Mapping the transport cycle of asymmetric ABC transporters.
EMSL Project ID
50692
Abstract
ATP binding cassette (ABC) transporters play vital roles in maintaining both the composition of cell membranes and the identity of molecules traveling across them. In particular, many ABC transporters have been identified that control the movement of hydrophobic molecules, making them particularly relevant to disease in the case of drug transporters or critical cell wall builders in bacteria. A unique class of ABC transporter, that we term “asymmetric transporters”, remains particularly understudied. These asymmetric transporters are unusual in that they maintain only one functional nucleotide binding domain (NBD) capable of hydrolyzing ATP, instead of the usual two. Here, we focus on two asymmetric bacterial ABC transporters, one from a gram negative bacterium and one from a gram positive bacterium. The first is E. coli TmrAB, a likely mediator of membrane biogenesis. The second is BmrCD - a drug resistance transporter from a gram positive organism. BmrCD was a large focus of our November PNCC data collection which yielded a large dataset and a 3D reconstruction. We now aim to improve the heterogeneity evident in our reconstruction to permit higher resolution reconstructions using a double mutant that will likely lock the transporter in one confirmation. We simultaneously plan to use this mutant to alter and lock BmrCD into an outward open confirmation different than that the one we currently have. Finally, we aim to determine a structure of TmrAB in a delipidated confirmation to assess the structural link between substrate binding and conformational state. The first TmrAB structure was the first published structure of any transporter using new direct electron detectors and was determined in the inward open, substrate bound state. We now seek to expand on this work and characterize the full structural cycle of TmrAB and BmrCD transport as they change confirmation to recognize and deliver cargo. The results from these investigations will give critical insights into this medically important class of transporters, particularly in areas of antibiotic resistance as well as providing insights into targeting processes by which bacteria use to build their protective cellular membranes.
Project Details
Start Date
2019-04-15
End Date
2019-06-13
Status
Closed
Released Data Link
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Principal Investigator
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