ABCMdb

PMID: 22923500

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., [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCC7 p.Phe342Ala 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 51 ABCC7 p.Phe342Ala 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 ABCC7 p.Phe342Ala 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 73 ABCC7 p.Phe342Ala 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 ABCC7 p.Phe342Ala 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 108 ABCC7 p.Phe337Cys In this example, externally-applied [Au(CN)2]- was reacted with F337C CFTR. Login to comment 115 ABCC7 p.Thr338Cys ABCC7 p.Phe337Cys Figure 3C and 3D contain the time courses for the reactions of [Au(CN)2]- with F337C and T338C CFTR pre-and post-activation. Login to comment 116 ABCC7 p.Thr338Cys In the case of T338C CFTR, different concentrations of [Au(CN)2]- were used to construct the time course (50 µM for the pre-activation rate; 5 µM for the post-activation rate), in order to compensate for the slower reaction rate in the pre-activation condition. Login to comment 125 ABCC7 p.Arg334Cys Figure 4 shows the time courses for the reactions of [Au(CN)2]- and MTSET+ with R334C CFTR channels. Login to comment 127 ABCC7 p.Thr338Cys When either Phe337 or Thr338 was replaced with cysteine, the EC50 for GlyH-101 blockade at 0 mV was only modestly increased (Table 1).1 Increasing the bulk of either cysteine side chain through alkylation with iodoacetamide further increased the EC50(0) b03;6-fold for position 338 but only 1.3-fold for position 337. Login to comment 130 ABCC7 p.Arg334Cys ABCC7 p.Lys95Cys Beck et al., (2008) also studied R334C CFTR channels expressed in Xenopus oocytes using a protocol similar to that employed here, but failed to detect increased reactivity toward externally-applied MTSEA+ in the activated state. Login to comment 138 ABCC7 p.Thr338Cys Figure 5A contains a representative time course showing the cumulative effect of 30 s and 1 min applications of 5 µM [Au(CN)2]- to an oocyte expressing T338C CFTR. Login to comment 140 ABCC7 p.Thr338Cys Inspection of the response to the first, 30s exposure reveals that the extent of modification of T338C conductance by [Au(CN)2]- was markedly reduced when the reagent was applied in the presence of the blocker. Login to comment 141 ABCC7 p.Thr338Cys ABCC7 p.Phe337Cys Figure 5C and 5D summarize the inhibition of F337C CFTR and T338C CFTR by [Au(CN)2]- in the presence and in the absence of GlyH-101. Login to comment 144 ABCC7 p.Thr338Cys ABCC7 p.Phe337Cys In Figure 5E and 5F the measured second order rate constants for covalent modification of F337C and T338C CFTR are plotted versus GlyH-101 concentration. Login to comment 148 ABCC7 p.Thr338Cys As a test for the role of charge-charge interactions we compared the protection of T338C CFTR against reaction with MTSET+ and MTSES- . Login to comment 149 ABCC7 p.Thr338Cys The results summarized in Figure 6 show that T338C is protected by GlyH-101 from negatively-charged MTSES- but not from positively-charged MTSET+ , indicating that the charge on the blocker is a major determinant of the protection effect at this position. Login to comment 151 ABCC7 p.Phe337Cys ABCC7 p.Ile1131Cys We also studied the reaction of I1131C CFTR with MTSES- . Login to comment 154 ABCC7 p.Ile1131Cys ABCC7 p.Ile1131Cys In fact, no occlusion of I1131C was observed with 10 µM GlyH-101, despite more than 80% block of the I1131C CFTR current. Login to comment 155 ABCC7 p.Ile1131Cys The rate of reaction between MTSES- and I1131C CFTR was actually somewhat greater in the presence of GlyH-101 (Figure 7A). Login to comment 156 ABCC7 p.Ile1131Cys The reaction rates of charged reagents with I1131C CFTR would be influenced by relative positions of nearby charges, which may change slightly in the event of binding of GlyH-101. Login to comment 158 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Lys95Cys ABCC7 p.Phe337Cys ABCC7 p.Phe337Cys ABCC7 p.Phe337Cys Figure 3, C and D, contains the time courses for the reactions of [Au(CN)2]afa; with the F337C and T338C CFTRs before