ABCMdb

ABCC7 p.His1402Ala

Predicted by SNAP2:	A: D (91%), C: D (85%), D: D (91%), E: D (91%), F: D (91%), G: D (91%), I: D (91%), K: D (91%), L: D (91%), M: D (91%), N: D (75%), P: D (95%), Q: D (80%), R: D (91%), S: D (85%), T: D (85%), V: D (91%), W: D (91%), Y: D (91%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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Publications
[hide] Localization of sequences within the C-terminal do... J Biol Chem. 2001 Jan 12;276(2):1291-8. Gentzsch M, Riordan JR
Localization of sequences within the C-terminal domain of the cystic fibrosis transmembrane conductance regulator which impact maturation and stability.
J Biol Chem. 2001 Jan 12;276(2):1291-8., 2001-01-12 [PMID:11022033]

Abstract [show]
Some disease-associated truncations within the 100-residue domain C-terminal of the second nucleotide-binding domain destabilize the mature protein (Haardt, M., Benharouga, M., Lechardeur, D., Kartner, N., and Lukacs, G. L. (1999) J. Biol. Chem. 274, 21873-21877). We now have identified three short oligopeptide regions in the C-terminal domain which impact cystic fibrosis transmembrane conductance regulator (CFTR) maturation and stability in different ways. A highly conserved hydrophobic patch (region I) formed by residues 1413-1416 (FLVI) was found to be crucial for the stability of the mature protein. Nascent chain stability was severely decreased by shortening the protein by 81 amino acids (1400X). This accelerated degradation was sensitive to proteasome inhibitors but not influenced by brefeldin A, indicating that it occurred at the endoplasmic reticulum. The five residues at positions 1400 to 1404 (region II) normally maintain nascent CFTR stability in a positional rather than a sequence-specific manner. A third modulating region (III) constituted by residues 1390 to 1394 destabilizes the protein. Hence the nascent form regains stability on further truncation back to residues 1390 or 1380, permitting some degree of maturation and a low level of cyclic AMP-stimulated chloride channel activity at the cell surface. Thus while not absolutely essential, the C-terminal domain strongly modulates the biogenesis and maturation of CFTR.

