ABCMdb

ABCC7 p.Gln220Arg

ClinVar:	c.658C>T , p.Gln220* D , Pathogenic c.659A>G , p.Gln220Arg ? , not provided
CF databases:	c.658C>T , p.Gln220* D , CF-causing c.659A>G , p.Gln220Arg (CFTR1) ? , Found in a patient with CBAVD.
Predicted by SNAP2:	A: N (57%), C: D (59%), D: N (66%), E: N (82%), F: D (75%), G: N (61%), H: N (53%), I: D (71%), K: D (53%), L: D (59%), M: D (71%), N: N (61%), P: D (63%), R: D (63%), S: N (57%), T: N (66%), V: N (57%), W: D (80%), Y: D (71%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: D, K: N, L: D, M: N, N: N, P: N, R: N, S: N, T: N, V: N, W: N, Y: N,

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Publications
[hide] Role of the extracellular loop in the folding of a... Biochemistry. 2007 Jun 19;46(24):7099-106. Epub 2007 May 22. Wehbi H, Rath A, Glibowicka M, Deber CM
Role of the extracellular loop in the folding of a CFTR transmembrane helical hairpin.
Biochemistry. 2007 Jun 19;46(24):7099-106. Epub 2007 May 22., 2007-06-19 [PMID:17516627]

Abstract [show]
The folding of membrane-spanning domains into their native functional forms depends on interactions between transmembrane (TM) helices joined by covalent loops. However, the importance of these covalent linker regions in mediating the strength of helix-helix associations has not been systematically addressed. Here we examine the potential structural impact of cystic fibrosis-phenotypic mutations in the extracellular loop 2 (ECL2) on interactions between the TM3 and TM4 helices of the cystic fibrosis transmembrane conductance regulator (CFTR) in constructs containing CFTR residues 194-241. When the effects of replacements in ECL2 (including the CF-phenotypic mutants E217G and Q220R) were evaluated in a library of wild-type and mutant TM3-ECL2-TM4 hairpin constructs, we found that SDS-PAGE gel migration rates differed over a range of nearly 40% +/- the wild-type position and that decreased migration rates correlate with increasing hairpin alpha-helical content as measured by circular dichroism spectra in sodium dodecyl sulfate micelles. The decreased mobility of TM3/4 constructs by introduction of non-native residues is interpreted in terms of an elongation or "opening" of the helical hairpin and concomitant destabilization of membrane-based helix-helix interactions. Our results support a role for short loop regions in dictating the stability of membrane protein folds and highlight the interplay between membrane-embedded helix-helix interactions and loop conformation in influencing the structure of membrane proteins.

