Structural Mechanism of Action of Binding Protein Independent Mutant MalG511 of Escherichia Coli Maltose Transporter

ATP-binding cassette (ABC) transporters couple the uphill transport of substances to the hydrolysis of ATP. Mutations in the E. coli maltose ABC transporter have been isolated that allow transport in the absence of periplasmic maltose binding protein (MBP), a protein that delivers maltose to the transporter with high affinity and stimulates the ATPase activity of the transporter. One such mutant, MalG511, shows an interesting biphasic behavior with low levels of MBP stimulating transport activity while higher levels (>50µM) inhibit transport activity. Remarkably, the rescuing effect of MBP suppressor mutants isolated at high, inhibitory MBP concentrations provided insight into regulatory mechanisms in the ABC transporter superfamily. Site directed spin labeling EPR spectroscopy was used to follow the catalytic cycle of MalG511 without any binding protein, with MBP and with suppressor MBP by attaching spin label at two positions in the MalK ATPase subunit (V16 and R129) that can be used to detect the closure of nucleotide-binding interface. MalG511 responded differently to the addition of ligands, including nucleotide and MBP, than wild type transporter. Notably, MalG511 can sample all conformational states (open, semi-open and closed interface) in the absence of MBP, as expected for a MBP-independent transporter. However, the closed state could be stabilized only in the presence of a transition state analogue (vanadate) suggesting that the mutations in MalG511 altered the conformational equilibrium of the transporter, favoring a semi-open over a closed conformation, even in the presence of ATP, plus or minus MBP. The addition of MBP, but not suppressor MBP, also resulted in a failure of the transporter to return to the fully open state at the end of a catalytic cycle, suggesting a mechanism for inhibition of transport at high MBP concentration.