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Sentences [show]
No. Sentence Comment
40 F1413A/ L1414A/V1415A/I1416A/E1417A, 5Ј-GCTGGAATGCCAACAAGCTGC- GGCCGCAGCAGAGAACAAAGTGCGG-3Ј and 5Ј-CCGCACTTTGTTC- TCTGCTGCGGCCGCAGCTTGTTGGCATTCCAGC-3Ј; F1413A- /L1414A/V1415A/I1416A, 5Ј-GCTGGAATGCCAACAAGCTGCGGCCG- CAGAAGAGAACAAAGTGCGG-3Ј and 5Ј-CCGCACTTTGTTCTCTTC- TGCGGCCGCAGCTTGTTGGCATTCCAGC-3Ј; Q1411A/Q1412A, 5Ј-G- GATAGAAGCAATGCTGGAATGCGCAGCATTTTTGGTCATAGAAG-3 and 5Ј-CTTCTATGACCAAAAATGCTGCGCATTCCAGCATTGCTTCT- ATCC-3; F1413A/L1414A, 5Ј-GCTGGAATGCCAACAAGCTGCGGTCA- TAGAAGAGAACAAAGTGCG-3Ј and 5Ј-CGCACTTTGTTCTCTTCTA- TGACCGCAGCTTGTTGGCATTCCAGC-3Ј; L1414A/V1415A, 5Ј-GCT- GGAATGCCAACAATTTGCGGCCATAGAAGAGAACAAAGTGCGG-3Ј and 5Ј-CCGCACTTTGTTCTCTTCTATGGCCGCAAATTGTTGGCATT- CCAGC-3Ј; V1415A/I1416A, 5Ј-GGAATGCCAACAATTTTTGGCCGCA- GAAGAGAACAAAGTGCGGCAG-3Ј and 5Ј-CTGCCGCACTTTGTTCT- CTTCTGCGGCCAAAAATTGTTGGCATTCC-3Ј; E1417A/E1418A, 5Ј- GCCAACAATTTTTGGTCATAGCAGCGAACAAAGTGCGGCAGTAC- G-3Ј and 5Ј-CGTACTGCCGCACTTTGTTCGCTGCTATGACCAAAAA- TTGTTGGC-3Ј; F1413A, 5Ј-GCAATGCTGGAATGCCAACAAGCTTTG- GTCATAGAAGAGAAC-3Ј and 5Ј-GTTCTCTTCTATGACCAAAGCTT- GTTGGCATTCCAGCATTGC-3Ј; L1414A, 5Ј-GCTGGAATGCCAACAA- TTTGCGGTCATAGAAGAGAACAAAGTGCG-3Ј and 5Ј-CGCACTTTG- TTCTCTTCTATGACCGCAAATTGTTGGCATTCCAGC-3Ј; V1415A, 5Ј- GGAATGCCAACAATTTTTGGCCATAGAAGAGAACAAAGTGCGGC- AG-3Ј and 5Ј-CTGCCGCACTTTGTTCTCTTCTATGGCCAAAAATTGT- TGGCATTCC-3Ј; I1416A, 5Ј-GGAATGCCAACAATTTTTGGTCGCAG- AAGAGAACAAAGTGCGGCAG-3Ј and 5Ј-CTGCCGCACTTTGTTCTC- TTCTGCGACCAAAAATTGTTGGCATTCC-3Ј; E1417A, 5Ј-GCCAACA- ATTTTTGGTCATAGCAGAGAACAAAGTGCGGCAGTACG-3Ј and 5Ј- CGTACTGCCGCACTTTGTTCTCTGCTATGACCAAAAATTGTTGGC- 3Ј; C1400A/E1401A/H1402A/R1403A/I1404A, 5Ј-GCACAGTAATTCTC- GCTGCAGCCGCGGCAGAAGCAATGCTGGAATGCC-3Ј and 5Ј-GGC- ATTCCAGCATTGCTTCTGCCGCGGCTGCAGCGAGAATTACTGTG- C-3Ј; ⌬1400-1404: 5Ј-GCATTTGCTGATTGCACAGTAATTCTCGAAG- CAATGCTGGAATGCC-3Ј and 5Ј-GGCATTCCAGCATTGCTTCGAGA- ATTACTGTGCAATCAGCAAATGC-3Ј; C1400A/E1401A, 5Ј-GATTGC- ACAGTAATTCTCGCTGCACACAGGATAGAAGCAATGC-3Ј and 5Ј-G- CATTGCTTCTATCCTGTGTGCAGCGAGAATTACTGTGCAATC-3Ј; H1402A/R1403A, 5Ј-CAGTAATTCTCTGTGAAGCCGCGATAGAAGC- AATGCTGGAATGCC-3Ј and 5Ј-GGCATTCCAGCATTGCTTCTATCG- CGGCTTCACAGAGAATTAC TG-3Ј; I1404A/E1405A, 5Ј-CTCTGTGA- ACACAGGGCAGCAGCAATGCTGGAATGCCAAC-3Ј and 5Ј-GTTGGC- ATTCCAGCATTGCTGCTGCCCTGTGTTCACAGAG-3Ј, Q1390A/A- 1391A/F1392A/A1393A/D1394A, 5Ј-GAAGAACTCTAAAAGCAGCAG- CTGCTGCTTGCACAGTAATTCTC-3Ј and 5Ј-GAGAATTACTGTGCA- AGCAGCAGCTGCTGCTTTTAGAGTTCTTC-3Ј. Login to comment

[hide] Aggregation of misfolded proteins can be a selecti... J Biol Chem. 2002 Sep 13;277(37):34462-70. Epub 2002 Jun 25. Milewski MI, Mickle JE, Forrest JK, Stanton BA, Cutting GR
Aggregation of misfolded proteins can be a selective process dependent upon peptide composition.
J Biol Chem. 2002 Sep 13;277(37):34462-70. Epub 2002 Jun 25., 2002-09-13 [PMID:12084728]

