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Cysteine accessibility probes timing and extent of NBD separation along the dimer interface in gating CFTR channels.

Chaves LA, Gadsby DC - J. Gen. Physiol. (2015)

Bottom Line: These results suggest that the target cysteines can be modified only in closed channels; that after modification the attached MTS adduct interferes with ATP-mediated opening; and that modification in the presence of ATP occurs rapidly once channels close, before they can reopen.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.

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Affiliation: The Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, New York, NY 10065.

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Structural comparison of heterodimeric, TM287/288 (A–C), and homodimeric, Sav1866 (D–F), ABC transporters. (A and D) Cartoon (left) and surface (right) representations of TM287/288 (A; PDB accession no. 3QF4) and Sav1866 (D; PDB accession no. 2ONJ) viewed along expected membrane plane (boundaries indicated by gray lines). Transmembrane domains equivalent to CFTR’s transmembrane domain 1 and transmembrane domain 2 are forest green and blue, respectively; NBDs equivalent to CFTR’s NBD1 and NBD2 are lime and cyan, respectively. Walker A motifs are shown in red, Walker B in orange, ABC signature in purple, bound AMPPNP as yellow spheres, and target residue positions as CPK spheres. (B and E) Magnified (∼2×) surface views of the boxed cytoplasmic regions in A and D. Interfacial residues equivalent to CFTR’s ABC signature-sequence targets S549 and S1347 are exposed in TM287/288 (B) but buried in Sav1866 (E). (C and F) NBD dimer interfaces viewed from the membrane, after 90° rotation from B and E views and removal of transmembrane domains, with solid (left) and transparent (right) surfaces. In Sav1866 (F), a signature sequence and AMPPNP are buried in the tight NBD dimer interface in each composite site, whereas in TM287/288 (C), both signature sequences are exposed and a single AMPPNP is bound by the CFTR equivalent NBD1 Walker A motif.
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fig1: Structural comparison of heterodimeric, TM287/288 (A–C), and homodimeric, Sav1866 (D–F), ABC transporters. (A and D) Cartoon (left) and surface (right) representations of TM287/288 (A; PDB accession no. 3QF4) and Sav1866 (D; PDB accession no. 2ONJ) viewed along expected membrane plane (boundaries indicated by gray lines). Transmembrane domains equivalent to CFTR’s transmembrane domain 1 and transmembrane domain 2 are forest green and blue, respectively; NBDs equivalent to CFTR’s NBD1 and NBD2 are lime and cyan, respectively. Walker A motifs are shown in red, Walker B in orange, ABC signature in purple, bound AMPPNP as yellow spheres, and target residue positions as CPK spheres. (B and E) Magnified (∼2×) surface views of the boxed cytoplasmic regions in A and D. Interfacial residues equivalent to CFTR’s ABC signature-sequence targets S549 and S1347 are exposed in TM287/288 (B) but buried in Sav1866 (E). (C and F) NBD dimer interfaces viewed from the membrane, after 90° rotation from B and E views and removal of transmembrane domains, with solid (left) and transparent (right) surfaces. In Sav1866 (F), a signature sequence and AMPPNP are buried in the tight NBD dimer interface in each composite site, whereas in TM287/288 (C), both signature sequences are exposed and a single AMPPNP is bound by the CFTR equivalent NBD1 Walker A motif.

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).


Cysteine accessibility probes timing and extent of NBD separation along the dimer interface in gating CFTR channels.

Chaves LA, Gadsby DC - J. Gen. Physiol. (2015)

Structural comparison of heterodimeric, TM287/288 (A–C), and homodimeric, Sav1866 (D–F), ABC transporters. (A and D) Cartoon (left) and surface (right) representations of TM287/288 (A; PDB accession no. 3QF4) and Sav1866 (D; PDB accession no. 2ONJ) viewed along expected membrane plane (boundaries indicated by gray lines). Transmembrane domains equivalent to CFTR’s transmembrane domain 1 and transmembrane domain 2 are forest green and blue, respectively; NBDs equivalent to CFTR’s NBD1 and NBD2 are lime and cyan, respectively. Walker A motifs are shown in red, Walker B in orange, ABC signature in purple, bound AMPPNP as yellow spheres, and target residue positions as CPK spheres. (B and E) Magnified (∼2×) surface views of the boxed cytoplasmic regions in A and D. Interfacial residues equivalent to CFTR’s ABC signature-sequence targets S549 and S1347 are exposed in TM287/288 (B) but buried in Sav1866 (E). (C and F) NBD dimer interfaces viewed from the membrane, after 90° rotation from B and E views and removal of transmembrane domains, with solid (left) and transparent (right) surfaces. In Sav1866 (F), a signature sequence and AMPPNP are buried in the tight NBD dimer interface in each composite site, whereas in TM287/288 (C), both signature sequences are exposed and a single AMPPNP is bound by the CFTR equivalent NBD1 Walker A motif.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig1: Structural comparison of heterodimeric, TM287/288 (A–C), and homodimeric, Sav1866 (D–F), ABC transporters. (A and D) Cartoon (left) and surface (right) representations of TM287/288 (A; PDB accession no. 3QF4) and Sav1866 (D; PDB accession no. 2ONJ) viewed along expected membrane plane (boundaries indicated by gray lines). Transmembrane domains equivalent to CFTR’s transmembrane domain 1 and transmembrane domain 2 are forest green and blue, respectively; NBDs equivalent to CFTR’s NBD1 and NBD2 are lime and cyan, respectively. Walker A motifs are shown in red, Walker B in orange, ABC signature in purple, bound AMPPNP as yellow spheres, and target residue positions as CPK spheres. (B and E) Magnified (∼2×) surface views of the boxed cytoplasmic regions in A and D. Interfacial residues equivalent to CFTR’s ABC signature-sequence targets S549 and S1347 are exposed in TM287/288 (B) but buried in Sav1866 (E). (C and F) NBD dimer interfaces viewed from the membrane, after 90° rotation from B and E views and removal of transmembrane domains, with solid (left) and transparent (right) surfaces. In Sav1866 (F), a signature sequence and AMPPNP are buried in the tight NBD dimer interface in each composite site, whereas in TM287/288 (C), both signature sequences are exposed and a single AMPPNP is bound by the CFTR equivalent NBD1 Walker A motif.
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).

Bottom Line: These results suggest that the target cysteines can be modified only in closed channels; that after modification the attached MTS adduct interferes with ATP-mediated opening; and that modification in the presence of ATP occurs rapidly once channels close, before they can reopen.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, New York, NY 10065.

Show MeSH
Related in: MedlinePlus