ABCMdb

ABCC7 p.Ser492Pro

ClinVar:	c.1475C>T , p.Ser492Phe D , Pathogenic
CF databases:	c.1475C>T , p.Ser492Phe D , CF-causing ; CFTR1: The nucleotide change destroys a DdeI site in the PCR product obtained with 10i3 and 10i5. We have found it on one CF chromosomes among 87 tested through DGGE and DNA sequencing. The patient is 20 years old and PS, he carries the [delta]F508 on the other chromosome.
Predicted by SNAP2:	A: D (66%), C: D (80%), D: D (91%), E: D (91%), F: D (63%), G: D (75%), H: D (95%), I: D (95%), K: D (91%), L: D (91%), M: D (95%), N: D (80%), P: D (66%), Q: D (85%), R: D (91%), T: D (75%), V: D (91%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: D, G: N, H: N, I: D, K: N, L: D, M: N, N: N, P: N, Q: N, R: N, T: N, V: N, W: D, Y: D,

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Publications
[hide] Allosteric modulation balances thermodynamic stabi... J Mol Biol. 2012 May 25;419(1-2):41-60. Epub 2012 Mar 8. Aleksandrov AA, Kota P, Cui L, Jensen T, Alekseev AE, Reyes S, He L, Gentzsch M, Aleksandrov LA, Dokholyan NV, Riordan JR
Allosteric modulation balances thermodynamic stability and restores function of DeltaF508 CFTR.
J Mol Biol. 2012 May 25;419(1-2):41-60. Epub 2012 Mar 8., [PMID:22406676]

Abstract [show]
Most cystic fibrosis is caused by a deletion of a single residue (F508) in CFTR (cystic fibrosis transmembrane conductance regulator) that disrupts the folding and biosynthetic maturation of the ion channel protein. Progress towards understanding the underlying mechanisms and overcoming the defect remains incomplete. Here, we show that the thermal instability of human DeltaF508 CFTR channel activity evident in both cell-attached membrane patches and planar phospholipid bilayers is not observed in corresponding mutant CFTRs of several non-mammalian species. These more stable orthologs are distinguished from their mammalian counterparts by the substitution of proline residues at several key dynamic locations in first N-terminal nucleotide-binding domain (NBD1), including the structurally diverse region, the gamma-phosphate switch loop, and the regulatory insertion. Molecular dynamics analyses revealed that addition of the prolines could reduce flexibility at these locations and increase the temperatures of unfolding transitions of DeltaF508 NBD1 to that of the wild type. Introduction of these prolines experimentally into full-length human DeltaF508 CFTR together with the already recognized I539T suppressor mutation, also in the structurally diverse region, restored channel function and thermodynamic stability as well as its trafficking to and lifetime at the cell surface. Thus, while cellular manipulations that circumvent its culling by quality control systems leave DeltaF508 CFTR dysfunctional at physiological temperature, restoration of the delicate balance between the dynamic protein's inherent stability and channel activity returns a near-normal state.

