Dynamics of subunit interactions in ECF transporters

• Applicant: Professor Dr. Thomas Eitinger
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2009 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 137770702

Energy-coupling factor (ECF)" transporters are composed of a substrate-specific (S) and a moderately conserved (T) integral membrane protein and of ABC ATPases. In contrast to canonocal ABC importers, ECF systems do not rely on extracytoplasmic solute-binding proteins. The oligomeric composition of A-, T- and S-units, the exact roles of the S- and T-components and the molecular mechanisms that couple dynamic interactions during ATP hydrolysis to substrate translocation are controversial or unknown, respectively. Crystal structure analysis identified three different S-units as substrate-loaded monomers. In vivo investigations, site-directed mutagenesis and crosslinking experiments, on the other hand, favor oligomeric states of the S- and potentially the T-units as the functional in vivo states. The focus of the present application is on biotin transporters (BioYMN). Our previous results favor a BioY dimer as the functional S-unit that has transport (in addition to substrate-binding) activity in its solitary state. An appropriate E. coli reporter strain will be used to clarify eventually the controversially discussed issue of BioY's transport function using a set of BioY homologs. The oligomeric states of the T-unit BioN and of BioY will be analyzed by fluorescence-spectroscopical and –imaging techniques using live cells as well as purified transporter complexes reconstituted in nanodiscs. EPR techniques will be applied to spin-labeled complexes in nanodiscs as a complementary approach. The dynamics of subunit interactions during ATP hydrolysis will be investigated by kinetic analysis, pull-down and EPR experiments. An ongoing structural biology approach in external collaboration aims at solving the 3D structure of an ECF holotransporter as a major step towards an understanding the subunit organization.

• DFG Programme: Research Grants