ABCMdb

PMID: 22722932

Hudson RP, Chong PA, Protasevich II, Vernon R, Noy E, Bihler H, An JL, Kalid O, Sela-Culang I, Mense M, Senderowitz H, Brouillette CG, Forman-Kay JD
Conformational changes relevant to channel activity and folding within the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 2012 Aug 17;287(34):28480-94. doi: 10.1074/jbc.M112.371138. Epub 2012 Jun 21., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC7 p.His620Gln Results: H620Q mutation associated with increased channel Po, and the corrector/potentiator CFFT-001 both lead to similar conformational shifts in NBD1. Login to comment 5 ABCC7 p.His620Gln NMR data on human F508del NBD1 indicate that an H620Q mutant, shown to increase channel open probability, and the dual corrector/potentiator CFFT-001 similarly disrupt interactions between beta-strands S3, S9, and S10 and the C-terminal helices H8 and H9, shifting a preexisting conformational equilibrium from helix to coil. Login to comment 7 ABCC7 p.His620Gln Decreases in Tm from differential scanning calorimetry with H620Q or CFFT-001 suggest direct compound binding to a less thermostable state of NBD1. Login to comment 38 ABCC7 p.Gly551Asp As potential therapies for CF, small-molecule compounds have been sought to address the underlying defects of F508del and G551D, a mutation that impairs gating. Login to comment 40 ABCC7 p.Gly551Asp As potential therapies for CF, small-molecule compounds have been sought to address the underlying defects of F508del and G551D, a mutation that impairs gating. Login to comment 43 ABCC7 p.Gly551Asp The compound VX-770 is a potentiator of G551D (24) and F508del-CFTR (25), as well as several other gating mutations (26, 27). Login to comment 45 ABCC7 p.Gly551Asp The compound VX-770 is a potentiator of G551D (24) and F508del-CFTR (25), as well as several other gating mutations (26, 27). Login to comment 48 ABCC7 p.His620Gln To probe the conformational effects of mutations and binding of small molecule modulators, we have applied a range of biophysical approaches to study H620Q and helix H9 variants of NBD1 and the interaction of the most soluble compound of a series of dual corrector/potentiator compounds provided by the Cystic Fibrosis Foundation, N-cyclohexyl-4-(6-methyl-3-pyridinyl) pyrimidine-2-amine, referred to here as CFFT-001. Login to comment 49 ABCC7 p.His620Gln NMR backbone resonance assignments (82%) carried out on a human F508del NBD1 lacking the RI and ending at residue 646 (F508del NBD1 èc;RIèc;RE) enable us to define conformational changes due to the H620Q mutation. Login to comment 50 ABCC7 p.His620Gln To probe the conformational effects of mutations and binding of small molecule modulators, we have applied a range of biophysical approaches to study H620Q and helix H9 variants of NBD1 and the interaction of the most soluble compound of a series of dual corrector/potentiator compounds provided by the Cystic Fibrosis Foundation, N-cyclohexyl-4-(6-methyl-3-pyridinyl) pyrimidine-2-amine, referred to here as CFFT-001. Login to comment 51 ABCC7 p.His620Gln ABCC7 p.His620Gln NMR backbone resonance assignments (82%) carried out on a human F508del NBD1 lacking the RI and ending at residue 646 (F508del NBD1 ⌬RI⌬RE) enable us to define conformational changes due to the H620Q mutation. Login to comment 52 ABCC7 p.His620Gln Differential scanning calorimetry (DSC) data show a reduction in NBD1 thermal melting temperature for H620Q or in the presence of CFFT-001, demonstrating direct binding of the compound to a less thermostable conformation. Login to comment 53 ABCC7 p.His620Gln Addition of CFFT-001 to F508del NBD1 ⌬RI⌬RE results in NBD1 conformational changes overlapping with those observed for the H620Q mutant protein that has higher open channel probability. Login to comment 54 ABCC7 p.His620Gln Differential scanning calorimetry (DSC) data show a reduction in NBD1 thermal melting temperature for H620Q or in the presence of CFFT-001, demonstrating direct binding of the compound to a less thermostable conformation. Login to comment 55 ABCC7 p.His620Gln EXPERIMENTAL PROCEDURES Protein Expression and Purification (NMR and DSC Studies)- Human NBD1 (387-646, èc;405-436) constructs with or without