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2.A.15 The Betaine/Carnitine/Choline Transporter (BCCT) Family

Proteins of the BCCT family are found in Gram-negative and Gram-positive bacteria and archaea. Their common functional feature is that they all transport molecules with a quaternary ammonium group [R-N (CH3)3]. The BCCT family proteins vary in length between 481 and 706 amino acyl residues and possess 12 putative transmembrane α-helical spanners (TMSs).  The x-ray structures (see next paragraph) reveal two 5 TMS repeats with the total number of TMSs being 10. These porters catalyze bidirectional uniport or are energized by pmf-driven or smf-driven proton or sodium ion symport, respectively, or else by substrate:substrate antiport. Some of these permeases exhibit osmosensory and osmoregulatory properties inherent to their polypeptide chains.

Schulze et al. (2010) reported the structures of the sodium-independent carnitine/butyrobetaine antiporter CaiT from Proteus mirabilis (PmCaiT) at 2.3 Å and from E. coli (EcCaiT) at 3.5 Å resolution. Most members of the BCCT family are Na+- or H+-dependent, whereas EcCaiT is Na+- and H+-independent. The three-dimensional architecture of CaiT resembles that of the Na+-dependent transporters LeuT and BetP, but in CaiT, a methionine sulphur takes the place of the Na+ to coordinate the substrate in the central transport site, accounting for Na+ independence. Both CaiT structures show the fully open, inward-facing conformation, and thus complete the set of functional states that describe the alternating access mechanism. 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 of up to 1.7, indicating that the extracellular site is regulatory. Schulze et al. (2010) proposed a mechanism whereby the occupied regulatory site increases the binding affinity of the transport site and initiates substrate translocation.

Most secondary-active transporters transport their substrates using an electrochemical ion gradient, but the carnitine transporter (CaiT) is an ion-independent, l-carnitine/gamma-butyrobetaine antiporter. Crystal structures of CaiT from E. coli and Proteus mirabilis revealed the inverted five-transmembrane-helix repeat similar to that in the amino acid/Na+ symporter, LeuT. Kalayil et al.(2013) showed that mutations of arginine 262 (R262) made CaiT Na+-dependent with increased transport activity in the presence of a membrane potential, in agreement with substrate/Na+ cotransport. R262 also plays a role in substrate binding by stabilizing the partly unwound TM1' helix.

Modeling CaiT from P. mirabilis in the outward-open and closed states on the corresponding structures of the related symporter BetP revealed alternating orientations of the buried R262 side chain, which mimic sodium binding and unbinding in the Na+-coupled substrate symporters. A similar mechanism may be operative in other Na+/H+-independent transporters, in which a positively

charged amino acid replaces the cotransported cation. The oscillation of the R262 side chain in CaiT indicates how a positive charge triggers the change between outward-open and inward-open conformations (Kalayil et al., 2013). 

The generalized transport reactions catalyzed by members of the BCCT family are:

Substrate (out) + nH+ (out) → Substrate (in) + nH+ (in)

Substrate (out) + Na+ (out) → Substrate (in) + Na+ (in)

Substrate1 (out) + Substrate2 (in) → Substrate1 (in) + Substrate2 (out)

Substrate (out) ⇌ Substrate (in)

Substrate = a quaternary amine

 

This family belongs to the: APC Superfamily.

References associated with 2.A.15 family:

