ABCMdb

ABCC7 p.Phe342Ala

Predicted by SNAP2:	A: D (59%), C: D (63%), D: D (85%), E: D (80%), G: D (80%), H: D (80%), I: D (59%), K: D (85%), L: N (66%), M: N (53%), N: D (71%), P: D (91%), Q: D (66%), R: D (85%), S: D (75%), T: D (75%), V: D (63%), W: D (71%), Y: N (82%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, G: D, H: N, I: N, K: D, L: N, M: N, N: N, P: D, Q: D, R: D, S: D, T: N, V: N, W: N, Y: N,

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Publications
[hide] Locating a Plausible Binding Site for an Open Chan... Mol Pharmacol. 2012 Aug 24. Norimatsu Y, Ivetac A, Alexander C, O'Donnell N, Frye L, Sansom MS, Dawson DC
Locating a Plausible Binding Site for an Open Channel Blocker, GlyH-101, in the Pore of the Cystic Fibrosis Transmembrane Conductance Regulator.
Mol Pharmacol. 2012 Aug 24., [PMID:22923500]

Abstract [show]
High-throughput screening has led to the identification of small-molecule blockers of the CFTR chloride channel, but the structural basis of blocker binding remains to be defined. We recently developed molecular models of the CFTR channel based on homology to the bacterial transporter, Sav1866, that could permit blocker binding to be analyzed in silico. The models accurately predicted the existence of a narrow region in the pore that is a likely candidate for the binding site of an open-channel pore blocker like GlyH-101, thought to act by entering the channel from the extracellular side. As a more stringent test of predictions of the CFTR pore model, we applied induced-fit, virtual ligand docking techniques to identify potential binding sites for GlyH-101 within the CFTR pore. The highest scoring, docked position was near two pore-lining residues, F337 and T338, and the rate of reaction of anionic thiol-directed reagents with cysteines substituted at either of these positions was slowed in the presence of the blocker, consistent with the predicted repulsive effect of the net negative charge on GlyH-101. When a bulky phenylalanine that forms part of the predicted binding pocket (F342) was replaced with alanine, the apparent affinity of the blocker increased by approximately 200 fold. A Molecular Mechanics-Generalized Born/Surface Area (MM-GB/SA) analysis of GlyH-101 binding predicted that substitution of F342 with alanine would substantially increase blocker affinity, primarily due to decreased intramolecular strain within the blocker-protein complex. This study suggests that GlyH-101 blocks the CFTR channel by binding within the pore bottleneck.

