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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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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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.
The authors declare no competing financial interests.
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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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