TABLE 1 EC50 at 0 mV (mean afe; S.E.M.) for GlyH-101 for wt and mutant CFTRs, with and without modification with iodoacetamide CFTR EC50 at 0 mV òe;M wt 1.1 afe; 0.11 (n afd; 4) K95C 1.4 afe; 0.35 (n afd; 4) F337C 1.8 afe; 0.06 (n afd; 3) F337C af9; iodoacetamide 2.4 afe; 0.29 (n afd; 3) T338C 3.7 afe; 0.27 (n afd; 3) T338C af9; iodoacetamide 24 afe; 2.6 (n afd; 3) Fig. 3. Login to comment 159 ABCC7 p.Phe337Cys ABCC7 p.Ile1131Cys The presence of negative charges near I1131 is consistent with the observed slow reaction of I1131C CFTR with MTSES- (20 M-1 sec-1 ; Figure 7) which is more than 100-fold less than that seen for a Cys at 338 (3.3 x 103 M-1 sec-1 ; Figure 6). Login to comment 160 ABCC7 p.Phe337Cys ABCC7 p.Phe337Cys ABCC7 p.Ile1131Cys As reported previously (Norimatsu et al., 2012) the macroscopic conductance of I1131C CFTR was increased by depositing the negatively charged sulfonic acid group via reaction with MTSES- . Login to comment 161 ABCC7 p.Arg352Cys ABCC7 p.Ile1131Cys This result is most likely a reflection of an increase in the open probability of I1131C CFTR channels similar to that observed for R352C CFTR by Bai et al., (2010). Login to comment 162 ABCC7 p.Phe337Cys The subsequent application of [Au(CN)2]afa; almost completely abolished F337C CFTR conductance. Login to comment 163 ABCC7 p.Thr338Cys ABCC7 p.Phe337Cys C and D, time courses of the decreases in normalized conductance as a result of F337C (C) and T338C (D) modifications with [Au(CN)2]afa; . Login to comment 164 ABCC7 p.Ser341Cys S341C CFTR is reactive toward channel permeant reagents, like [Ag(CN)2]- , but the reactions are not irreversible at this position, a requirement for implementation of the post-GlyH-101 washout protocol. Login to comment 165 ABCC7 p.Phe337Cys For the F337C CFTR, the abscissa represents cumulative [Au(CN)2]afa; exposure time. Login to comment 166 ABCC7 p.Thr338Cys For the T338C CFTR, the abscissa represents cumulative [Au(CN)2]afa; exposure (exposure time afb; [Au(CN)2]afa; concentration used). Login to comment 167 ABCC7 p.Phe337Cys The reaction rate for the F337C CFTR before activation of the channels was b03;20 times slower than the rate after activation. Login to comment 168 ABCC7 p.Thr338Cys The rate for the T338C CFTR was almost 30 times slower before activation. Login to comment 170 ABCC7 p.Phe342Ala 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 171 ABCC7 p.Phe337Cys The second-order reaction rate constants for the F337C CFTR before and after activation were 1.5 and 26 Mafa;1 safa;1 , respectively. Login to comment 172 ABCC7 p.Thr338Cys The second-order reaction rate constants for the T338C CFTR before and after activation were 1.2 afb; 102 and 3.0 afb; 103 Mafa;1 safa;1 , respectively. Login to comment 174 ABCC7 p.Thr338Cys ABCC7 p.Phe342Ala 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 ABCC7 p.Phe342Ala 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 ABCC7 p.Phe342Ala The estimated parameter values for wt and F342A are collected in Table 2. Login to comment 178 ABCC7 p.Phe342Ala ABCC7 p.Phe342Ala 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 ABCC7 p.Phe342Ala 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 ABCC7 p.Phe342Ala 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 ABCC7 p.Phe342Ala 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 ABCC7 p.Arg334Cys ABCC7 p.Phe342Ala 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 188 ABCC7 p.Arg334Cys Beck et al. (2008) studied R334C CFTR channels expressed in X. laevis oocytes by using a protocol similar to that used here, but they failed to detect increased reactivity toward externally applied 2-aminoethylmethanethiosulfonate in the activated state. Login to comment 196 ABCC7 p.Arg334Cys Time courses of reactions of R334C (an engineered cysteine at position 334) with 10 òe;M [Au(CN)2]afa; (A) or 250 nM MTSETaf9; (B). Login to comment 197 ABCC7 p.Phe342Ala ABCC7 p.Phe342Ala ABCC7 p.Phe342Ala 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 ABCC7 p.Phe342Ala 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 ABCC7 p.Phe342Ala 