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Chen, C. and G.A. Beattie. (2008). Pseudomonas syringae BetT is a low-affinity choline transporter that is responsible for superior osmoprotection by choline over glycine betaine. J. Bacteriol. 190(8): 2717-2725. 18156257
Eichler, K., F. Bourgis, A. Buchet, H.P. Kleber, and M.A. Mandrand-Berthelot. (1994). Molecular characterization of the cai operon necessary for carnitine metabolism in Escherichia coli. Mol. Microbiol. 13: 775-786. 7815937
Ge, L., C. Perez, I. Waclawska, C. Ziegler, and D.J. Muller. (2011). Locating an extracellular K+-dependent interaction site that modulates betaine-binding of the Na+-coupled betaine symporter BetP. Proc. Natl. Acad. Sci. USA 108: E890-898. 21987793
Gärtner, R.M., C. Perez, C. Koshy, and C. Ziegler. (2011). Role of Bundle Helices in a Regulatory Crosstalk in the Trimeric Betaine Transporter BetP. J. Mol. Biol. 414: 327-336. 22024596
Hohle, T.H. and M.R. O'Brian. (2009). The mntH gene encodes the major Mn2+ transporter in Bradyrhizobium japonicum and is regulated by manganese via the Fur protein. Mol. Microbiol. 72: 399-409. 19298371
Jung, H., M. Buchholz, J. Clausen, M. Nietschke, A. Revermann, R. Schmid, and K. Jung. (2002). CaiT of Escherichia coli, a new transporter catalyzing L-carnitine/γ-butyrobetaine exchange. J. Biol. Chem. 277: 39251-39258. 12163501
Kalayil, S., S. Schulze, and W. Kühlbrandt. (2013). Arginine oscillation explains Na+ independence in the substrate/product antiporter CaiT. Proc. Natl. Acad. Sci. USA 110: 17296-17301. 24101465
Kappes, R., B. Kempf, and E. Bremer. (1996). Three transport systems for the osmoprotectant glycine betaine operate in Bacillus subtilis: Characterization of OpuD. J. Bacteriol. 178: 5071-5079. 8752321
Kempf, B. and E. Bremer. (1998). Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments. Arch. Microbiol. 170: 319-330. 9818351
Khafizov, K., C. Perez, C. Koshy, M. Quick, K. Fendler, C. Ziegler, and L.R. Forrest. (2012). Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP. Proc. Natl. Acad. Sci. USA. [Epub: Ahead of Print] 23047697
Lamark, T., I. Kaasen, M.W. Eshoo, P. Falkenberg, J. McDougall, and A.R. Strom. (1991). DNA sequence and analysis of the betgenes encoding the osmoregulatory choline-glycine betaine pathway of Escherichia coli. Mol. Microbiol. 5: 1049-1064. 1956285
Peter, H., B. Weil, A. Burkovski, R. Krämer, and S. Morbach. (1998). Corynebacterium glutamicumis equipped with four secondary carriers for compatible solutes: identification, sequencing, and characterization of the proline/ectoine uptake system, ProP, and the ectoine/proline/glycine betaine carrier, EctP. J. Bacteriol. 180: 6005-6012. 9811661
Ressl, S., A.C. Terwisscha van Scheltinga, C. Vonrhein, V. Ott, and C. Ziegler. (2009). Molecular basis of transport and regulation in the Na+/betaine symporter BetP. Nature 458: 47-52. 19262666
Rübenhagen, R., H. Rönsch, H. Jung, R. Krämer, and S. Morbach. (2000). Osmosensor and osmoregulator properties of the betaine carrier BetP from Corynebacterium glutamicumin proteoliposomes. J. Biol. Chem. 275: 735-741. 10625602
Saier, M.H., Jr., B.H. Eng, S. Fard, J. Garg, D.A. Haggerty, W.J. Hutchinson, D.L. Jack, E.C. Lai, H.J. Liu, D.P. Nusinew, A.M. Omar, S.S. Pao, I.T. Paulsen, J.A. Quan, M. Sliwinski, T.-T. Tseng, S. Wachi, and G.B. Young. (1999). Phylogenetic characterization of novel transport protein families revealed by genome analyses. Biochim. Biophys. Acta 1422: 1-56. 10082980
Schulze, S., S. Köster, U. Geldmacher, A.C. Terwisscha van Scheltinga, and W. Kühlbrandt. (2010). Structural basis of Na+-independent and cooperative substrate/product antiport in CaiT. Nature 467: 233-236. 20829798
Tang, L., L. Bai, W.H. Wang, and T. Jiang. (2010). Crystal structure of the carnitine transporter and insights into the antiport mechanism. Nat Struct Mol Biol 17: 492-496. 20357772
Tsai, C.J., K. Khafizov, J. Hakulinen, L.R. Forrest, L.R. Forrest, R. Krämer, W. Kühlbrandt, and C. Ziegler. (2011). Structural asymmetry in a trimeric Na+/betaine symporter, BetP, from Corynebacterium glutamicum. J. Mol. Biol. 407: 368-381. 21281647