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Mutant cycles at CFTR's non-canonical ATP-binding site support little interface separation during gating.

Szollosi A, Muallem DR, Csanády L, Vergani P - J. Gen. Physiol. (2011)

Bottom Line: Mutation T460S accelerated closure in hydrolytic conditions and in the nonhydrolytic K1250R background; mutation L1353M did not affect these rates.Analysis of the double mutant showed additive effects of mutations, suggesting that energetic coupling between the two residues remains unchanged during the gating cycle.These results provide independent support for a gating model in which ATP-bound composite site 1 remains closed throughout the gating cycle.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary.

ABSTRACT
Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily. ABC proteins share a common molecular mechanism that couples ATP binding and hydrolysis at two nucleotide-binding domains (NBDs) to diverse functions. This involves formation of NBD dimers, with ATP bound at two composite interfacial sites. In CFTR, intramolecular NBD dimerization is coupled to channel opening. Channel closing is triggered by hydrolysis of the ATP molecule bound at composite site 2. Site 1, which is non-canonical, binds nucleotide tightly but is not hydrolytic. Recently, based on kinetic arguments, it was suggested that this site remains closed for several gating cycles. To investigate movements at site 1 by an independent technique, we studied changes in thermodynamic coupling between pairs of residues on opposite sides of this site. The chosen targets are likely to interact based on both phylogenetic analysis and closeness on structural models. First, we mutated T460 in NBD1 and L1353 in NBD2 (the corresponding site-2 residues become energetically coupled as channels open). Mutation T460S accelerated closure in hydrolytic conditions and in the nonhydrolytic K1250R background; mutation L1353M did not affect these rates. Analysis of the double mutant showed additive effects of mutations, suggesting that energetic coupling between the two residues remains unchanged during the gating cycle. We next investigated pairs 460-1348 and 460-1375. Although both mutations H1348A and H1375A produced dramatic changes in hydrolytic and nonhydrolytic channel closing rates, in the corresponding double mutants these changes proved mostly additive with those caused by mutation T460S, suggesting little change in energetic coupling between either positions 460-1348 or positions 460-1375 during gating. These results provide independent support for a gating model in which ATP-bound composite site 1 remains closed throughout the gating cycle.

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Spatial arrangement of target residues on opposite sides of CFTR’s composite site 1 in homology models. In a homology model of CFTR’s NBD dimer (Mornon et al., 2008), T460 (red) in the Walker A motif of NBD1 is close to NBD2 residues H1348 (gold; in the signature sequence), L1353 (blue; at the end of the signature sequence), and H1375 (magenta; in the D-loop).
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fig1: Spatial arrangement of target residues on opposite sides of CFTR’s composite site 1 in homology models. In a homology model of CFTR’s NBD dimer (Mornon et al., 2008), T460 (red) in the Walker A motif of NBD1 is close to NBD2 residues H1348 (gold; in the signature sequence), L1353 (blue; at the end of the signature sequence), and H1375 (magenta; in the D-loop).

Mentions: Two positions, T460 in NBD1 (in Walker A sequence; Fig. 1, red) and L1353 in NBD2 (just downstream of the signature sequence; Fig. 1, blue), were identified by phylogenetic statistical coupling analysis (Lockless and Ranganathan, 1999), using sequence alignments of thousands of NBD sequences, which suggested that the two (the equivalent T460 and L1353 in site 1, and T1246 and R555 in site 2 of CFTR) had been conserved as a pair during evolution (Vergani et al., 2005). In homology models of the CFTR NBD1/NBD2 heterodimer (Mense et al., 2006; Mornon et al., 2008; Serohijos et al., 2008), the two α carbons are separated by <10 Å (Fig. 1). Indeed, energetic coupling between R555 and T1246 in site 2 was shown to be state dependent (Vergani et al., 2005). Unlike R555 and T1246, T460 and L1353 in site 1 cannot form a hydrogen bond, consistent with a distinct fine structure and functional role of the degenerate site. However, within an alignment of 182 full-length sequences of “asymmetric” transporters in which sequence abnormalities suggest a functionally degenerate site 1 (including most sequences of the organic anion conjugates, anions, and drugs group of the ABCISSE database; see Dassa and Bouige, 2001, and supplemental AsymmetricABCs.txt), the residues at these positions are well conserved (>94% of sequences have a Valine-Leucine or a Threonine-Leucine pair). It is therefore likely that some interaction between them is maintained.