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128 (c) Western blot of WT and ΔF508 CFTR expressed in HEK-293 cells and ΔF508 modified with I539T (ΔF/T), S422P/S434P/S492P/A534P (ΔF/4P), I539T/S492P (ΔF/PT), I539T/S492P/A534P (ΔF/2PT), and I539T/S422P/S434P/S492P/A534P (ΔF/4PT). Login to comment
136 Thermostable binding of the nucleotide by the WT, which is lost in ΔF508 CFTR,19 was not restored after rescue in cells grown at 27 °C [(r)ΔF panel], but was to some extent by I539T (ΔF/T panel), to a greater extent when S492P was added (ΔF/PT panel), still further with A534P also added (ΔF/2PT panel), and to near the WT level with proline replacements at residues S492 and A534 as well (ΔF/4PT panel). Login to comment
140 The addition of the S492P substitution generated a dominant full-conductance behavior (ΔF/PT), with a Po approximately half of the WT human channel at that temperature. Login to comment
143 Addition of S492P to form ΔF/PT resulted in WT-like full-conductance gating at temperatures up to approximately 37 °C, above which there was a transition to the ffm (middle tracing). Login to comment
144 Thus, as observed in the 35 °C fixed temperature recording (Fig. 6a), introduction of the Q-loop proline (S492P) had a significant stabilizing effect. Login to comment
178 HEK293 cells were transiently transfected with Cys-less CFTR or Cys-less ΔF508-CFTR in the presence or absence of the 4PT mutations (S422P/S434P/S492P/A534P/I539T), with the Cys pair V510C/G1069C introduced at the CL4/NBD1 interface. Login to comment
216 Introduction of a proline at S492 in the context of I539T increases the folding transition temperature of ΔF508 NBD1 to ~327 K, consistent with the observed decrease in thermal fluctuations upon S492P substitution (Fig. 9b, orange broken line). Login to comment
230 The apparent additive effects of several substituted prolines acting independently have been observed in other proteins as well.47 Molecular dynamics simulations reveal that the SDR of NBD1 is stabilized by the S492P substitution, and the stability further increases with each proline substitution. Login to comment

[hide] Correctors of DeltaF508 CFTR restore global confor... FASEB J. 2013 Feb;27(2):536-45. doi: 10.1096/fj.12-216119. Epub 2012 Oct 26. He L, Kota P, Aleksandrov AA, Cui L, Jensen T, Dokholyan NV, Riordan JR
Correctors of DeltaF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein.
FASEB J. 2013 Feb;27(2):536-45. doi: 10.1096/fj.12-216119. Epub 2012 Oct 26., [PMID:23104983]

Abstract [show]
Most cystic fibrosis is caused by the deletion of a single amino acid (F508) from CFTR and the resulting misfolding and destabilization of the protein. Compounds identified by high-throughput screening to improve DeltaF508 CFTR maturation have already entered clinical trials, and it is important to understand their mechanisms of action to further improve their efficacy. Here, we showed that several of these compounds, including the investigational drug VX-809, caused a much greater increase (5- to 10-fold) in maturation at 27 than at 37 degrees C (<2-fold), and the mature product remained short-lived (T(1/2) approximately 4.5 h) and thermally unstable, even though its overall conformational state was similar to wild type, as judged by resistance to proteolysis and interdomain cross-linking. Consistent with its inability to restore thermodynamic stability, VX-809 stimulated maturation 2-5-fold beyond that caused by several different stabilizing modifications of NBD1 and the NBD1/CL4 interface. The compound also promoted maturation of several disease-associated processing mutants on the CL4 side of this interface. Although these effects may reflect an interaction of VX-809 with this interface, an interpretation supported by computational docking, it also rescued maturation of mutants in other cytoplasmic loops, either by allosteric effects or via additional sites of action. In addition to revealing the capabilities and some of the limitations of this important investigational drug, these findings clearly demonstrate that DeltaF508 CFTR can be completely assembled and evade cellular quality control systems, while remaining thermodynamically unstable. He, L., Kota, P., Aleksandrov, A. A., Cui, L., Jensen, T., Dokholyan, N. V., Riordan, J. R. Correctors of DeltaF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein.