Abstract [show]
Intracellular aggregation of misfolded proteins is observed in a number of human diseases, in particular, neurologic disorders in which expanded tracts of polyglutamine residues play a central role. A variety of other proteins are prone to aggregation when mutated, indicating that this process is a common pathologic mechanism for inherited disorders. However, little is known about the relationship between the sequence of aggregating peptides and the specificity of intracellular accumulation. Here we demonstrate that substitution of two residues eliminates aggregation of a 111-amino acid peptide derived from the C-terminal portion of the cystic fibrosis transmembrane conductance regulator (CFTR). We also show that fusion to a reporter protein considerably alters the subcellular distribution of aggregating peptide. When fused to green fluorescent protein, the peptide containing amino acids 1370-1480 of CFTR accumulates in large perinuclear or nuclear aggregates. The same CFTR fragment devoid of green fluorescent protein localizes predominantly to discrete accumulations associated with mitochondria. Importantly, both types of accumulation are dependent on the presence of the same two amino acids within the CFTR sequence. Co-expression studies show that both CFTR-derived proteins can co-localize in large cytoplasmic/nuclear aggregates. However, neither CFTR construct accumulates in intracellular inclusions formed by N-terminal fragment of huntingtin. In addition to unique accumulation patterns, each aggregating peptide shows differences in association with chaperone proteins. Thus, our results indicate that the process of intracellular aggregation can be a selective process determined by the composition of the aggregating peptides.

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Sentences [show]
No. Sentence Comment
90 Only 25% (52/208) of cells expressing GFP fusion protein bearing single H1402A substitution and 27% (41/153) of R1403A mutants showed intracellular protein accumulation, whereas the corresponding number for the wild type sequence was 91% (132/145). Login to comment
93 Cells expressing the double mutant H1402A,R1403A showed no aggregation at all (0/108). Login to comment
97 However, the introduction of the ⌬ag deletion or the H1402A,R1403A double mutation significantly decreased the amount of the fusion protein in the insoluble fraction to approximately 31 and 36%, respectively. Login to comment
108 B-E, the effect of different mutations within the ag region, including ⌬ag (B), V1397E (C), T1396A (D), and double mutation H1402A,R1403A (E) on the aggregation of C terminus of CFTR fused to GFP (shown in green) in transiently transfected human airway epithelial (IB3-1) cells. Login to comment
120 Additionally, the double mutation H1402A,R1403A prevented aggregation of HA-tagged CFTR C terminus (Fig. 2E). Login to comment
146 E, lack of aggregation of HA-CFTR 1370-1480 construct carrying the H1402A and R1403A mutations. Login to comment
160 Similarly, the non-aggregating GFP-CFTR 1370-1480 H1402A,R1403A construct was included in giant aggregates formed by HA-CFTR 1370-1480 (Fig. 4D). Login to comment
195 C, colocalization of the aggregating GFP-CFTR 1370-1480 construct (green) and the non-aggregating HA-CFTR 1370-1480 H1402A,R1403A construct (red) in giant aggregates. Login to comment
196 D, co-localization of the non-aggregating GFP-CFTR 1370-1480 H1402A,R1403A construct (green) with the aggregating HA-CFTR 1370-1480 construct (red) in giant aggregates. Login to comment

[hide] The H-loop in the second nucleotide-binding domain... Cell Physiol Biochem. 2010;25(2-3):169-80. Epub 2010 Jan 12. Kloch M, Milewski M, Nurowska E, Dworakowska B, Cutting GR, Dolowy K
The H-loop in the second nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator is required for efficient chloride channel closing.
Cell Physiol Biochem. 2010;25(2-3):169-80. Epub 2010 Jan 12., [PMID:20110677]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a cAMP-activated chloride channel. The recent model of CFTR gating predicts that the ATP binding to both nucleotide-binding domains (NBD1 and NBD2) of CFTR is required for the opening of the channel, while the ATP hydrolysis at NBD2 induces subsequent channel closing. In most ABC proteins, efficient hydrolysis of ATP requires the presence of the invariant histidine residue within the H-loop located in the C-terminal part of the NBD. However, the contribution of the corresponding region (H-loop) of NBD2 to the CFTR channel gating has not been examined so far. Here we report that the alanine substitution of the conserved dipeptide HR motif (HR-->AA) in the H-loop of NBD2 leads to prolonged open states of CFTR channel, indicating that the H-loop is required for efficient channel closing. On the other hand, the HR-->AA substitution lead to the substantial decrease of CFTR-mediated current density (pA/pF) in transfected HEK 293 cells, as recorded in the whole-cell patch-clamp analysis. These results suggest that the H-loop of NBD2, apart from being required for CFTR channel closing, may be involved in regulating CFTR trafficking to the cell surface.