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Sentences [show]
No. Sentence Comment
51 The structure of F342A CFTR was generated by replacing F342 of wt CFTR from the predicted complex with alanine in Maestro (version 9.1, Schrödinger LLC). Login to comment
52 GlyH-101 was docked to the structure of the F342A CFTR using the IFD protocol and the structure of the resulting complex was used for MM-GB/SA analysis (see Binding Free Energy Calculation for more details). Login to comment
170 The EC50 of GlyH-101 is reduced approximately 200-fold in F342A CFTR The binding pose predicted for GlyH-101 drew our attention to another residue predicted to lie within the pore bottleneck, F342. Login to comment
174 We analyzed GlyH-101 binding to wt and F342A CFTR channels using the MM-GB/SA method as described by Guimaraes and Cardozo, (2008). Login to comment
175 In order to predict the structure of the F342A CFTR/GlyH-101 complex, the phenylalanine at position 342 of wt CFTR was mutated to alanine in silico (see Methods) and GlyH-101 was docked to the mutant CFTR using IFD (supplemental PDB file 3). Login to comment
177 The estimated parameter values for wt and F342A are collected in Table 2. Login to comment
178 The calculation predicts that the free energy of binding of GlyH-101, ΔGbind, is more negative for F342A CFTR than for wt CFTR, that is F342A CFTR binds GlyH-101 more tightly than does wt CFTR. Login to comment
179 Decomposition of the total binding free energy according to the terms listed in Equation 1 reveals that the predicted energy difference arises mainly from reduction of the strain energy of the F342A CFTR (EPTN), consistent with the notion that the side chain of F342 in the wt CFTR channel causes an unfavorable steric clash with GlyH-101. Login to comment
181 The calculated free energies of GlyH-101 binding were -7.43 and -8.54 kcal/mol for wt CFTR and -11.8 and -12.6 kcal/mol for F342A CFTR with and without the membrane respectively. Login to comment
182 The experimental results collected in Figure 8A show that dose-dependent block of F342A CFTR by GlyH-101, as predicted by the MM-GB/SA analysis, exhibited an EC50(0) that was dramatically reduced (5.2 ± 0.83 nM; n = 3) compared to that of wt CFTR (1.1 ± 0.11 µM; n = 4; Figure 1C), consistent with a substantial increase in binding affinity for the blocker that was brought about by eliminating the bulky phenylalanine side chain. Login to comment
183 The substantially higher apparent affinity of F342A CFTR for GlyH-101 implies a dramatic slowing of the off-rate for the bound blocker. Login to comment
197 Using their data, the k`on for wt CFTR was calculated to be 1.6 x 107 M-1 s-1 , a value similar to the k`on of F342A CFTR (2.3 × 107 M-1 s-1 ) determined in the current study, again indicating that the difference in EC50 between wt and F342A CFTR is due to the slow off rate of GlyH-101 for F342A CFTR. Login to comment
198 The binding free energy based on the observed EC50, estimated as RTln(EC50), is -8.12 kcal/mol for wt CFTR and -11.3 kcal/mol for F342A CFTR. Login to comment
199 These empirical values are qualitatively consistent with the computed free energies, obtained with the MM-GB/SA technique using the proposed model of GlyH-101 binding (Figure 2), for wt CFTR (-7.43 kcal/mol) and F342A CFTR (-11.8 kcal/mol). Login to comment
211 Finally, when a predicted steric clash between the side chain of F342 and the naphthalene tail of the bound blocker was removed by mutating the residue to an alanine (F342A), the apparent blocker affinity was increased by more than 200-fold, a change that was consistent with free energies of binding estimated using a MM-GB/SA approach. Login to comment
7 A molecular mechanics-generalized Born/surface area analysis of GlyH-101 binding predicted that substitution of Phe342 with alanine would substantially increase blocker affinity, primarily because of decreased intramolecular strain within the blocker-protein complex. Login to comment
73 The structure of the F342A CFTR was generated with Maestro 9.1 by replacing Phe342 of the wt CFTR in the predicted complex with alanine. Login to comment
74 GlyH-101 was docked to the structure of the F342A CFTR by using the IFD protocol, and the structure of the resulting complex was used for MM-GB/SA analysis (see Calculation of Binding Free Energy for more details). Login to comment
262 The GlyH-101 EC50 Is Reduced Approximately 200-Fold with F342A CFTR. Login to comment