Mutant cycles at CFTR's non-canonical ATP-binding site support little interface separation during gating.

Szollosi A, Muallem DR, Csanády L, Vergani P - J. Gen. Physiol. (2011)

Spatial arrangement of target residues on opposite sides of CFTR’s composite site 1 in homology models. In a homology model of CFTR’s NBD dimer (Mornon et al., 2008), T460 (red) in the Walker A motif of NBD1 is close to NBD2 residues H1348 (gold; in the signature sequence), L1353 (blue; at the end of the signature sequence), and H1375 (magenta; in the D-loop).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3105517&req=5

fig1: Spatial arrangement of target residues on opposite sides of CFTR’s composite site 1 in homology models. In a homology model of CFTR’s NBD dimer (Mornon et al., 2008), T460 (red) in the Walker A motif of NBD1 is close to NBD2 residues H1348 (gold; in the signature sequence), L1353 (blue; at the end of the signature sequence), and H1375 (magenta; in the D-loop).
Mentions: Two positions, T460 in NBD1 (in Walker A sequence; Fig. 1, red) and L1353 in NBD2 (just downstream of the signature sequence; Fig. 1, blue), were identified by phylogenetic statistical coupling analysis (Lockless and Ranganathan, 1999), using sequence alignments of thousands of NBD sequences, which suggested that the two (the equivalent T460 and L1353 in site 1, and T1246 and R555 in site 2 of CFTR) had been conserved as a pair during evolution (Vergani et al., 2005). In homology models of the CFTR NBD1/NBD2 heterodimer (Mense et al., 2006; Mornon et al., 2008; Serohijos et al., 2008), the two α carbons are separated by <10 Å (Fig. 1). Indeed, energetic coupling between R555 and T1246 in site 2 was shown to be state dependent (Vergani et al., 2005). Unlike R555 and T1246, T460 and L1353 in site 1 cannot form a hydrogen bond, consistent with a distinct fine structure and functional role of the degenerate site. However, within an alignment of 182 full-length sequences of “asymmetric” transporters in which sequence abnormalities suggest a functionally degenerate site 1 (including most sequences of the organic anion conjugates, anions, and drugs group of the ABCISSE database; see Dassa and Bouige, 2001, and supplemental AsymmetricABCs.txt), the residues at these positions are well conserved (>94% of sequences have a Valine-Leucine or a Threonine-Leucine pair). It is therefore likely that some interaction between them is maintained.

Bottom Line: Mutation T460S accelerated closure in hydrolytic conditions and in the nonhydrolytic K1250R background; mutation L1353M did not affect these rates.Analysis of the double mutant showed additive effects of mutations, suggesting that energetic coupling between the two residues remains unchanged during the gating cycle.These results provide independent support for a gating model in which ATP-bound composite site 1 remains closed throughout the gating cycle.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary.

ABSTRACT
Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily. ABC proteins share a common molecular mechanism that couples ATP binding and hydrolysis at two nucleotide-binding domains (NBDs) to diverse functions. This involves formation of NBD dimers, with ATP bound at two composite interfacial sites. In CFTR, intramolecular NBD dimerization is coupled to channel opening. Channel closing is triggered by hydrolysis of the ATP molecule bound at composite site 2. Site 1, which is non-canonical, binds nucleotide tightly but is not hydrolytic. Recently, based on kinetic arguments, it was suggested that this site remains closed for several gating cycles. To investigate movements at site 1 by an independent technique, we studied changes in thermodynamic coupling between pairs of residues on opposite sides of this site. The chosen targets are likely to interact based on both phylogenetic analysis and closeness on structural models. First, we mutated T460 in NBD1 and L1353 in NBD2 (the corresponding site-2 residues become energetically coupled as channels open). Mutation T460S accelerated closure in hydrolytic conditions and in the nonhydrolytic K1250R background; mutation L1353M did not affect these rates. Analysis of the double mutant showed additive effects of mutations, suggesting that energetic coupling between the two residues remains unchanged during the gating cycle. We next investigated pairs 460-1348 and 460-1375. Although both mutations H1348A and H1375A produced dramatic changes in hydrolytic and nonhydrolytic channel closing rates, in the corresponding double mutants these changes proved mostly additive with those caused by mutation T460S, suggesting little change in energetic coupling between either positions 460-1348 or positions 460-1375 during gating. These results provide independent support for a gating model in which ATP-bound composite site 1 remains closed throughout the gating cycle.

Show MeSH
Related in: MedlinePlus