CHARACTERIZING THE ATPase ACTIVITY OF HOMODIMERIC AND HETERODIMERIC ABC TRANSPORTERS USING HIGH FIELD HYPERFINE SPECTROSCOPIES

Multidrug ATP binding cassette (ABC) exporters extrude cytotoxic molecules across cell membranes. The energy for this translocational process, which comes from ATP hydrolysis, is transduced into the motion of the substrate through conformational rearrangements of the protein; this process was examined for specific steps in the ATPase cycle of the transporters by using DEER spectroscopy coupled with site-directed spin labeling to track conformational changes [1]. Here we focus on the ATPase activity of homodimeric (MsbA) and heterodimeric (BmrCD) ABC exporters using Mn²⁺ as a probe, substituting for the essential Mg²⁺ cofactor, and following the coupled ³¹P nuclei using hyperfine spectroscopic techniques, ENDOR and EDNMR. While in MsbA the two monomeric units constituting the transporter are equivalent, both hydrolyzing ATP with the same rate, in BmrCD the sequence of the BmrD monomer shows a modification which involves a catalytic residue of the nucleotide binding domain; this is expected to impair ATP turnover of BmrD with respect to BmrC, resulting in an asymmetric ATP hydrolysis mechanism. The two exporters were first probed in specific states of their ATPase cycle by incubating the proteins with ADP and ATP analogues; ³¹P spectra revealed the hydrolysis state of the nucleoside phosphate [2]. Upon incubation with ATP and subsequent freeze quenching, the ATPase kinetics under single turnover conditions were followed by monitoring the ADP/ATP ratio through ³¹P-EDNMR spectroscopy; this showed that the hydrolysis rate is considerably faster for MsbA.

References

[1] S. Mishra, B. Verhalen, R. A. Stein, P.-C. Wen, E. Tajkhorshid, H. S. Mchaourab, Elife 2014, 3:e02740.

[2] I. Kaminker, A. Sushenko, A. Potapov, S. Daube, B. Akabayov, I. Sagi, D. Goldfarb, J. Am. Chem. Soc. 2011, 133, 15514.