Probing the ATP Hydrolysis
Cycle of the ABC Multidrug
Transporter LmrA by Pulsed EPR Spectroscopy
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Abstract
Members of the ATP binding cassette (ABC) transporter
superfamily
translocate various types of molecules across the membrane at the
expense of ATP. This requires cycling through a number of catalytic
states. Here, we report conformational changes throughout the catalytic
cycle of LmrA, a homodimeric multidrug ABC transporter from <i>L. lactis.</i> Using site-directed spin labeling and pulsed
electron–electron double resonance (PELDOR/DEER) spectroscopy,
we have probed the reorientation of the nucleotide binding domains
and transmembrane helix 6 which is of particular relevance to drug
binding and part of the dimerization interface. Our data show that
LmrA samples a very large conformational space in its apo state, which
is significantly reduced upon nucleotide binding. ATP binding but
not hydrolysis is required to trigger this conformational change,
which results in a relatively fixed orientation of both the nucleotide
binding domains and transmembrane helices 6. This orientation is maintained
throughout the ATP hydrolysis cycle until the protein cycles back
to its apo state. Our data present strong evidence that switching
between two dynamically and structurally distinct states is required
for substrate translocation