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 201 ABCC7 p.Arg334Cys For both [Au(CN)2]afa; and MTSETaf9; , the reaction rate for the R334C CFTR before activation of the channels was faster than the rate after activation. Login to comment 204 ABCC7 p.Thr338Cys and 1-min applications of 5 òe;M [Au(CN)2]afa; to an oocyte expressing T338C CFTR. Login to comment 205 ABCC7 p.Thr338Cys A similar procedure, in which [Au(CN)2]afa; was applied during brief exposures of the oocyte to GlyH-101 and the extent of the reaction was estimated from the conductance recovered after washout of the blocker, is illustrated in Fig. 5B. Inspection of the response to the first, 30-s exposure revealed that the extent of modification of T338C CFTR conductance by [Au(CN)2]afa; was markedly reduced when the reagent was applied in the presence of the blocker. Login to comment 206 ABCC7 p.Thr338Cys ABCC7 p.Phe337Cys Figure 5, C and D, summarizes the inhibition of the F337C and T338C CFTRs by [Au(CN)2]afa; in the presence and absence of GlyH-101. Login to comment 209 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Phe337Cys Second, alkylation of T338C CFTR with IAM, which results in covalent addition of an acetamide moiety predicted by the model to create a steric clash with GlyH-101, significantly reduced the apparent binding affinity of GlyH-101. Login to comment 210 ABCC7 p.Thr338Cys ABCC7 p.Phe337Cys In contrast, alkylation of F337C CFTR with IAM is not predicted by the molecular model to cause a steric clash and does not markedly alter GlyH-101 block. Login to comment 211 ABCC7 p.Thr338Cys ABCC7 p.Phe342Ala 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 215 ABCC7 p.Thr338Cys ABCC7 p.Phe337Cys C and D, F337C (C) and T338C (D) CFTR channels were protected by 10 òe;M GlyH-101 from reactions with [Au(CN)2]afa; . Login to comment 217 ABCC7 p.Thr338Cys ABCC7 p.Phe337Cys The F337C CFTR was reacted with 600 òe;M [Au(CN)2]afa; and the T338C CFTR was reacted with 5 òe;M [Au(CN)2]afa; in the presence and absence of 10 òe;M GlyH-101. Login to comment 220 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Glu1371Gln ABCC7 p.Phe337Cys The state-dependent reactivity of T338C CFTR observed in the current study is consistent with the finding of Beck et al., (2008) that MTSES- reacts slightly faster with a high open probability mutant T338C/E1371Q CFTR than with T338C/wt CFTR.2 Mornon et al., (2009) created a homology model of CFTR based on the inward-facing conformation of a prokaryotic transporter, MsbA (Ward et al., 2007) (PDB code: 3B5X). Login to comment 224 ABCC7 p.Thr338Cys ABCC7 p.Phe337Cys A conformational change of this sort would be consistent with the state-dependent reactivity of F337C and T338C CFTR observed in the current study. Login to comment 225 ABCC7 p.Arg334Cys ABCC7 p.Thr338Cys ABCC7 p.Phe337Cys The MsbA-based model of Mornon et al., (2009) also predicts that the side chain of R334 protrudes into the external aqueous environment, and when R334 is mutated to a cysteine in the MsbA-based model of Mornon et al., (2009) using Maestro (version 9.1, Schrödinger LLC), the reactive thiolate is clearly accessible from the extracellular solution (Figure 9C), consistent with the closed state reactivity of R334C observed in the current study and by Zhang et al., (2005). Login to comment 226 ABCC7 p.Arg334Cys ABCC7 p.Thr338Cys ABCC7 p.Phe337Cys On the other hand, the mechanism that renders R334C CFTR unreactive in the conducting state of CFTR is not clear. Login to comment 228 ABCC7 p.Arg334Cys The R334C mutation may cause a significant conformational change to the CFTR protein (not captured in our MD simulation), as suggested by Zhou et al., (2007), that renders the engineered cysteine inaccessible in the conducting state of CFTR. Login to comment 231 ABCC7 p.Thr338Cys As a test for the role of charge-charge interactions, we compared the protection of the T338C CFTR against reactions with MTSETaf9; and MTSESafa; . Login to comment 232 ABCC7 p.Thr338Cys The results summarized in Fig. 6 showed that the T338C CFTR was protected by GlyH-101 from negatively