Uncoupling substrate transport from ATP hydrolysis in the maltose ATP binding cassette transporter

Jinming Cui, Purdue University

Abstract

The bacterial ATP-binding cassette (ABC) importers mediate nutrient uptake and some can become essential for survival under restricted growth conditions, such as in the human body. Although ABC importers exhibit remarkable versatility in the substrates they can transport, they appear to share a similar multisubunit architecture and mechanism of coupling the energy from ATP hydrolysis to the active transport of substrates across the membrane. The Escherichia coli maltose/maltodextrin transporter, a well characterized model system, consists of a periplasmic maltose binding protein (MBP) and a multisubunit membrane transporter MalFGK2. The structure of the MBP-MalFGK2 complex in the transition state has been determined [Oldham, M. L., et al. (2007) Crystal structure of a catalytic intermediate of the maltose transporter. Nature 450, 515-522], and it revealed that a periplasmic loop of the transmembrane subunit MalG (scoop loop) is inserted into the MBP maltose-binding site, and maltose is bound to the transmembrane maltose-binding site in MalF. To probe the function of the MalG scoop loop and the MalF maltose-binding site, a MalG-scoop mutant, with four residues deleted in the scoop loop, and a MalF-G380D mutant, with a substitution of the Gly 380 residue by Asp at the MalF maltose-binding site, were made. Both mutants generated a novel futile cycling phenotype: while MBP could still stimulate the ATPase activity of MalFGK2, maltose was not transported. The MalG-scoop mutant limited the reach of the loop from MalFGK2 into MBP, allowing maltose to remain in the MBP maltose-binding site. The MalF-G380D mutant contains a side chain substitution in the MalF maltose-binding site that prevented maltose from binding to MalFGK 2. Both mutants still allowed the catalytic cycling of ATP hydrolysis, but disrupted the transfer of maltose from MBP to MalFGK2 during the catalytic cycle. Another feature revealed in the MBP-MalFGK2 crystal is the insertion of the C-terminal tail of MalG into the ATP-binding MalK dimer interface in the cytoplasm. A MalG-tail mutant, with a seven-residue deletion at the C-terminus, was made. This MalG-tail mutant lost all ATPase activity indicating that the MalG tail insertion is important for the formation of the catalytic transition state. The involvement of the scoop loop in the translocation of substrate was further demonstrated by electron paramagnetic resonance (EPR). Spin labels were attached to MBP near the maltose-binding site and mobility changes monitored in response to protein and ligand additions that promoted conformational changes associated with the translocation cycle. The spin labels sensed the insertion of the loop in the wild type and the retention of maltose in the MBP binding site when the loop was shortened in the MalG-scoop mutant. These results enriched our understanding of the coupling mechanism by which substrate is efficiently transferred from the binding protein to the transporter during the catalytic cycle; and offered new insight into the mechanism of transport in ABC systems generally. A better understanding of the mechanism of maltose transport has the potential to impact human health directly because maltodextrin transport has been implicated in virulence in some bacteria. The growth of Streptococcus pneumoniae in the human host has been proposed to be dependent on glycogen breakdown into maltodextrin, followed by uptake by the maltodextrin transporter, an E. coli MalFGK2 homologue [Abbott, D.W., et.al. (2010) The molecular basis of glycogen breakdown and transport in Streptococcus pneumoniae. Mol Microbiol. 77, 183-99]. It may be possible to design inhibitors that disrupt the coupling of the maltodextrin transporter in S. pneumoniae, thereby arrest the bacterial growth. The S. pneumoniae transporter genes have been cloned, and the first inhibitor has been tested.

Degree

Ph.D.

Advisors

Davidson, Purdue University.

Subject Area

Biochemistry

COinS