267 We analyzed GlyH-101 binding to wt and F342A CFTR channels by using the MM-GB/SA method described by Guimara dc;es and Cardozo (2008). Login to comment
268 To predict the structure of the F342A CFTR-GlyH-101 complex, the phenylalanine at position 342 of the wt CFTR was mutated to alanine in silico (see Materials and Methods) and GlyH-101 was docked to the mutant CFTR by using IFD techniques (Supplemental PDB File 3). Login to comment
270 The estimated parameter values for wt and F342A CFTRs are presented in Table 2. Login to comment
271 The calculations suggested that the free energy of binding of GlyH-101, èc;Gbind, is more negative for the F342A CFTR than for the wt CFTR, that is, the F342A CFTR binds GlyH-101 more tightly than does the wt CFTR. Login to comment
285 TABLE 2 GlyH-101 binding free energy, èc;Gbind, and its components estimated through MM-GB/SA analysis for wt and F342A CFTRs, without and with a membrane Analyses were performed with eq. 1, èc;Gbind afd; èc;Elig af9; èc;Gsolv afa; Tèc;Sconf af9; Evdw af9; Ees af9; èc;EPTN, where èc;Elig and èc;EPTN are changes (upon ligand binding) in the intramolecular strains of the ligand and protein, respectively, èc;Gsolv is the total desolvation penalty for the ligand and protein, Tèc;Sconf is the ligand conformational penalty, and Evdw and Ees are the van der Waals and electrostatic energies, respectively. Login to comment
286 CFTR èc;Elig èc;EPTN Evdw af9; Ees af9; èc;Gsolv afa;Tèc;Sconf èc;Gbind kcal/mol kcal/mol kcal/mol kcal/mol kcal/mol Without membrane wt 4.68 6.89 afa;21.2 1.05 afa;8.54 F342A 3.90 4.04 afa;21.6 1.05 afa;12.6 wt afa; F342A 0.78 2.85 0.42 0.0 4.0 With membrane wt 4.68 6.88 afa;19.8 1.05 afa;7.23 F342A 3.92 4.05 afa;20.4 1.05 afa;11.4 wt afa; F342A 0.76 2.82 0.54 0.0 4.1 the F342A CFTR (EPTN), consistent with the notion that the side chain of Phe342 in the wt CFTR channel causes an unfavorable steric clash with GlyH-101. Login to comment
288 The calculated free energies of GlyH-101 binding were afa;7.43 and afa;8.54 kcal/mol for the wt CFTR and afa;11.8 and afa;12.6 kcal/mol for the F342A CFTR with and without the membrane, respectively. Login to comment
289 The experimental results presented in Fig. 8A showed that dose-dependent blockade of the F342A CFTR by GlyH-101, as predicted by the MM-GB/SA analysis, exhibited an EC50(0) that was dramatically reduced (5.2 afe; 0.83 nM; n afd; 3), compared with that of the wt CFTR (1.1 afe; 0.11 òe;M; n afd; 4) (Fig. 1C), which is consistent with a substantial increase in binding affinity for the blocker with elimination of the bulky phenylalanine side chain. Login to comment
290 The substantially higher apparent affinity of the F342A CFTR for GlyH-101 suggests dramatic slowing of the off-rate for the bound blocker. Login to comment
304 With the data reported by those authors, the kb18; on for the wt CFTR was calculated to be 1.6 afb; 107 Mafa;1 safa;1 , a value similar to the kb18; on for the F342A CFTR (2.3 afb; 107 Mafa;1 safa;1 ) determined in the current study, which indicates that the difference in EC50 values between the wt and F342A CFTRs is attributable to the slow off-rate of GlyH-101 with the F342A CFTR. Login to comment
305 The binding free energy values based on the observed EC50 values, estimated as RTln(EC50), were afa;8.12 kcal/mol for the wt CFTR and afa;11.3 kcal/mol for the F342A CFTR. Login to comment
306 These empirical values are qualitatively consistent with the computed free energies obtained by applying the MM-GB/SA technique to the proposed model of GlyH-101 binding (Fig. 2) for the wt CFTR (afa;7.43 kcal/mol) and the F342A CFTR (afa;11.8 kcal/mol). Login to comment
319 Finally, when a predicted steric clash between the side chain of Phe342 and the naphthalene tail of the bound blocker was removed through mutation of the residue to an alanine (F342A), the apparent blocker affinity was increased more than 200-fold, a change consistent with the free energies of binding estimated using a MM-GB/SA approach. Login to comment
322 F342A CFTR transmembrane currents. Login to comment
323 F342A CFTR channels were expressed in Xenopus laevis oocytes, and transmembrane currents were measured at clamped voltages by using a two-electrode, voltage-clamp technique. Login to comment
324 A, the voltage dependence of the GlyH-101 EC50 for the F342A CFTR is consistent with the Woodhull model (Woodhull, 1973; Tikhonov and Magazanik, 1998), in a manner similar to that of wt CFTR (Fig. 1). Login to comment