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85 Therefore, it serves as a basis for the comparison of the influence of the VX-809 compound with that of a known stabilizing second-site mutation, S492P (24). Login to comment
88 This behavior strongly contrasted that of the èc;F508/I539T variant with the stabilizing S492P mutation added, where transport capacity increased, and full conductance state persisted up to 35&#b0;C (compare tracings in Fig. 2Biii, iv). Login to comment
98 B) Single-channel recordings of èc;F508/I539T CFTR (èc;F/T) rescued by 3 òe;M VX-809 at 35&#b0;C (i) and 25&#b0;C (ii) and of èc;F508/I539T/S492P CFTR (èc;F/PT) as an example of an already known (24) alternative type of èc;F508/I539T, thermally stabilized by proline substitutions at 35&#b0;C (iii) and 25&#b0;C (iv). Login to comment
101 Two sets of 4 independent experiments of 35 and 38 min total time were used to estimate transport capacity ᐹॹᐺ afd; 0.85 afe; 0.26 at 25&#b0;C (iii) and ᐹॹᐺ afd; 3.14 afe; 0.32 at 35&#b0;C (iv) for èc;F508/I539T/S492P CFTR. Login to comment
122 4PT, S422P/S434P/S492P/ A534P/I539T. Login to comment
148 A) èc;F508 with NBD1-stabilizing mutations: 4S, I539T/G550E/R553M/R555K; èc;RI, deletion of amino acid residues 404-435; 4PT, S422P/S434P/S492P/A534P/I539T. Login to comment

[hide] Restoration of NBD1 thermal stability is necessary... J Mol Biol. 2015 Jan 16;427(1):106-20. doi: 10.1016/j.jmb.2014.07.026. Epub 2014 Jul 30. He L, Aleksandrov AA, An J, Cui L, Yang Z, Brouillette CG, Riordan JR
Restoration of NBD1 thermal stability is necessary and sufficient to correct F508 CFTR folding and assembly.
J Mol Biol. 2015 Jan 16;427(1):106-20. doi: 10.1016/j.jmb.2014.07.026. Epub 2014 Jul 30., [PMID:25083918]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) (ABCC7), unique among ABC exporters as an ion channel, regulates ion and fluid transport in epithelial tissues. Loss of function due to mutations in the cftr gene causes cystic fibrosis. The most common cystic-fibrosis-causing mutation, the deletion of F508 (DeltaF508) from the first nucleotide binding domain (NBD1) of CFTR, results in misfolding of the protein and clearance by cellular quality control systems. The DeltaF508 mutation has two major impacts on CFTR: reduced thermal stability of NBD1 and disruption of its interface with membrane-spanning domains (MSDs). It is unknown if these two defects are independent and need to be targeted separately. To address this question, we varied the extent of stabilization of NBD1 using different second-site mutations and NBD1 binding small molecules with or without NBD1/MSD interface mutation. Combinations of different NBD1 changes had additive corrective effects on F508 maturation that correlated with their ability to increase NBD1 thermostability. These effects were much larger than those caused by interface modification alone and accounted for most of the correction achieved by modifying both the domain and the interface. Thus, NBD1 stabilization plays a dominant role in overcoming the DeltaF508 defect. Furthermore, the dual target approach resulted in a locked-open ion channel that was constitutively active in the absence of the normally obligatory dependence on phosphorylation by protein kinase A. Thus, simultaneous targeting of both the domain and the interface, as well as being non-essential for correction of biogenesis, may disrupt normal regulation of channel function.

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Sentences [show]
No. Sentence Comment
45 2PT, S492P/A534P/I539T; 4PT, 2PT + S422P/S434P; 3SS, G550E/R553M/R555K; 4SS, 3SS + I539T; ƊRI, deletion of RI amino acids 404-435; combo, ƊRI + 2PT + 3SS. Login to comment
65 1-WT; 2-S492P; 3-I539T; 4. Login to comment
66 S492P/I539T; 5-G550E/R553Q/R555K; 6-combo. Login to comment
72 2PT, S492P/ A534P/I539T; 3PT, 2PT + S495P. Login to comment
93 In addition to the S492P substitution found in three non-mammalian species that are relatively insensitive to the destabilizing influence of the ƊF508 mutation [13], we also included a second Q-loop proline substitution, S495P present in shark CFTR. Login to comment
95 S492P or I539T alone slightly increased the Tm of NBD1 (ƊTm = 2-3 &#b0;C) similar to the affect of the solubilization mutations F494N and Q637R combined. Login to comment
96 The S492P and I539T substitutions had additive affects such that ƊTm increased to 4.4 &#b0;C, and ƊTm was further increased to 8.4 &#b0;C when the additional mutations A534P/G550E/R553M/R555K were introduced. Login to comment
100 The effect of this single mutation was in contrast to that of S492P, which only increased maturation substantially when present together with I539T. Login to comment
123 Figure 3e shows that both BIA and BEIA further strongly increased maturation of the NBD1 stabilized variant ƊF508/2PT (S492P/A534P/I539T). Login to comment

