The two ATP binding sites of cystic fibrosis transmembrane conductance regulator (CFTR) play distinct roles in gating kinetics and energetics

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC (ATP binding cassette) transporter family, is a chloride channel whose activity is controlled by protein kinase-dependent phosphorylation. Opening and closing (gating) of the phosphorylated CFTR is coupled to ATP binding and hydrolysis at CFTR's two nucleotide binding domains (NBD1 and NBD2). Recent studies present evidence that the open channel conformation reflects a head-to-tail dimerization of CFTR's two NBDs as seen in the NBDs of other ABC transporters (Vergani et al., 2005). Whether these two ATP binding sites play an equivalent role in the dynamics of NBD dimerization, and thus in gating CFTR channels, remains unsettled. Based on the crystal structures of NBDs, sequence alignment, and homology modeling, we have identified two critical aromatic amino acids (W401 in NBD1 and Y1219 in NBD2) that coordinate the adenine ring of the bound ATP. Conversion of the W401 residue to glycine (W401G) has little effect on the sensitivity of the opening rate to [ATP], but the same mutation at the Y1219 residue dramatically lowers the apparent affinity for ATP by >50-fold, suggesting distinct roles of these two ATP binding sites in channel opening. The W401G mutation, however, shortens the open time constant. Energetic analysis of our data suggests that the free energy of ATP binding at NBD1, but not at NBD2, contributes significantly to the energetics of the open state. This kinetic and energetic asymmetry of CFTR's two NBDs suggests an asymmetric motion of the NBDs during channel gating. Opening of the channel is initiated by ATP binding at the NBD2 site, whereas separation of the NBD dimer at the NBD1 site constitutes the rate-limiting step in channel closing.

Figure 1.

Tryptophan 401 and tyrosine 1219 residues interact with the adenine ring of ATP in the human CFTR NBD1 and NBD2, respectively. (A) Interactions between ATP and key amino acids in the NBD1 binding pocket, adopted from the monomeric crystal structure of the human F508A NBD1-ATP complexes (pdb code: 1xmi, chain A) (left). A similar picture of modeled NBD2 binding pocket is shown on the right. Residues of interest are represented by sticks, including those that interact with adenine ring (W401 in NBD1 and Y1219 in NBD2), the Walker A lysines (K464 in NBD1 and K1250 in NBD2), and the catalytic Walker B glutamate residue, E1371. The protein backbone atoms are plotted in thin lines and colored in gray. The figures were prepared by PYMOL. In other members of the ABC family, there is either an E or a D at the position equivalent to E1371 of CFTR NBD2, however, it is a serine in CFTR's NBD1 that is believed to be the structural basis for CFTR NBD1's inability to hydrolyze ATP (Lewis et al., 2004; Lewis et al., 2005). Since ATP is found to be associated with the Walker A and B motifs in all crystal structures of NBDs resolved so far (e.g., Hung et al., 1998; Karpowich et al., 2001; Yuan et al., 2001; Lewis et al., 2004), for the sake of clarity, we define the NBD1 ATP-binding site (or NBD1 site) as the binding pocket containing Walker A and Walker B motifs in the NBD1 sequence. An equivalent definition is applied to the NBD2 site. (B) Sequence alignment of the N-terminal part of the NBD1 and NBD2 of CFTR from 10 species (chosen randomly out of 36). Aromatic residues studied in this paper are highlighted in yellow. Conserved residues are highlighted in gray. Note that a stretch of ∼30 amino acids (from 404 to 435) is present in NBD1 (i.e., regulatory insertion) but absent in NBD2 (dashed line). (C) Representative bracketed current traces at different [ATP] for constructing ATP dose–response relationships. Current induced by various [ATP] was normalized to 2.75 mM ATP in the case of WT and W401G and to 20 mM ATP in the case of Y1219G. Horizontal bars represent 50 s. (D) ATP dose–response relationships of WT (black), W401G (red), and Y1219G (green). Solid lines are Michaelis-Menten fits to the data. The K_1/2 values are 0.09 ± 0.02 mM, 0.11 ± 0.02 mM, and 4.72 ± 1.12 mM for WT, W401G, and Y1219G, respectively. Each data point represents the average from three to eight experiments.

Figure 2.

Effects of mutations at W401 and Y1219 on the opening rate. (A) Normalized ATP dose–response relationships of WT (black line, Michaelis-Menten fit from Fig. 1 D), Y1219W (brown), Y1219F (pink), Y1219I (blue), and Y1219G (green line, Michaelis-Menten fit from Fig. 1 D). Solid lines are the Michaelis-Menten fits to the data. K_1/2 values are 0.13 ± 0.02 mM (Y1219W), 0.46 ± 0.06 mM (Y1219F), and 0.94 ± 0.20 mM (Y1219I), respectively. (B) Representative single-channel current traces of WT, W401G, Y1219G, and Y1219I in response to [ATP] as marked. (C) Relationships between channel opening rates and [ATP] for WT (black), W401G (red), Y1219I (blue), and Y1219G (green). Solid lines are Michaelis-Menten fits to the data of WT (black) and Y1219I (blue). The maximal opening rate and K_1/2 values are 2.42 ± 0.11 s⁻¹ and 0.11 ± 0.02 mM for WT, and 2.60 ± 0.11 s⁻¹ and 1.73 ± 0.26 mM for Y1219I, respectively. (D) Relationships between channel opening rates and [ATP] for ΔR-CFTR (black), ΔR-Y1219I (blue), and ΔR-Y1219G (green). K_1/2 from Michaelis-Menten fits (solid lines) are 0.16 ± 0.04 mM and 1.27 ± 0.16 mM for ΔR-CFTR and ΔR-Y1219I, respectively. (Data for ΔR-CFTR were obtained from Bompadre et al., 2005a.)

Figure 3.

Effect of mutations at W401 and Y1219 residues on channel open time. (A) Mean open times of WT and W401G in the presence of 2.75 mM ATP are 441.3 ± 49.4 ms (n = 13) and 248.7 ± 11.3 ms (n = 22), respectively. The mean open time of Y1219G in the presence of 20 mM ATP is 399.1 ± 40.4 ms (n = 5). *** indicates P < 0.001 between WT and W401G. (B) Representative current relaxation traces upon withdrawal of 1 mM ATP plus PKA for E1371S, W401G/E1371S, triple/E1371S, Y1219G/E1371S, and W401G/Y1219G/E1371S. Solid lines above the traces indicate the duration of application of 1 mM ATP plus PKA. Horizontal scale bars represent 200 s. (C) Mean relaxation time constants upon withdrawal of 1 mM ATP plus PKA. ** indicates P < 0.01 and *** indicates P < 0.001 (compared with E1371S).

Figure 4.

Effects of P-ATP on the relaxation time constant. (A) Two representative single-channel current traces of WT-CFTR in the presence of P-ATP (red) or ATP (black). The mean open time for P-ATP or ATP opened channels are 583.9 ± 60.6 ms (n = 6) and 441.3 ± 49.4 ms (n = 13), respectively. (B) Representative current relaxation traces of E1371S, W401G/E1371S, triple/E1371S, and Y1219G/E1371S after withdrawal of 1 mM ATP plus PKA or 50 μM P-ATP plus PKA. Horizontal scale bars represent 200 s. (C) The ratio of the relaxation time constant upon withdrawal of 50 μM P-ATP plus PKA to that upon withdrawal of 1 mM ATP plus PKA from the same patch was calculated for E1371S and various mutants in the E1371S background. The mean values are taken from 3–11 experiments. *** indicates P < 0.001 and **** indicates P < 0.0001 (compared with E1371S).