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3 When the effects of replacements in ECL2 (including the CF-phenotypic mutants E217G and Q220R) were evaluated in a library of wild-type and mutant TM3-ECL2-TM4 hairpin constructs, we found that SDS-PAGE gel migration rates differed over a range of nearly 40% +/- the wild-type position and that decreased migration rates correlate with increasing hairpin R-helical content as measured by circular dichroism spectra in sodium dodecyl sulfate micelles. Login to comment
57 Two CF-phenotypic missense mutations, E217G and Q220R, have been documented in the ECL2 region (contributed to the Cystic Fibrosis Mutation Database at www.genet.sickkids.on.ca/cftr/ by Zielenski et al. and Fe´rec). Login to comment
59 E217G and Q220R point mutants were constructed initially in the TM3/4 WT background, and their migration on SDS-PAGE was compared to WT following the gel shift assay developed in our laboratory (33). Login to comment
61 We noted that the E217G and Q220R substitutions in the WT background each showed slower migration relative to WT on SDS-PAGE (Figure 2), initially implying that replacement of these ECL2 positions has rendered the TM3/4 WT construct less compact (vide infra). Login to comment
78 (A) SDS-PAGE of TM3/4 WT and TM3/4 WT with E217G and with Q220R. Login to comment
83 The Q220E replacement migrates faster than TM3/4 WT, the Q220G and Q220N mutants migrate at equivalent rates to TM3/4 WT, while the Q220W, Q220K, and Q220R substitutions migrate more slowly. Login to comment
89 The Q220K and Q220W hairpins migrated identically to Q220R (p ) 0.259 and p ) 0.101, respectively) and to one another (p ) 0.341), making it unlikely that the positive charge on the Arg side chain per se reduced hairpin compactness. Login to comment
95 Thus, the mutants that migrated more slowly than TM3/4 WT on SDS-PAGE gels, among them Q220R and E217S, generally displayed increased helicity compared to TM3/4 WT (Table 1, Figure 4A). Login to comment
97 When the changes in TM3/4 WT hairpin migration were compared to changes in overall hairpin helicity, a strong correlation (R ) 0.79) was observed (Figure 5), leading us to propose that increases in non-native R-helix structure within ECL2 might Table 1: Migration Behavior on SDS-PAGE Gels of Single and Double Mutants in the Loop Region of CFTR TM3/4 Constructs % change in apparent MW on SDS-PAGE mutant vs TM3/4 WT in WT loop mutantsa vs TM3/4 V232D in V232D loop mutantsa Pb E217G 6.8 ( 0.7 E217S 11.1 ( 3.4 5.4 ( 1.4 0.056 Q220R 15.2 ( 1.1 Q220G 0.3 ( 0.4 Q220N 2.1 ( 1.3 0.5 ( 0.3 0.108 Q220K 14.1 ( 1.0 Q220W 13.1 ( 1.3 11.5 ( 0.9 0.157 Q220E -11.1 ( 1.1 -4.0 ( 0.3 <0.001 S222G 12.0 ( 2.1 1.1 ( 0.6 0.001 S222E -0.3 ( 2.4 1.3 ( 0.5 0.512 E217G/S222G 12.4 ( 1.9 E217S/S222E 26.1 ( 4.5 averagec 10.4 ( 7.3 4.0 ( 4.2 0.067 a Values are the percentage difference vs TM3/4 WT or TM3/4 V232D migration of SDS-PAGE gels. Login to comment
146 While there may be a potential charge factor in the migration patterns for certain Q220 mutations (Q220E, Q220K, and Q220R; Figure 3), the migration rates of other ECL2 mutants cannot be rationalized as simple functions of adding or subtracting single charges. Login to comment
147 For example, S222E and WT migrate at approximately the same rate, but Q220E moves at -11% vs WT; both S222G and E217G/S222G are at +12%; Q220K, Q220R, and Q220W are each at +13-15%. Login to comment
163 While conformational changes induced by ECL2 mutants in the TM3/4 hairpin should be minimized by additional intrachain contacts imposed by the full-length CFTR molecule, our results suggest that the molecular mechanism of disease introduced by the CF-phenotypic ECL2 mutants E217G and Q220R may involve introduction of non-native R-helical loop structure that destabilizes the CFTR fold and leads to aberrant function in the resting state and/or during substrate transport. Login to comment

[hide] Misfolding of the cystic fibrosis transmembrane co... Biochemistry. 2008 Feb 12;47(6):1465-73. Epub 2008 Jan 15. Cheung JC, Deber CM
Misfolding of the cystic fibrosis transmembrane conductance regulator and disease.
Biochemistry. 2008 Feb 12;47(6):1465-73. Epub 2008 Jan 15., 2008-02-12 [PMID:18193900]

Abstract [show]
Understanding the structural basis for defects in protein function that underlie protein-based genetic diseases is the fundamental requirement for development of therapies. This situation is epitomized by the cystic fibrosis transmembrane conductance regulator (CFTR)-the gene product known to be defective in CF patients-that appears particularly susceptible to misfolding when its biogenesis is hampered by mutations at critical loci. While the primary CF-related defect in CFTR has been localized to deletion of nucleotide binding fold (NBD1) residue Phe508, an increasing number of mutations (now ca. 1,500) are being associated with CF disease of varying severity. Hundreds of these mutations occur in the CFTR transmembrane domain, the site of the protein's chloride channel. This report summarizes our current knowledge on how mutation-dependent misfolding of the CFTR protein is recognized on the cellular level; how specific types of mutations can contribute to the misfolding process; and describes experimental approaches to detecting and elucidating the structural consequences of CF-phenotypic mutations.

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145 When we examined the potential structural impact of CF-phenotypic mutations in extracellular loop 2 (ECL2) (including E217G and Q220R) in a library of wild type and mutant TM3-ECL2- TM4 hairpin constructs, we found that SDS-PAGE gel migration rates differed over a range of nearly 40% +/- the wild type position, and that decreased migration rates FIGURE 6: Disruption of helix-helix interactions by increased R-helical structure in the extracellular loop. Login to comment