[hide] Deletion of Phenylalanine 508 in the First Nucleot... J Biol Chem. 2015 Sep 18;290(38):22862-78. doi: 10.1074/jbc.M115.641134. Epub 2015 Jul 6. Chong PA, Farber PJ, Vernon RM, Hudson RP, Mittermaier AK, Forman-Kay JD
Deletion of Phenylalanine 508 in the First Nucleotide-binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator Increases Conformational Exchange and Inhibits Dimerization.
J Biol Chem. 2015 Sep 18;290(38):22862-78. doi: 10.1074/jbc.M115.641134. Epub 2015 Jul 6., [PMID:26149808]

Abstract [show]
Deletion of Phe-508 (F508del) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) results in destabilization of the domain, intramolecular interactions involving the domain, and the entire channel. The destabilization caused by F508del manifests itself in defective channel processing and channel gating defects. Here, we present NMR studies of the effect of F508del and the I539T stabilizing mutation on NBD1 dynamics, with a view to understanding these changes in stability. Qualitatively, F508del NMR spectra exhibit significantly more peak broadening than WT spectra due to the enhanced intermediate time scale (millisecond to microsecond) motions in the mutant. Unexpectedly, studies of fast (nanosecond to picosecond) motions revealed that F508del NBD1 tumbles more rapidly in solution than WT NBD1. Whereas F508del tumbles at a rate nearly consistent with the monomeric state, the WT protein tumbles significantly more slowly. Paramagnetic relaxation enhancement experiments confirm that NBD1 homodimerizes in solution in the expected head-to-tail orientation. NMR spectra of WT NBD1 reveal significant concentration-dependent chemical shift perturbations consistent with NBD1 dimerization. Chemical shift analysis suggests that the more rapid tumbling of F508del is the result of an impaired ability to dimerize. Based on previously published crystal structures and NMR spectra of various NBD1 mutants, we propose that deletion of Phe-508 affects Q-loop conformational sampling in a manner that inhibits dimerization. These results provide a potential mechanism for inhibition of channel opening by F508del and support the dimer interface as a target for cystic fibrosis therapeutics.

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No. Sentence Comment
386 Other published Q-loop segment suppressor mutations such as S492P and S495P (21, 29) are also likely to modulate NBD dimerization. Login to comment

[hide] Thermal stability of purified and reconstituted CF... Protein Expr Purif. 2015 Dec;116:159-66. doi: 10.1016/j.pep.2015.09.018. Epub 2015 Sep 15. Aleksandrov LA, Jensen TJ, Cui L, Kousouros JN, He L, Aleksandrov AA, Riordan JR
Thermal stability of purified and reconstituted CFTR in a locked open channel conformation.
Protein Expr Purif. 2015 Dec;116:159-66. doi: 10.1016/j.pep.2015.09.018. Epub 2015 Sep 15., [PMID:26384709]

