Tseng PY et al. (2011), Binding of ATP to the CBS domains in the C-term...

XB-ART-43608

J Gen Physiol 2011 Apr 01;1374:357-68. doi: 10.1085/jgp.201010495.

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Binding of ATP to the CBS domains in the C-terminal region of CLC-1.

Tseng PY , Yu WP , Liu HY , Zhang XD , Zou X , Chen TY .

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The common gating of CLC-1 has been shown to be inhibited by intracellular adenosine triphosphate (ATP) in acidic pH conditions. Such modulation is thought to be mediated by direct binding of ATP to the cystathionine β-synthase (CBS) domains at the C-terminal cytoplasmic region of CLC-1. Guided by the crystal structure of the C-terminal domain of CLC-5, we constructed a homology model of CLC-1's C terminus and mutated critical amino acid residues lining the potential ATP-binding site. The CLC-1 mutations V634A and E865A completely abolished the ATP inhibition of CLC-1, consistent with the loss of ATP binding seen with the corresponding mutations in CLC-5. Mutating two other residues, V613 and V860, also disrupted the ATP modulation of CLC-1. However, placing aromatic amino acids at position 634 increases the apparent ATP affinity. Mutant cycle analyses showed that the modulation effects of ATP and cytidine triphosphate on wild-type CLC-1 and the V634F mutant were nonadditive, suggesting that the side chain of amino acid at position 634 interacts with the base moiety of the nucleotide. The mutation effects of V634F and V613A on the ATP modulation were also nonadditive, which is consistent with the assertion suggested from the homology model that these two residues may both interact with the bound nucleotide. These results provide evidence for a direct ATP binding for modulating the function of CLC-1 and suggest an overall conserved architecture of the ATP-binding sites in CLC-1 and CLC-5. This study also demonstrates that CLC-1 is a convenient experimental model for studying the interaction of nucleotides/nucleosides with the CBS domain.

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Species referenced: Xenopus
Genes referenced: clcn1 clcn5

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	Figure 1. Homology modeling of the C-terminal domain of CLC-1. (A) Homology model of the CBS domain of CLC-1 shown in ribbon presentation. The two segments of CLC-1 sequence corresponding to CLC-5 are colored in red and yellow, whereas the segment that is missing in CLC-5 is illustrated with a dotted black line. ATP is shown in stick presentation (in CPK color). Key amino acids examined in this study are colored in yellow (V613), orange (V634), blue (V860), and purple (E865). (B) Sequence alignment of CLC-0, CLC-1, CLC-5, and AtCLC-a for the region encompassing the four residues shown in A.
	Figure 2. Effects of alanine mutations for four residues on the ATP modulation of CLC-1. (left and middle) Recording traces of WT CLC-1 and the four mutants. The recordings in the absence (left) and in the presence (middle) of 3 mM ATP were obtained from the same patch (pHi 6.8). (right) Steady-state Poc-V curves of WT CLC-1 and the four alanine mutants shown in left and middle panels. Black squares, red circles, and green triangles represent the data in control (before ATP), 3 mM ATP, and washout, respectively.
	Figure 3. Changes of the V1/2 of the Poc-V curves of various mutants in response to 3 mM ATP at pHi 6.8. The Poc-V curves of each mutant in the absence and in the presence of 3 mM ATP were constructed from the same patch, and the V1/2 of the Poc-V curves were obtained by fitting the data points to a Boltzmann equation. The difference in V1/2 between control and 3 mM ATP (ÎV1/2) was first determined from each patch and then averaged (n = 14 for WT CLC-1 and n = 4â7 for various mutants). The label n.c. indicates that the mutant did not show functional current in at least two runs of expression in Xenopus oocytes. Error bars represent SEM.
	Figure 4. The V634F mutation increases the apparent affinity for the ATP modulation. Representative recording traces were obtained from an excised patch of the oocyte expressing the V634F mutant in control (0 ATP) and in various concentrations of ATP as indicated. The averaged Poc-V curves of the V634F mutant in various ATP concentrations are shown in the bottom right panel. Error bars represent SEM. n = 7.
	Figure 5. Shift of the V1/2 of the Poc-V curve (ÎV1/2) in various mutants of V634 as a function of the ATP concentration. Data points were fitted to Eq. 1 to determine the apparent K1/2 and V1/2,max. (A) Mutants of V634 in which the apparent ATP affinity is reduced. For comparison, the ATP doseâresponse curve of WT CLC-1 is also shown. (B) ATP dose-dependent curves of WT CLC-1, V634F, V634Y, and V634W. Error bars represent SEM. n = 4â8.
	Figure 6. Interaction of nucleotides with residue V634 in the ATP-binding site explored by double mutant cycle analyses. (A) Mutant cycle analysis of the interaction of the nucleotide with residue V634. The ATP and CTP doseâresponse curves of WT CLC-1 and the V634F mutant were first constructed using the parameter Poc(ATP)/Poc(control) as the dependent parameter. The values of Ko and Kc (Table III) of ATP/CTP modulations were determined by fitting the data points to Eq. 2, and the Kc values were used to calculate the free energy differences of single or double manipulations (bar graph) according to Eq. 3 and the coupling constant, Î©, according to Eq. 4. n = 6â8. (B) Mutant cycle analysis of the nucleotide interaction with V634 using ATP and dATP. The ligand doseâresponse curves of WT CLC-1 and V634F were constructed using Poc(ATP)/Poc(control) as the dependent parameter. Ko and Kc were determined (Table III), and the Î© value and the free energy differences were calculated accordingly. n = 4â8.
	Figure 7. Nonadditive effects of mutations on the ATP modulations of CLC-1. The parameter Poc(ATP)/Poc(control) was used as the response to ATP modulation. (A) Mutant cycle analysis of the effects of V634F and V613A mutations on the ATP modulation. ATP doseâresponse curves of single point mutants and the double mutants were constructed to determine the values of Ko and Kc. The fitted Kc values were used to calculate the free energy differences of single or double mutants (bar graph) according to Eq. 3 and the coupling constant, Î©, according to Eq. 4. In the context of Eq. 2, the V634F mutation corresponds to the change from X1 to X2, whereas the V613A mutation represents the change from Y1 to Y2. (B) Double mutant cycle analysis for V634F/E865D mutations. The values of Î© at different voltages are shown in Fig. S2. Error bars represent SEM. n = 4â8.

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