Phe-508 and containing the H620Q mutation or deletion of helix H9(636-646) were expressed as His6-SUMO fusions at 16 &#b0;C in BL21(DE3) Codon Plus cells grown in minimal media with [15 N]NH4Cl and/or [13 C]glucose, for NMR studies, or LB for DSC studies, and purified as described previously (31, 32). Login to comment 57 ABCC7 p.His620Gln EXPERIMENTAL PROCEDURES Protein Expression and Purification (NMR and DSC Studies)- Human NBD1 (387-646, ⌬405-436) constructs with or without Phe-508 and containing the H620Q mutation or deletion of helix H9(636-646) were expressed as His6-SUMO fusions at 16 °C in BL21(DE3) Codon Plus cells grown in minimal media with [15 N]NH4Cl and/or [13 C]glucose, for NMR studies, or LB for DSC studies, and purified as described previously (31, 32). Login to comment 97 ABCC7 p.His620Gln Repeated measurements for the H620Q F508del sample gave identical Tm values to the first decimal place; the general reproducibility of NBD1 Tm values is within Ϯ0.3 °C. Login to comment 116 ABCC7 p.His620Gln H620Q Variant Reduces Helicity of H8 and H9 at the C Terminus of F508del NBD1 ⌬RI⌬RE-To address possible conformational changes within NBD1 that may be relevant to channel activity and/or misfolding, we focused on the C-terminal beta-strands (S9 and S10) which have been shown to affect processing, activity and pharmacology of CFTR (56). Login to comment 117 ABCC7 p.His620Gln In particular, an H620Q variant (in S9) originally identified in CF patients has an increased Po in single channels (56-58). Login to comment 118 ABCC7 p.His620Gln ABCC7 p.His620Gln ABCC7 p.His620Gln ABCC7 p.His620Gln Fig. 2, A and B, presents an overlay of NMR spectra for F508del NBD1 ⌬RI⌬RE and its H620Q variant (F508del H620Q NBD1 ⌬RI⌬RE). Login to comment 119 ABCC7 p.His620Gln Arrows indicate a subset of peaks that shift upon H620Q mutation, including Glu-621, Gly-622, Gln-634, Leu-636, Ser-641, Leu-644, and Met-645. Login to comment 134 ABCC7 p.His620Gln Chemical shifts in the absence of the mutation demonstrate that these helices are in equilibrium with a coil conformation (supplemental Fig. S4C) (59) and the chemical shift perturbations for the residues of the H8/H9 helices upon replacing His-620 with a Gln reflect a change in the equilibrium further toward the coil conformation. Login to comment 141 ABCC7 p.His620Gln H620Q variant reduces helicity of H8 and H9 at the C terminus of F508del NBD1 èc;RIèc;RE. Login to comment 142 ABCC7 p.His620Gln ABCC7 p.His620Gln ABCC7 p.His620Gln H620Q variant reduces helicity of H8 and H9 at the C terminus of F508del NBD1 ⌬RI⌬RE. Login to comment 143 ABCC7 p.His620Gln ABCC7 p.His620Gln A, overlay of 15 N-1 H correlation spectra at 500 MHz for F508del NBD1 ⌬RI⌬RE (black; background) and the H620Q variant, F508del H620Q NBD1 ⌬RI⌬RE (red; foreground). Login to comment 144 ABCC7 p.His620Gln Asterisks show a separate subset of peaks that shift in the H620Q variant but do not shift upon addition of CFFT-001. Login to comment 145 ABCC7 p.His620Gln Asterisks show a separate subset of peaks that shift in the H620Q variant but do not shift upon addition of CFFT-001. Login to comment 146 ABCC7 p.His620Gln C, same overlay as in B, with a third layer showing the addition of compound to the H620Q variant (green; foreground). Login to comment 147 ABCC7 p.His620Gln C, same overlay as in B, with a third layer showing the addition of compound to the H620Q variant (green; foreground). Login to comment 148 ABCC7 p.His620Gln E, ribbon diagram of WT NBD1 èc;RIèc;RE (2PZE) with unassigned residues in cyan, and assigned residues that do not shift upon H620Q mutation are shown in light gray. Login to comment 149 ABCC7 p.His620Gln E, ribbon diagram of WT NBD1 ⌬RI⌬RE (2PZE) with unassigned residues in cyan, and assigned residues that do not shift upon H620Q mutation are shown in light gray. Login to comment 160 ABCC7 p.His620Gln Comparison of this set of peaks with a subset of those resulting from the H620Q mutation (compare Fig. 2 with Fig. 4) shows significant similarities, including both the identity of perturbed resonances and the direction of the chemical shift changes. Login to comment 161 ABCC7 p.His620Gln ABCC7 p.His620Gln Comparison of this set of peaks