Comparative analysis of the dynamics of the binding protein-dependent histidine ABC import system and the ECF-type biotin transporter

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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 transport mechanism is largely based on structural and functional analysis of the maltose transporter of E. coli/Salmonella. Due to some unique features of this system it is questionable whether the derived transport model holds for other type I ABC importers. Thus, we will study conformational changes of the similarly well-characterized histidine transporter, HisJ/LAO-QMP2, of S. typhimurium. We will focus on investigating changes in affinity of the transporter for its cognate binding protein during the transport cycle using microscale thermophoresis and surface plasmon resonance as experimental tools. These studies are aimed to better understand conformational changes of the transmembrane domains that are initiated by the binding protein and in turn trigger ATP hydrolysis at the opposite side of the membrane. Furthermore, by taking advantage of our expertise on canonical ABC importers, we will study conformational changes of the BioYMN transporter, a member of the newly discovered subfamily of ECF-transporters. In particular, we will apply sitedirected chemical cross-linking on cysteine variants incorporated into nano-discs to elucidate the dynamics of the ATPase BioM and the interaction of BioM and BioN dependent on substrate and/or nucleotides.