Dynamics and regulation of ABC transporters elucidated by EPR

Abstract

ATP-binding cassette transporters (ABC transporters) harness energy from hydrolyzing ATP to transport many different substrates across cell membranes. ABC transporters occur in all three kingdoms of life and are commonly divided into four families: the type I and type II importers, which appear in prokaryotes only, the exporters, which exist ubiquitously, and the energy coupling factor (ECF) type transporters. ABC importers of type I and II transport lots of different substrates into the cell, with a corresponding substrate binding protein (SBP) being involved. Exporters usually are less substrate speci c than importers and transport products from metabolism or lethal substances outside of the cytosol. Their function makes them very important for cell viability in general and for multidrug resistance properties in particular. All ABC transporters consist of two transmembrane domains (TMDs) which form the translocation pathway and two nucleotide binding domains (NBDs) which hydrolyze ATP and thus deliver the energy for switching the transporter from an inward to an outward facing confirmation or vice versa. Although several high resolution X-ray structures of quite a number of transporters are known, essential characteristics of the transport mechanism, the substrate translocation pathway, conformational changes during the nucleotide cycle, regulatory mechanisms etc. are still not completely understood. In this thesis I provide three case studies performed on one ABC importer (MalFGK2) and two exporters (MsbA and TM287/288) with electron paramagnetic resonance spectroscopy (EPR) in combination with site-directed spin labeling (SDSL) of specific, engineered cysteines in the transporters. The probably best studied importer is the type I maltose importer from E. coli MalFGK2 in conjunction with its corresponding substrate binding protein MalE. Crystal structures of the pretranslocation, ATP- and vanadate trapped state exist and have largely contributed to the general understanding of the transport mechanism. However, the response of the transporter to the presence of liganded or unliganded MalE as well as the stimulation of ATPase activity even if unliganded MalE is present are still hardly understood. In this thesis I present results which show that the transporter binds MalE in the apo- and ADPstate independently on the presence of substrate. Different periplasmic conformations of the transport complex are adopted depending on whether maltose is present or not. In the ATP-state, the NBDs are driven into a fully closed conformation no matter if MalE/maltose or MalE alone are present, triggering the hydrolysis of ATP, thus generating a futile cycle in the latter case. All together, the results could widen the knowledge about the conformational plasticity of the type I ABC transporters. In case of the homodimeric, lipid A and lipopolysaccharides transporter MsbA from E. coli which is homologous to the multidrug exporter LmrA of L. lactis and mammalian multidrug resistance transporters such as P-glycoprotein, three structures showing the transporter in the apo, closed apo and AMP-PNP [Adenosine 5'-(β; γ-imido)triphosphate] state are known. The apo and closed apo structures exist at a rather low resolution, namely only showing the Cα-atoms. The outward facing, AMP-PNP-bound structure is similar to the one of Sav1866. Interestingly, in this thesis I show that a 1:1 stoichiometric binding of an in vitro selected designed ankyrin repeat protein (DARPin) to MsbA was observed. This revealed an unforeseen asymmetric nature of the homodimeric transporter MsbA. In fact, by means of EPR, I show that the binding epitope of the DARPin could be mapped to the TMDs, lying outside of the twofold symmetry axis. Furthermore, no differences concerning the pronounced switch from inward to outward facing conformation in presence of the DARPin were observed. This and additional biochemical experiments showed that the transporter's ability to translocate substrate is not impaired by binding of the DARPin. Moreover, a stimulation of the ATPase activity is observed if DARPins are present, suggesting that the catalytic cycle is accelerated if MsbA is in an asymmetric state. Crystal structures of the heterodimeric ABC exporter TM287/288 from T. maritima showed an asymmetry in the NBDs in the apo and AMP-PNP-bound state. The current model for the transport mechanism of TM287/288 emanates from ATP being bound and hydrolyzed to the consensus site in each transport cycle and ATP being exchanged from time to time at the degenerate site. However, in the presence of AMP-PNP, only one molecule of this nucleotide analog was crystallized bound to the degenerate NBD site, and, in contrast to many other exporters, the two NBDs were found to be in close contact, with the overall structure of the transporter trapped in an inward-facing state. Interestingly, also in the crystal structure obtained without nucleotides, the NBDs were found to be in close contact, in contrast to apo states of other transporters like MsbA or ABCB10. By comparing the apo and the AMP-PNP-bound conformations by EPR experiments performed in frozen solution, the peculiar behaviour of this heterodimeric exporter in the presence and absence of AMP-PNP was confirmed.