with a subset of those resulting from the H620Q mutation (compare Fig. 2 with Fig. 4) shows significant similarities, including both the identity of perturbed resonances and the direction of the chemical shift changes. Login to comment 162 ABCC7 p.His620Gln These similarities are consistent with a common effect of the H620Q substitution and CFFT-001 on H8 and H9, although additional peak shifts reflecting other conformational changes in the mutant are present as well (Fig. 2A). Login to comment 164 ABCC7 p.His620Gln Similar to the H620Q variant, residues on both surfaces of these helices are affected by CFFT-001, as opposed to one surface of the helix as one might expect for a direct drug interaction with the helix. Login to comment 165 ABCC7 p.His620Gln Similar to the H620Q variant, residues on both surfaces of these helices are affected by CFFT-001, as opposed to one surface of the helix as one might expect for a direct drug interaction with the helix. Login to comment 196 ABCC7 p.His620Gln Interestingly, titration of the compound into the F508del H620Q NBD1 èc;RIèc;RE leads to resonances of H9 shifting more toward coil (Fig. 2C, arrows). Login to comment 197 ABCC7 p.His620Gln ABCC7 p.His620Gln Interestingly, titration of the compound into the F508del H620Q NBD1 ⌬RI⌬RE leads to resonances of H9 shifting more toward coil (Fig. 2C, arrows). Login to comment 198 ABCC7 p.His620Gln Thus, the compound further pushes the conformational equilibrium shift already present in the H620Q variant, in a qualitatively additive fashion, and the mutation does not inhibit compound binding. Login to comment 225 ABCC7 p.His620Gln In all of these binding modes, interaction of CFFT-001 with the surface of strands S3, S9, and S10 is not expected to be significantly perturbed by mutation of H620Q, which is adjacent to but not directly at the proposed binding surface (Fig. 6 and supplemental Fig. S6). Login to comment 227 ABCC7 p.His620Gln In all of these binding modes, interaction of CFFT-001 with the surface of strands S3, S9, and S10 is not expected to be significantly perturbed by mutation of H620Q, which is adjacent to but not directly at the proposed binding surface (Fig. 6 and supplemental Fig. S6). Login to comment 228 ABCC7 p.His620Gln The data in Fig. 7A illustrate that both the H620Q variant and the deletion of H9 reduce the thermal stability of NBD1. Login to comment 229 ABCC7 p.His620Gln The midpoint of thermal denaturation, Tm, is reduced by b03;1-2 degrees in each, comparing F508del NBD1 èc;RIèc;RE (Tm in 2 mM ATP afd; 49.4 &#b0;C and 5 mM ATP afd; 51.0 &#b0;C) to either F508del H620Q NBD1 èc;RIèc;RE (bottom curve, Tm in 2 mM ATP afd; 47.1 &#b0;C) or F508del NBD1 èc;RIèc;H9 (middle curve, Tm in 5 mM ATP afd; 50.4 &#b0;C). Login to comment 230 ABCC7 p.His620Gln The data in Fig. 7A illustrate that both the H620Q variant and the deletion of H9 reduce the thermal stability of NBD1. Login to comment 231 ABCC7 p.His620Gln The midpoint of thermal denaturation, Tm, is reduced by ϳ1-2 degrees in each, comparing F508del NBD1 ⌬RI⌬RE (Tm in 2 mM ATP ϭ 49.4 °C and 5 mM ATP ϭ 51.0 °C) to either F508del H620Q NBD1 ⌬RI⌬RE (bottom curve, Tm in 2 mM ATP ϭ 47.1 °C) or F508del NBD1 ⌬RI⌬H9 (middle curve, Tm in 5 mM ATP ϭ 50.4 °C). Login to comment 242 ABCC7 p.His620Gln Overall, the DSC data are in agreement with the effects of deletion of H9, an H620Q variant, and addition of CFFT-001 on NBD1 inferred from NMR data for these constructs. Login to comment 244 ABCC7 p.His620Gln Overall, the DSC data are in agreement with the effects of deletion of H9, an H620Q variant, and addition of CFFT-001 on NBD1 inferred from NMR data for these constructs. Login to comment 248 ABCC7 p.His620Gln A, DSC traces for WT and F508del NBD1 èc;RIèc;RE (upper curves) in the absence (solid lines) and presence (dashed lines) of CFFT-001; buffer includes 2 mM ATP. DSC traces for F508del NBD1 èc;RIèc;H9 (middle curves) in the absence (solid line) and presence (dashed line) of CFFT-001; buffer includes 5 mM ATP. DSC traces for F508del H620Q NBD1 èc;RIèc;RE (lower curve); buffer includes 2 mM ATP. Login to comment 250 ABCC7 p.His620Gln A, DSC traces for WT and F508del NBD1 ⌬RI⌬RE (upper curves) in the absence (solid