Abstract
The Escherichia coli MalE-MalFGK2 complex is one of the best characterized members of the large and ubiquitous family of ATP-binding cassette (ABC) transporters. It is composed of a membrane-spanning heterodimer, MalF-MalG; a homodimeric ATPase, MalK2; and a periplasmic maltose receptor, MalE. Opening and closure of MalK2 is coupled to conformational changes in MalF-MalG and the alternate exposition of the substrate-binding site to either side of the membrane. To further define this alternate access mechanism and the impact of ATP, MalE, and maltose on the conformation of the transporter during the transport cycle, we have reconstituted MalFGK2 in nanodiscs and analyzed its conformations under 10 different biochemical conditions using negative stain single-particle EM. EM map results (at 15-25 Å resolution) indicate that binding of ATP to MalK2 promotes an asymmetric, semi-closed conformation in accordance with the low ATPase activity of MalFGK2 In the presence of MalE, the MalK dimer becomes fully closed, gaining the ability to hydrolyze ATP. In the presence of ADP or maltose, MalE·MalFGK2 remains essentially in a semi-closed symmetric conformation, indicating that release of these ligands is required for the return to the initial state. Taken together, this structural information provides a rationale for the stimulation of MalK ATPase activity by MalE as well as by maltose.
Highlights
The majority of the particles (ϳ79%) were in the semi-closed symmetric conformation (Table 1 and Fig. 4E). The dominance of this conformation suggests that release of maltose from the transporter is necessary to permit return of MalK2 to the open resting state. This single-particle EM analysis has defined the conformational changes of MalK2 in response to the three factors—nucleotide, MalE, and maltose—individually and cooperatively
The resolution of the EM maps was between 15 and 25 Å, which is sufficient for the detection of the large scale conformational changes of MalK2
The differences observed between the EM maps are consistent with the differences between simulated maps of MalK2 in the open, closed, and semi-closed conformations (Fig. S6)
Summary
In nanodiscs (as in proteoliposomes), the ATPase activity of MalFGK2 is coupled to MalE and maltose [12]. We have determined the role of nucleotide, MalE, and maltose on the closure and reopening of MalK2 in the context of the full transporter. We have characterized the conformations of MalFGK2 in nanodiscs under 10 different biochemical conditions using single-particle EM combined with a systematic analysis of the conformational heterogeneity in any given states (for a complete summary, see Table 1). Whereas MalK2 can dynamically sample the closed state, complete stabilization in that conformation requires binding of open state MalE. In the presence of ADP or maltose, most of MalE1⁄7MalFGK2 remains in a semiclosed conformation, indicating that release ADP and maltose contributes to the return of the transporter to the resting state
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