charged MTSESafa; but not from positively charged MTSETaf9; , indicating that the charge on the blocker is a major determinant of the protective effect at this position. Login to comment 234 ABCC7 p.Ile1131Cys We also studied the reaction of the I1131C CFTR with MTSESafa; . Login to comment 237 ABCC7 p.Ile1131Cys ABCC7 p.Ile1131Cys No occlusion of I1131C was observed with 10 òe;M GlyH-101, despite more than 80% blockade of the I1131C CFTR current. Login to comment 238 ABCC7 p.Ile1131Cys The rate of reaction between MTSESafa; and the I1131C CFTR was actually somewhat greater in the presence of GlyH-101 (Fig. 7A). Login to comment 239 ABCC7 p.Ile1131Cys The rates of reactions of charged reagents with the I1131C CFTR would be influenced by the relative positions of nearby charges, which might change slightly upon GlyH-101 binding. Login to comment 242 ABCC7 p.Ile1131Cys The presence of negative charges near Ile1131 is consistent with the observed slow reaction of the I1131C CFTR with MTSESafa; (20 Mafa;1 safa;1 ) (Fig. 7), which is more than 100-fold less than the rate seen with a cysteine at position 338 (3.3 afb; 103 Mafa;1 safa;1 ) (Fig. 6). Login to comment 243 ABCC7 p.Ile1131Cys As reported previously (Norimatsu et al., 2012), the macroscopic conductance of the I1131C CFTR was increased by deposition of the negatively charged sulfonic acid group through reaction with MTSESafa; . Login to comment 244 ABCC7 p.Arg352Cys ABCC7 p.Ile1131Cys This result is most likely a reflection of an increase in the open probability of I1131C CFTR channels, similar to that observed for the R352C CFTR (Bai et al., 2010). Login to comment 247 ABCC7 p.Ser341Cys The S341C CFTR is reactive toward channel-permeant reagents such as [Ag(CN)2]afa; but the reactions at this position are not irreversible, which is a requirement for implementation of the post-GlyH-101 washout protocol. Login to comment 250 ABCC7 p.Thr338Cys GlyH-101 protection of T338C (an engineered cysteine at position 338) from MTSESafa; but not from MTSETaf9; . Login to comment 251 ABCC7 p.Thr338Cys A, the T338C CFTR was reacted with 5 òe;M MTSESafa; in the presence and absence of 10 òe;M GlyH-101. Data points represent mean afe; S.E.M. (n afd; 3). Login to comment 255 ABCC7 p.Thr338Cys The second-order reaction rate constants for MTSESafa; in the presence and absence of 10 òe;M GlyH-101 were 1.2 afb; 103 and 3.3 afb; 103 Mafa;1 safa;1 , respectively. B, the T338C CFTR was reacted with 50 òe;M MTSETaf9; in the presence and absence of 10 òe;M GlyH-101. Data points represent mean afe; S.E.M. (n afd; 3). Login to comment 262 ABCC7 p.Phe342Ala The GlyH-101 EC50 Is Reduced Approximately 200-Fold with F342A CFTR. Login to comment 267 ABCC7 p.Phe342Ala 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 ABCC7 p.Phe342Ala 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 ABCC7 p.Phe342Ala The estimated parameter values for wt and F342A CFTRs are presented in Table 2. Login to comment 271 ABCC7 p.Phe342Ala ABCC7 p.Phe342Ala 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 273 ABCC7 p.Ile1131Cys A, time courses of the reactions of the I1131C CFTR with 1 mM MTSESafa; in the presence and absence of 10 òe;M GlyH-101. Data points represent mean afe; S.E.M. (n afd; 3). Login to comment 274 ABCC7 p.Ile1131Cys Covalent labeling of the I1131C CFTR with MTSESafa; resulted in increases in conductance. Login to comment 278 ABCC7 p.Ile1131Cys The EC50 at 0 mV for GlyH-101 blockade for the I1131C CFTR was 0.86 afe; 0.016 òe;M (n afd; 3). Login to comment 279 ABCC7 p.Ser341Cys B, time courses of the reactions of the S341C CFTR with 1 mM NEM in the presence and absence of 10 òe;M GlyH-101. Data points represent mean afe; S.E.M. (n afd; 3). Login to comment 280 ABCC7 p.Ser341Cys Covalent labeling of the S341C CFTR with NEM resulted in reductions in conductance. Login to comment 284 ABCC7 p.Ser341Cys The EC50 at 0 mV for GlyH-101 blockade for the S341C CFTR was 0.89 afe; 0.056 òe;M (n afd; 3). Login to comment 285 ABCC7 p.Phe342Ala 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 ABCC7 p.Phe342Ala ABCC7 p.Phe342Ala