lines) and presence (dashed lines) of CFFT-001; buffer includes 2 mM ATP. DSC traces for F508del NBD1 ⌬RI⌬H9 (middle curves) in the absence (solid line) and presence (dashed line) of CFFT-001; buffer includes 5 mM ATP. DSC traces for F508del H620Q NBD1 ⌬RI⌬RE (lower curve); buffer includes 2 mM ATP. Login to comment 262 ABCC7 p.His620Gln NMR has enabled us to probe these effects at a residue-specific level and demonstrate that the H620Q substitution associated with higher channel open probability and a dual corrector/potentiator compound give rise to similar conformational changes within NBD1. Login to comment 264 ABCC7 p.His620Gln ABCC7 p.His620Gln NMR has enabled us to probe these effects at a residue-specific level and demonstrate that the H620Q substitution associated with higher channel open probability and a dual corrector/potentiator compound give rise to similar conformational changes within NBD1. Login to comment 266 ABCC7 p.His620Gln ABCC7 p.His620Gln The significance of the DSC results for the H620Q and H9 deletion can be better appreciated in the context of our current understanding of the NBD1 thermal unfolding pathway derived from a comprehensive analysis of previous DSC data on WT and F508del NBD1 (32, 64). Login to comment 268 ABCC7 p.His620Gln The H620Q and ⌬H9 mutations also may reduce the thermodynamic stability of NBD1, or accelerate the rate of aggregation, or both. Login to comment 281 ABCC7 p.His620Gln The H620Q variant, originally identified in CF patients demonstrating pancreatic insufficiency, has previously been shown to increase the Po of single CFTR channels, indicating an effect on the gating properties of CFTR (56-58). Login to comment 282 ABCC7 p.His620Gln Both the H620Q variant and the CFFT-001 compound cause a shift of helices H8 and H9 from a preexisting helix-coil conformational equilibrium toward the coil state. These results suggest that perturbations affecting this conformational equilibrium coincide with conditions that promote channel opening and/or impede channel closing. Login to comment 283 ABCC7 p.His620Gln ABCC7 p.His620Gln The H620Q variant, originally identified in CF patients demonstrating pancreatic insufficiency, has previously been shown to increase the Po of single CFTR channels, indicating an effect on the gating properties of CFTR (56-58). Login to comment 284 ABCC7 p.His620Gln Both the H620Q variant and the CFFT-001 compound cause a shift of helices H8 and H9 from a preexisting helix-coil conformational equilibrium toward the coil state. These results suggest that perturbations affecting this conformational equilibrium coincide with conditions that promote channel opening and/or impede channel closing. Login to comment 285 ABCC7 p.His620Gln ABCC7 p.His620Gln ABCC7 p.His620Pro Because the H620Q variant displays additional peak shifts relative to those observed for compound binding, there are certainly other consequences of the mutation. Login to comment 287 ABCC7 p.His620Gln ABCC7 p.His620Pro Although H620Q does not affect maturation/processing, an H620P mutation does, highlighting the importance of this position and the nature of the residue in it. Login to comment 292 ABCC7 p.His620Gln Thus, we hypothesize that conformational changes, as elicited by H620Q or CFFT-001, result in a shift in an underlying conformational equilibrium of regulatory interactions that are normally involved in gating. Login to comment 294 ABCC7 p.His620Gln ABCC7 p.His620Gln Thus, we hypothesize that conformational changes, as elicited by H620Q or CFFT-001, result in a shift in an underlying conformational equilibrium of regulatory interactions that are normally involved in gating. Login to comment 295 ABCC7 p.His620Gln It is possible that H620Q and CFFT-001 act to force an "unnatural" gating; however, the likelihood of this is low, considering that the portion of NBD1 affected appears to be a regulatory hot spot based on our sequence analysis and the observed effects of mutations here (56). Login to comment 296 ABCC7 p.His620Gln This should promote NBD dimerization and lead to an enhanced open probability, increased channel activity for H620Q mutant channels and the potentiating effect of CFFT-001. Login to comment 297 