Cysteine accessibility probes timing and extent of NBD separation along the dimer interface in gating CFTR channels.
Bottom Line: Modification while channels were opening and closing in the presence of ATP caused macroscopic CFTR current to decline at the same speed as when the unmodified channels shut upon sudden ATP withdrawal.We conclude that, in every CFTR channel gating cycle, the NBD dimer interface separates simultaneously at both composite sites sufficiently to allow MTS reagents to access both signature-sequence serines.Relatively rapid modification of S1347C channels by larger reagents-MTS-glucose, MTS-biotin, and MTS-rhodamine-demonstrates that, at the noncatalytic composite site, this separation must exceed 8 Å.
Affiliation: The Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, New York, NY 10065.Show MeSH
Related in: MedlinePlus
License 1 - License 2
Mentions: Accumulated functional and structural information has clarified the mechanistic underpinnings of ABC transporter catalytic cycles. Most structural detail has come from homodimeric bacterial transporters, or isolated NBDs, in which the two composite ATP sites are identical. Present evidence suggests that in homodimers, the ATP hydrolysis mechanism is the same in both catalytic sites, and the same in ABC importers (e.g., Oldham and Chen, 2011b) as in exporters (e.g., Smith et al., 2002), in accord with strict conservation of the key NBD sequence motifs. In ATP-bound tight NBD homodimers, each nucleotide contacts Walker A and B sequences in the head of one NBD and the ABC signature sequence, LSGGQ, in the other NBD tail (Fig. 1 F). The Walker A motif, or P loop (Fig. 1, red), curves around, and positions, the ATP phosphate chain; the glutamate immediately following the Walker B hydrophobic residues is the catalytic base that polarizes the attacking water molecule; and the signature sequence (Fig. 1, purple) serine and second glycine both contact the γ phosphate (Hung et al., 1998; Moody et al., 2002; Smith et al., 2002; Chen et al., 2003; Verdon et al., 2003; Dawson and Locher, 2006; Oldham and Chen, 2011b).
Affiliation: The Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, New York, NY 10065.