Publications

• Structural basis of Na(+)-independent and cooperative substrate/product antiport in CaiT.

  Schulze S., Koster S., Geldmacher U., Terwisscha van Scheltinga A.C., Kuhlbrandt W.

  Transport of solutes across biological membranes is performed by specialized secondary transport proteins in the lipid bilayer, 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-A and fr ... >> More

  Transport of solutes across biological membranes is performed by specialized secondary transport proteins in the lipid bilayer, 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-A and from Escherichia coli (EcCaiT) at 3.5-A 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(+) 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(+) 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 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 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. << Less

  Nature 467:233-236(2010) [PubMed] [EuropePMC]

• CaiT of Escherichia coli, a new transporter catalyzing L-carnitine/gamma-butyrobetaine exchange.

  Jung H., Buchholz M., Clausen J., Nietschke M., Revermann A., Schmid R., Jung K.

  l-Carnitine is essential for beta-oxidation of fatty acids in mitochondria. Bacterial metabolic pathways are used for the production of this medically important compound. Here, we report the first detailed functional characterization of the caiT gene product, a putative transport protein whose fun ... >> More

  l-Carnitine is essential for beta-oxidation of fatty acids in mitochondria. Bacterial metabolic pathways are used for the production of this medically important compound. Here, we report the first detailed functional characterization of the caiT gene product, a putative transport protein whose function is required for l-carnitine conversion in Escherichia coli. The caiT gene was overexpressed in E. coli, and the gene product was purified by affinity chromatography and reconstituted into proteoliposomes. Functional analyses with intact cells and proteoliposomes demonstrated that CaiT is able to catalyze the exchange of l-carnitine for gamma-butyrobetaine, the excreted end product of l-carnitine conversion in E. coli, and related betaines. Electrochemical ion gradients did not significantly stimulate l-carnitine uptake. Analysis of l-carnitine counterflow yielded an apparent external K(m) of 105 microm and a turnover number of 5.5 s(-1). Contrary to related proteins, CaiT activity was not modulated by osmotic stress. l-Carnitine binding to CaiT increased the protein fluorescence and caused a red shift in the emission maximum, an observation explained by ligand-induced conformational alterations. The fluorescence effect was specific for betaine structures, for which the distance between trimethylammonium and carboxyl groups proved to be crucial for affinity. Taken together, the results suggest that CaiT functions as an exchanger (antiporter) for l-carnitine and gamma-butyrobetaine according to the substrate/product antiport principle. << Less

  J. Biol. Chem. 277:39251-39258(2002) [PubMed] [EuropePMC]

• Crystal structure of the carnitine transporter and insights into the antiport mechanism.

  Tang L., Bai L., Wang W.H., Jiang T.

  CaiT is a membrane antiporter that catalyzes the exchange of L-carnitine with gamma-butyrobetaine across the Escherichia coli membrane. To obtain structural insights into the antiport mechanism, we solved the crystal structure of CaiT at a resolution of 3.15 A. We crystallized CaiT as a homotrimer ... >> More

  CaiT is a membrane antiporter that catalyzes the exchange of L-carnitine with gamma-butyrobetaine across the Escherichia coli membrane. To obtain structural insights into the antiport mechanism, we solved the crystal structure of CaiT at a resolution of 3.15 A. We crystallized CaiT as a homotrimer complex, in which each protomer contained 12 transmembrane helices and 4 l-carnitine molecules outlining the transport pathway across the membrane. Mutagenesis studies revealed a primary binding site at the center of the protein and a secondary substrate-binding site at the bottom of the intracellular vestibule. These results, together with the insights obtained from structural comparison with structurally homologous transporters, provide mechanistic insights into the association between substrate translocation and the conformational changes of CaiT. << Less

  Nat. Struct. Mol. Biol. 17:492-496(2010) [PubMed] [EuropePMC]

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