ABCC7 p.Phe342Ala ABCC7 p.Phe342Ala ABCC7 p.Phe342Ala 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 ABCC7 p.Phe342Ala 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 ABCC7 p.Phe342Ala 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 ABCC7 p.Phe342Ala 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 ABCC7 p.Phe342Ala ABCC7 p.Phe342Ala ABCC7 p.Phe342Ala 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 ABCC7 p.Phe342Ala 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 ABCC7 p.Phe342Ala 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 317 ABCC7 p.Thr338Cys Second, alkylation of the T338C CFTR with iodoacetamide, which results in covalent addition of an acetamide moiety that is predicted by the model to create a steric clash with GlyH-101, significantly reduced the apparent binding affinity of GlyH-101. Login to comment 318 ABCC7 p.Phe337Cys In contrast, alkylation of the F337C CFTR with iodoacetamide was not predicted by the molecular model to cause a steric clash and did not alter GlyH-101 blockade markedly. Login to comment 319 ABCC7 p.Phe342Ala 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 ABCC7 p.Phe342Ala F342A CFTR transmembrane currents. Login to comment 323 ABCC7 p.Phe342Ala 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 ABCC7 p.Phe342Ala 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 346 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Glu1371Gln The state-dependent reactivity of the T338C CFTR that was observed in the current study is consistent with the finding by Beck et al. (2008) that MTSESafa; reacted slightly faster with a mutant with high open probability (T338C/ E1371Q CFTR) than with the T338C/wt CFTR.2 Mornon et al. (2009) created a homology model of the CFTR that was based on the inward-facing conformation of the prokaryotic transporter MsbA (PDB code 3B5X) (Ward et al., 2007). Login to comment 350 ABCC7 p.Thr338Cys ABCC7 p.Phe337Cys A conformational change of this sort would be consistent with the state-dependent reactivity of the F337C and T338C CFTRs observed in the current study. Login to comment 352 ABCC7 p.Thr338Cys ABCC7 p.Glu1371Gln Wang and Linsdell (2012) studied reactions of the T338C/E1371Q CFTR with MTSESafa; and [Au(CN)2]afa; and suggested that the reaction of an engineered cysteine at position 338 with externally applied reagents was favored in the closed state. Login to comment 353 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Glu1371Gln However, this observation conflicts with that by Beck et al. (2008), who reported that MTSESafa; reacted slightly faster with a double-mutant with high open probability (T338C/E1371Q CFTR) than with the T338C/wt CFTR. Login to comment 354 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Glu1371Gln We observed reaction rates for MTSESafa; with T338C/wt and T338C/E1371Q CFTRs that were similar to those observed by Beck et al. (2008) (data not shown), which supports the idea that the T338C CFTR reacts in the open state. Login to comment 356 ABCC7 p.Arg334Cys Cells expressing the R334C CFTR were preincubated with MTSESafa; with and without an activating cocktail of forskolin and IBMX, and the reaction, which was inferred on the basis of changes in the rectification ratio of the CFTR I-V curve, was observed only with cells exposed to the activating cocktail. Login to comment 368 ABCC7 p.Arg334Cys with Maestro 9.1 (Schro &#a8;dinger) in the MsbA-based model, the reactive thiolate is clearly accessible from the extracellular solution (Fig. 9C), which is consistent with the closed-state reactivity of R334C observed in the current study and in the study by Zhang et al. (2005). Login to comment 369 ABCC7 p.Arg334Cys The mechanism that renders the R334C CFTR unreactive in the conducting state is not clear. Login to comment 371 ABCC7 p.Arg334Cys The R334C mutation may cause significant conformational changes in the CFTR protein (that are not captured in our MD simulation), as suggested by Zhou et al. (2007), which render the engineered cysteine inaccessible in the conducting state of the CFTR. Login to comment