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Sentences [show]
No. Sentence Comment
30 Mutations within the H-loop of NBD2, including the deletion of the entire ag region (Δ1395-1403) and the double alanine substitution H1402A, R1403A (HR→AA), were created in the CFTR-containing pBQ4.7 vector (a gift from J. Rommens and L.-C. Tsui) using the site-directed mutagenesis system "Transformer" (Becton Dickinson). Login to comment

[hide] PDZ-binding motifs are unable to ensure correct po... FEBS Lett. 2005 Jan 17;579(2):483-7. Milewski MI, Lopez A, Jurkowska M, Larusch J, Cutting GR
PDZ-binding motifs are unable to ensure correct polarized protein distribution in the absence of additional localization signals.
FEBS Lett. 2005 Jan 17;579(2):483-7., [PMID:15642363]

Abstract [show]
The C-terminal PDZ-binding motifs are required for polarized apical/basolateral localization of many membrane proteins. To determine the specificity of the PDZ-binding motifs in establishing cellular distribution, we utilized a 111-amino acid region from the C-terminus of cystic fibrosis transmembrane conductance regulator (CFTR) that is able to direct apical localization of fused reporter proteins. Substitution of the C-terminal PDZ-binding motif of CFTR with corresponding motifs necessary for basolateral localization of other membrane proteins did not lead to the redistribution of the fusion protein to the basolateral membrane. Instead, some fusion proteins remained localized to the apical membrane, whereas others showed no specific distribution. The specificity of the PDZ-based interactions was substantially increased when specific amino acids located upstream of the classical PDZ-binding motifs were included. However, even the presence of a longer C-terminal motif from a basolateral protein could not ensure basolateral distribution of the fusion protein. Our results indicate that the C-terminal PDZ-binding motifs are not the primary signals for polarized protein distribution, although they are required for targeting and/or stabilization of protein at the given location.

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No. Sentence Comment
25 Also, the introduction of the H1402A and R1403A mutations, reducing the aggregation rate of the CFTR C-terminus, was previously described [9]. Login to comment
64 A diagram illustrating the structure and subcellular localization of the GFP fusion proteins containing either the full-length CFTR protein or its C-terminal fragment (a.a. 1370-1480) with two alanine substitutions (H1402A and R1403A), eliminating the aggregation of the CFTR C-terminus. Login to comment
93 (B) Apical localization of the GFP-CFTR 1370-1480 H1402A, R1403A fusion protein containing the C-terminal M-K-D-T-R-L> motif. Login to comment
94 (C) Predominant cytoplasmic distribution of the GFP-CFTR 1370-1480 H1402A, R1403A fusion protein containing the extended Q-K-E-TC-L> PDZ-binding motif of LET23. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Cold Spring Harb Perspect Med. 2013 Feb 1;3(2):a009514. doi: 10.1101/cshperspect.a009514. Hunt JF, Wang C, Ford RC
Cystic fibrosis transmembrane conductance regulator (ABCC7) structure.
Cold Spring Harb Perspect Med. 2013 Feb 1;3(2):a009514. doi: 10.1101/cshperspect.a009514., [PMID:23378596]

Abstract [show]
Structural studies of the cystic fibrosis transmembrane conductance regulator (CFTR) are reviewed. Like many membrane proteins, full-length CFTR has proven to be difficult to express and purify, hence much of the structural data available is for the more tractable, independently expressed soluble domains. Therefore, this chapter covers structural data for individual CFTR domains in addition to the sparser data available for the full-length protein. To set the context for these studies, we will start by reviewing structural information on model proteins from the ATP-binding cassette (ABC) transporter superfamily, to which CFTR belongs.