Abstract [show]
CFTR is unique among ABC transporters as the only one functioning as an ion channel and from a human health perspective because mutations in its gene cause cystic fibrosis. Although considerable advances have been made towards understanding CFTR's mechanism of action and the impact of mutations, the lack of a high-resolution 3D structure has hindered progress. The large multi-domain membrane glycoprotein is normally present at low copy number and when over expressed at high levels it aggregates strongly, limiting the production of stable mono-disperse preparations. While the reasons for the strong self-association are not fully understood, its relatively low thermal stability seems likely to be one. The major CF causing mutation, DeltaF508, renders the protein very thermally unstable and therefore a great deal of attention has been paid to this property of CFTR. Multiple second site mutations of CFTR in NBD1 where F508 normally resides and small molecule binders of the domain increase the thermal stability of the mutant. These manipulations also stabilize the wild-type protein. Here we have applied DeltaF508-stabilizing changes and other modifications to generate wild-type constructs that express at much higher levels in scaled-up suspension cultures of mammalian cells. After purification and reconstitution into liposomes these proteins are active in a locked-open conformation at temperatures as high as 50 degrees C and remain monodisperse at 4 degrees C in detergent or lipid for at least a week. The availability of adequate amounts of these and related stable active preparations of homogeneous CFTR will enable stalled structural and ligand binding studies to proceed.

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No. Sentence Comment
20 Abbreviations used: CFTR, cystic fibrosis transmembrane conductance regulator; ABC, ATP-binding cassette; NBD1, N-terminal nucleotide-binding domain; CF, cystic fibrosis; CHO, Chinese hamster ovary; HEK, human embryonic kidney; BHK, baby hamster kidney; Tm, melting temperature; Ti, inactivation temperature; RI, Regulatory Insertion (residues 404-435); 2PT, variant with NBD1 mutations S492P, A534P and I539T; Q loop, residues contacting the gamma-phosphate of ATP; SDR, structurally divers region; DMNG, Decyl Maltose Neopentyl Glycol; MALS, multi-angle light scattering analysis; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanola mine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPS, 1,2-dioleoyl-sn- glycero-3-phospho-L-serine; SUV, small unilamellar vesicles; LMV, large multilamellar vesicles; LULV, large unilamellar vesicles; PKA, protein kinase A; RIPA, radioimmunoprecipitation assay; ER, endoplasmic reticulum; RAMP, gradual increase with constant slope. Login to comment
107 As seen in Fig. 2a the ''2PT" variant with NBD1 mutations S492P, A534P and I539T and the DRI variant, from which the Regulatory Insertion (residues 404-435) was deleted both increased expression levels substantially compared to the wild type. Login to comment
109 Channel open probability was substantially reduced in 2PT due to the introduction of the two prolines into the mobile Q loop (S492P) and SDR (A534P) regions of NBD1 (third tracing). Login to comment

[hide] Exploiting species differences to understand the C... Biochem Soc Trans. 2015 Oct 1;43(5):975-82. doi: 10.1042/BST20150129. Bose SJ, Scott-Ward TS, Cai Z, Sheppard DN
Exploiting species differences to understand the CFTR Cl- channel.
Biochem Soc Trans. 2015 Oct 1;43(5):975-82. doi: 10.1042/BST20150129., [PMID:26517912]

Abstract [show]
The anion channel cystic fibrosis transmembrane conductance regulator (CFTR) is a unique ATP-binding cassette (ABC) transporter. CFTR plays a pivotal role in transepithelial ion transport as its dysfunction in the genetic disease cystic fibrosis (CF) dramatically demonstrates. Phylogenetic analysis suggests that CFTR first appeared in aquatic vertebrates fulfilling important roles in osmosensing and organ development. Here, we review selectively, knowledge of CFTR structure, function and pharmacology, gleaned from cross-species comparative studies of recombinant CFTR proteins, including CFTR chimeras. The data argue that subtle changes in CFTR structure can affect strongly channel function and the action of CF mutations.

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120 Hypothesizing that structural differences between human and chicken CFTR account for the thermostability of F508del chicken CFTR, Aleksandrov et al. [41] demonstrated that the F508del revertant I539T and four proline residues at key positions within NBD1 (S422P, S434P, S492P and A534P) were responsible for rescuing the processing, plasma membrane stability and function of human CFTR. Login to comment