ABCC7 p.His620Gln It is possible that H620Q and CFFT-001 act to force an "unnatural" gating; however, the likelihood of this is low, considering that the portion of NBD1 affected appears to be a regulatory hot spot based on our sequence analysis and the observed effects of mutations here (56). Login to comment 303 ABCC7 p.His620Gln Addition of compound or the H620Q mutation shifts this equilibrium by reducing H9 helicity and contacts with the NBD1, subsequently leading to release of the RE/R region from the dimerization interface, relieving the inhibition and facilitating NBD1/NBD2 heterodimerization and channel opening. Login to comment 305 ABCC7 p.His620Gln Addition of compound or the H620Q mutation shifts this equilibrium by reducing H9 helicity and contacts with the NBD1, subsequently leading to release of the RE/R region from the dimerization interface, relieving the inhibition and facilitating NBD1/NBD2 heterodimerization and channel opening. Login to comment 306 ABCC7 p.Gly622Asp ABCC7 p.Gly628Arg Mutations of residues in these C-terminal strands of NBD1, specifically G622D and G628R, have been demonstrated to perturb the pharmacological effects of dual "MPB (benzo(c)- quinolizinium)" compounds, characterized by their ability to both activate CFTR and rescue defective trafficking (56). Login to comment 308 ABCC7 p.Gly622Asp ABCC7 p.Gly628Arg Mutations of residues in these C-terminal strands of NBD1, specifically G622D and G628R, have been demonstrated to perturb the pharmacological effects of dual "MPB (benzo(c)- quinolizinium)" compounds, characterized by their ability to both activate CFTR and rescue defective trafficking (56). Login to comment 313 ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys A similar argument can be made for an underlying allosteric mechanism of suppression by the "3M" (G550E/R553M/R555K) mutations (17) which can improve CFTR processing in the absence of F508; even though they do not directly address the structural changes at the Phe-508 site, these mutations apparently change the equilibrium distribution of conformations accessed by NBD1 to be more similar to that of wild-type. Login to comment 314 ABCC7 p.His620Gln The large number of substitutions in NBD1 that can suppress the F508del mutation supports such a general allosteric view of NBD1 with the structural changes we observe in the H620Q variant and upon CFFT-001 binding being one part of the NBD1 conformational equilibria. Login to comment 315 ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys A similar argument can be made for an underlying allosteric mechanism of suppression by the "3M" (G550E/R553M/R555K) mutations (17) which can improve CFTR processing in the absence of F508; even though they do not directly address the structural changes at the Phe-508 site, these mutations apparently change the equilibrium distribution of conformations accessed by NBD1 to be more similar to that of wild-type. Login to comment 316 ABCC7 p.His620Gln The large number of substitutions in NBD1 that can suppress the F508del mutation supports such a general allosteric view of NBD1 with the structural changes we observe in the H620Q variant and upon CFFT-001 binding being one part of the NBD1 conformational equilibria. Login to comment 318 ABCC7 p.His620Gln Because the H620Q mutation and CFFT-001 are both associated with an increase in the open probability of CFTR channels and because H8/H9 lead directly into the RE/R region within the context of full-length CFTR, we have hypothesized that this conformational shift at H8/H9 releases the R region from the NBD1/ NBD2 dimerization interface, allowing heterodimers to form and thereby enhancing channel open probability and possibly processing. Login to comment 320 ABCC7 p.His620Gln Because the H620Q mutation and CFFT-001 are both associated with an increase in the open probability of CFTR channels and because H8/H9 lead directly into the RE/R region within the context of full-length CFTR, we have hypothesized that this conformational shift at H8/H9 releases the R region from the NBD1/ NBD2 dimerization interface, allowing heterodimers to form and thereby enhancing channel open probability and possibly processing. Login to comment