ECF Transporters – A New Class of ATP-Binding Cassette-Containing Transport Systems

ECF (“energy-coupling factor”) transporters are a recently discovered family of membrane transporters, frequently of vitamins, whose members are abundant among prokaryotes including many Gram-positive pathogens with restricted biosynthetic capacities. They consist of highly diverse transmembrane substrate-capture (“S”) proteins that interact with an energizing module composed of a conserved transmembrane protein (“T”) and pairs of ABC domains (“A”). About half of the ECF systems are known or predicted to depend on a dedicated AT-module, but in more than 100 Grampositive bacteria and in certain archaea, up to to 12 different S-components are predicted to compete for the same AT (= EcfAA?T)-module. This shared use of the EcfAA?T module was confirmed experimentally for bacterial folate, pantothenate, riboflavin, and thiamine transporters. In the previous funding period, a major focus was set on basic characterization of a biotin transporter (BioMNY). This work identified BioY as the S-component that functions as a high-capacity transporter in its solitary form but is converted into an ATP-dependent high-affinity system in the presence of BioM (A) and BioN (T). Protocols were developed that allow purification of the solitary BioY, and BioMNY and BioMN complexes in milligram amounts. In the new period, advanced biophysical approaches including electron crystallography, atomic force spectroscopy, fluorescence and EPR techniques will be applied to analyze the mechanism, structure and dynamics of the biotin transporter and related systems with a dedicated AT-module, and to get insight into the molecular principles behind promiscuity among the majority of ECF transporters.