Protein-Protein interactions and conformational changes of an ATP-binding-cassette (ABC) transporter: the maltose transport system of Salmonella typhimurium
Final Report Abstract
ATP-binding cassette (ABC) transporters are membrane proteins that mediate the uptake or export of an enormous variety of substrates at the expense of ATP. They are found in all organisms from bacteria to man and dysfunction is often associated with disease. ABC transporters share a common modular organization comprising two transmembrane domains (TMDs) that form the translocation pore and two nucleotide-binding domains (NBDs) that hydrolyse ATP. Canonical ABC import systems require an additional protein component, an extracytoplasmic solute binding protein for function. It is suggested that alternating access of the translocation pore to the intra- and extracellular space achieves a net transport of substrate. The alternation of the TMDs between an outward-facing and an inward-facing conformation is energized by the NBDs’ catalytic cycle. The latter comprises NBD-dimer closure upon ATP-binding, hydrolysis of ATP in the closed conformer and reopening towards a semiclosed, ADP-bound state. The maltose ABC-importer of Escherichia coli/Salmonella typhimurium consists of two transmembrane subunits, MalF and MalG, a homodimer of the nucleotide-binding subunits, MalK2, and the periplasmic maltose-binding protein, MalE. The transporter is specifically inhibited by unphosphorylated enzyme IIAGlc of the glucose-specific phosphotransferase system in the context of global carbon regulation of enterobacteria (‘inducer exclusion’). The wealth of available biochemical, biophysical and structural data has made the maltose transporter a paradigm for binding protein-dependent ABC import systems. We have gained further insight into the dynamics of the system by mapping conformational changes of each of the subunits that are associated with the individual steps of substrate translocation. The methodology to achieve this goal included peptide arrays, site-specific chemical cross-linking, fluorescence spectroscopy, NMR and continuous wave and pulsed EPR. Our results point to a crucial role of the large periplasmic loop (P2) of MalF in communicating availability of substrate to the ATP-hydrolysing subunits, thereby initiating the transport cycle. These and other data have led to a more detailed model of maltose transport which also has implications for binding protein-dependent ABC transporters in general. Furthermore, we propose that inhibition of the transporter by EIIAGlc is caused by blocking dissociation of ADP from the MalK subunits.
Publications
- (2006) ATP induces conformational changes of periplasmic loop regions of the maltose ATP-binding cassette transporter. J. Biol. Chem. 281, 3856-3865
Daus, M.L., Landmesser, H., Schlosser, A., Müller, P., Herrmann, A., and Schneider, E.
- (2006) Topography of the surface of the signal transducing protein EIIAGlc that interacts with the MalK subunits of the maltose ATP-binding cassette transporter (MalFGK2) of Salmonella typhimurium. J. Biol. Chem. 281, 12833-12840
Blüschke, B., Volkmer-Engert, R., and Schneider, E.
- (2007) ATP-driven MalK dimer closure and re-opening and conformational changes of the ‘EAA’ motifs are crucial for function of the maltose ATP- binding cassette transporter (MalFGK2). J. Biol. Chem. 282, 22387-22396
Daus, M. L., Grote, M., Müller, P., Doebber, M., Herrmann, A., Steinhoff, H.-J. Dassa, E., and Schneider, E.
- (2008) A comparative EPR study of the nucleotide-binding domains’ catalytic cycle in the assembled maltose ABC-importer. Biophys. J. 95, 2924-2938
Grote, M., Bordignon, E., Polyhach, Y., Jeschke, G., Steinhoff, H.-J., and Schneider, E.
- (2009) The MalF-P2 loop of the ATP-binding cassette (ABC) transporter MalFGK2 from Escherichia coli / Salmonella enterica serovar Typhimurium interacts with maltose binding protein (MalE) throughout the catalytic cycle. J. Bacteriol., 191, 754-761
Daus, M.L. Grote, M., and Schneider, E.
- (2009) Transmembrane signaling in the maltose ABC transporter MalFGK2-E. Periplasmic MalF-P2 loop communicates substrate availability to the ATP-bound MalK dimer. J. Biol. Chem. 284, 17521-17526
Grote, M., Polyhach, Y., Jeschke, G., Steinhoff, H.-J., Schneider, E., and Bordignon, E.
- The periplasmic loop P2 of the MalF subunit of the maltose ATP binding cassette transporter is sufficient to bind the maltose binding protein MalE. Biochemistry 48, 2216- 2225
Jacso, T., Grote, M., Daus, M.L., Schmieder, P., Keller, S., Schneider, E., and Reif, B.