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Sentences [show]
No. Sentence Comment
289 Improvements in the yield of soluble protein were obtained by introducing the hydrolytically inactivating H1402A mutation in ATPase active site of hNBD2 plus a series of "solubilizing" mutations on its surface (Q1280E/ Y1307N/W1310H/Q1411D) (X Zhao, S Atwell, JF Hunt, et al., unpubl.). Login to comment
291 The H1402A mutation as well as at least some of the "solubilizing" mutations seem likely to stabilize the native conformation of the domain thermodynamically, as observed for the solubilizing surface mutations that improved the stability and yield J.F. Hunt et al. 18 Cite this article as Cold Spring Harb Perspect Med 2012;3:a009514 www.perspectivesinmedicine.org by Cold Spring Harbor Laboratory Press at SEMMELWEIS UNIV OF MEDICINE on December 5, of preparations of hNBD1 (as described above). Login to comment
293 Nonetheless, indirect evidence supports the hypothesis that the H1402A mutation stabilizes hNBD2. Login to comment
295 Electrophysiological studies of the H1402A and E1371Q mutations in intact human CFTR support these inferences concerning catalytic geometry by showing that the H1402A (Kloch et al. 2010) and E1371Q (Vergani et al. 2005) mutations both greatly increase the lifetime of the open state of the CFTR chloride channel, presumably because they block ATP hydrolysis and stabilize ATP-sandwich heterodimer formed by hNBD1 and hNBD2. Login to comment
297 However, despite their similar influence on the gating properties of intact CFTR, the H1402A and E1371Q mutations have dramatically different effects on the stabilityand yield of hNBD2 during purification, with the first greatly improving yield compared to the second (X Zhao, S Atwell, JF Hunt, et al., unpubl.). Login to comment
298 In silico energetic calculations suggest that the H1402A mutation stabilizes isolated hNBD2 bound to Mg-ATP, while the E1371Q mutation destabilizes it (P Kumar, C Wang, JF Hunt, et al., unpubl.). Login to comment

[hide] Regulatory R region of the CFTR chloride channel i... Proc Natl Acad Sci U S A. 2013 Nov 19;110(47):E4427-36. doi: 10.1073/pnas.1315104110. Epub 2013 Nov 4. Bozoky Z, Krzeminski M, Muhandiram R, Birtley JR, Al-Zahrani A, Thomas PJ, Frizzell RA, Ford RC, Forman-Kay JD
Regulatory R region of the CFTR chloride channel is a dynamic integrator of phospho-dependent intra- and intermolecular interactions.
Proc Natl Acad Sci U S A. 2013 Nov 19;110(47):E4427-36. doi: 10.1073/pnas.1315104110. Epub 2013 Nov 4., [PMID:24191035]

Abstract [show]
Intrinsically disordered proteins play crucial roles in regulatory processes and often function as protein interaction hubs. Here, we present a detailed characterization of a full-length disordered hub protein region involved in multiple dynamic complexes. We performed NMR, CD, and fluorescence binding studies on the nonphosphorylated and highly PKA-phosphorylated human cystic fibrosis transmembrane conductance regulator (CFTR) regulatory region, a approximately 200-residue disordered segment involved in phosphorylation-dependent regulation of channel trafficking and gating. Our data provide evidence for dynamic, phosphorylation-dependent, multisite interactions of various segments of the regulatory region for its intra- and intermolecular partners, including the CFTR nucleotide binding domains 1 and 2, a 42-residue peptide from the C terminus of CFTR, the SLC26A3 sulphate transporter and antisigma factor antagonist (STAS) domain, and 14-3-3beta. Because of its large number of binding partners, multivalent binding of individually weak sites facilitates rapid exchange between free and bound states to allow the regulatory region to engage with different partners and generate a graded or rheostat-like response to phosphorylation. Our results enrich the understanding of how disordered binding segments interact with multiple targets. We present structural models consistent with our data that illustrate this dynamic aspect of phospho-regulation of CFTR by the disordered regulatory region.

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Sentences [show]
No. Sentence Comment
117 Q1411D; L1436D; and H1402A) designed by SGX Inc. We find similar patterns of R region binding to NBD2 as for NBD1 (Fig. 1E), identifying NBD2 as a direct binding partner for R region and implying that the NBDs have similar binding properties. Login to comment
239 The NBD2 domain of human CFTR (aa 1193-1445, Q1280E; Y1307N; Q1411D; H1402A; and L1436D), a construct with solubilizing mutations designed by SGX, Inc., was encoded on a pET-SUMO plasmid (Invitrogen). Login to comment