Dynamics and regulation of ABC transporters elucidated by EPR
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2016
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Doctoral Thesis
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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.
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Examiner : Jeschke, Gunnar
Examiner : Bordignon, Enrica
Examiner : Riek, Roland
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ETH Zurich
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Subject
EPR; DEER; ABC transporter; biomolecular structure; membrane protein; nucleotide binding
Organisational unit
03810 - Jeschke, Gunnar / Jeschke, Gunnar