manuscript.

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Correspondence to Werner Kühlbrandt.

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The authors declare no competing financial interests.

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This file contains Supplementary Background Notes, Supplementary Tables 1- 4, Supplementary Figures 1- 9 with legends and additional references. (PDF 22457 kb)

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Schulze, S., Köster, S., Geldmacher, U. et al. Structural basis of Na+-independent and cooperative substrate/product antiport in CaiT. Nature 467, 233–236 (2010). https://doi.org/10.1038/nature09310

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