Analysis of coupling between substrate binding and ATP hydrolysis in canonical homo- and heterodimeric amino acid ABC import systems

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Canonical ABC transport systems mediating the uptake of solutes in prokaryotes are composed of an extracytoplasmic solute binding protein, two pore-forming subunits and an ATPase dimer. Our current knowledge on the mechanism of Type I importers is largely based on structural, biochemical and biophysical data of the maltose transporter (MalE-FGK2) of E. coli/Salmonella. Until recently, this was also the only system for which a (single) substrate binding site had been identified within the transmembrane domains. Novel crystal structures of a homo-dimeric amino acid transporter (ArtI-Q2N2) in complex with two substrate molecules raise some important questions concerning the general mechanism of Type I importers: (i) are two substrate molecules required to trigger ATP hydrolysis in a homo-dimeric transporter? (ii) do only homo-dimeric amino acid transporters contain two substrate binding sites or does this hold also for hetero-dimeric systems? and (iii) can a hetero-dimeric transporter be converted into a homo-dimeric system? We propose to address these questions in the next and final funding period as follows, using again the well-characterized hetero-dimeric histidine transporter, HisJ/LAO-QMP2, of S. Typhimurium and the homo-dimeric arginine transporter, ArtJ-(MP)2 of Geobacillus stearothermophilus as model systems. Current investigations on the interactions of the binding protein HisJ and the HisQMP2 transporter during the transport cycle studied by EPR(DEER) spectroscopy will be continued. Furthermore, we will apply site-directed mutagenesis combined with ATPase and transport assays to study the functional consequences of inactivating one of two substrate binding sites in the Art(MP)2 transporter. Complementary, we will analyse the functional consequences of introducing a second binding site (in HisQ) in the HisQMP2 transporter in addition to that proposed for HisM. Finally, by random mutagenesis, we hope to isolate suppressor mutations in HisJ/HisQM that allow homo-dimeric variants (HisM2P2 and HisQ2P2) to transport substrate coupled to ATP hydrolysis. We are confident that the results of these investigations the feasibility of which has been proven by initial experiments, will expand our knowledge on the mechanism of ABC transporters.