[hide] Differential contribution of TM6 and TM12 to the p... Pflugers Arch. 2012 Mar;463(3):405-18. Epub 2011 Dec 13. Cui G, Song B, Turki HW, McCarty NA
Differential contribution of TM6 and TM12 to the pore of CFTR identified by three sulfonylurea-based blockers.
Pflugers Arch. 2012 Mar;463(3):405-18. Epub 2011 Dec 13., [PMID:22160394]

Abstract [show]
Previous studies suggested that four transmembrane domains 5, 6, 11, 12 make the greatest contribution to forming the pore of the CFTR chloride channel. We used excised, inside-out patches from oocytes expressing CFTR with alanine-scanning mutagenesis in amino acids in TM6 and TM12 to probe CFTR pore structure with four blockers: glibenclamide (Glyb), glipizide (Glip), tolbutamide (Tolb), and Meglitinide. Glyb and Glip blocked wildtype (WT)-CFTR in a voltage-, time-, and concentration-dependent manner. At V (M) = -120 mV with symmetrical 150 mM Cl(-) solution, fractional block of WT-CFTR by 50 muM Glyb and 200 muM Glip was 0.64 +/- 0.03 (n = 7) and 0.48 +/- 0.02 (n = 7), respectively. The major effects on block by Glyb and Glip were found with mutations at F337, S341, I344, M348, and V350 of TM6. Under similar conditions, fractional block of WT-CFTR by 300 muM Tolb was 0.40 +/- 0.04. Unlike Glyb, Glip, and Meglitinide, block by Tolb lacked time-dependence (n = 7). We then tested the effects of alanine mutations in TM12 on block by Glyb and Glip; the major effects were found at N1138, T1142, V1147, N1148, S1149, S1150, I1151, and D1152. From these experiments, we infer that amino acids F337, S341, I344, M348, and V350 of TM6 face the pore when the channel is in the open state, while the amino acids of TM12 make less important contributions to pore function. These data also suggest that the region between F337 and S341 forms the narrow part of the CFTR pore.

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Sentences [show]
No. Sentence Comment
151 The surprising finding that mutations at six adjacent positions Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT ** ** ** ** ** ** * * * 0.8 0.6 0.4 0.2 0 Fractional block by Glyb50 μM Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT ** ** ** ** ** ** ** ** * * * * * * ** ** Fractional block by Tolb300 μM 0.8 0.6 0.4 0.2 0 Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT * ** ** ** ** ** ** ** ** Fractional block by Glip200 μM 0.8 0.6 0.4 0.2 0 Fig. 3 Alanine-scanning in TM6 to identify the amino acids that interact with the three blockers. Login to comment
166 Double asterisks indicate significantly different compared to WT-CFTR (p<0.01) Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT 0.3 0.2 0.1 0 * * ** ** 0.4 Initial block by 50 μM Glyb Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT 0.4 0.3 0.2 0.1 0 ** ** * Initial block by 200 μM Glip Fig. 5 Initial block of WT-CFTR and selected TM6 mutants by 50 μM Glyb (left) and 200 μM Glip (right) in symmetrical 150 mM Cl- solution. Data are shown only for those mutants which exhibited significant changes in steady-state fractional block according to Fig. 3 (bars show mean±SEM, n=5-10). Login to comment
193 Probable orientation of drugs in the pore Glyb and Glip are identical molecules along most of their lengths, differing only in the substituents on the ring at the Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT 0.8 0.6 0.2 0 ** ** ** ** Time-dependent block by 50 μμM Glyb Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT ** ** * ** * Time-dependent block by 200 μM Glip 0.4 0.8 0.6 0.2 00.4 Fig. 6 Time-dependent block of WT-CFTR and selected TM6 mutants by 50 μM Glyb (left) and 200 μM Glip (right) in symmetrical 150 mM Cl- solution. Data are shown only for those mutants which exhibited significant changes in fractional block according to Fig. 3 (bars show mean±SEM, n=5-10). Login to comment