ABCMdb

ABCC7 p.Ile231Asp

Predicted by SNAP2:	A: D (63%), C: N (57%), D: D (80%), E: D (75%), F: D (66%), G: D (75%), H: D (75%), K: D (80%), L: N (78%), M: N (66%), N: D (71%), P: D (75%), Q: D (71%), R: D (80%), S: N (66%), T: N (72%), V: N (87%), W: D (80%), Y: D (75%),
Predicted by PROVEAN:	A: N, C: D, D: D, E: D, F: N, G: D, H: D, K: D, L: N, M: N, N: D, P: D, Q: D, R: D, S: D, T: N, V: N, W: D, Y: D,

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[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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93 Earlier we had observed that TM3/4 constructs with lesions within their TM segments (e.g., WT vs I231D and V232D) display superimposible curves (38). Login to comment

[hide] Positional dependence of non-native polar mutation... Biochim Biophys Acta. 2008 Jan;1778(1):79-87. Epub 2007 Sep 15. Wehbi H, Gasmi-Seabrook G, Choi MY, Deber CM
Positional dependence of non-native polar mutations on folding of CFTR helical hairpins.
Biochim Biophys Acta. 2008 Jan;1778(1):79-87. Epub 2007 Sep 15., [PMID:17949679]

Abstract [show]
Mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) cause CF disease by altering the biosynthesis, maturation, folding and ion conductance of this protein. Our laboratory has focused on expression and structural analysis of the CFTR transmembrane (TM) domains using two-TM segments (i.e., helix-loop-helix constructs) which we term 'helical hairpins'; these represent the minimal model of tertiary contacts between two helices in a membrane. Previous studies on a library of TM3/4 hairpins of the first CFTR TM domain suggested that introduction of non-native polar residues into TM4 can compromise CFTR function through side chain-side chain H-bonding interactions with native Q207 in TM3 [Choi, M. Y., Cardarelli, L., Therien, A. G., and Deber, C. M. Non-native interhelical hydrogen bonds in the cystic fibrosis transmembrane conductance regulator domain modulated by polar mutations, Biochemistry 43 (2004) 8077-8083]. In the present work, we combine gel shift assays with a series of NMR experiments for comparative structural characterization of the wild type TM3/4 hairpin and its mutants V232D, I231D, Q207N/V232E. Over 95% of the backbone resonances of a 15N,13C-labelled V232D-TM3/4 construct in the membrane-mimetic environment of perfluorooctanoate (PFO) micelles were successfully assigned, and the presence and boundaries of helical segments within TM3 and TM4 were defined under these conditions. Comparative analysis of 15N and 1H chemical shift variations among HSQC spectra of WT-, V232D-, I231D- and Q207N/V232E-TM3/4 indicated that hairpin conformations vary with the position of a polar mutation (i.e., V232D and I231D vs. WT), but remain similar when hairpins with identically-positioned polar partners are compared (i.e., V232D vs. Q207N-V232E). The overall findings suggest that a polar mutation in a TM helix can potentially distort native interfacial packing determinants in membrane proteins such as CFTR, with consequences that may lead to disease.

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3 In the present work, we combine gel shift assays with a series of NMR experiments for comparative structural characterization of the wild type TM3/4 hairpin and its mutants V232D, I231D, Q207N/V232E. Login to comment
4 Over 95% of the backbone resonances of a 15 N,13 C-labelled V232D-TM3/4 construct in the membrane-mimetic environment of perfluorooctanoate (PFO) micelles were successfully assigned, and the presence and boundaries of helical segments within TM3 and TM4 were defined under these conditions. Login to comment
5 Comparative analysis of 15 N and 1 H chemical shift variations among HSQC spectra of WT-, V232D-, I231D- and Q207N/V232E-TM3/4 indicated that hairpin conformations vary with the position of a polar mutation (i.e., V232D and I231D vs. WT), but remain similar when hairpins with identically-positioned polar partners are compared (i.e., V232D vs. Q207N-V232E). Login to comment
13 CF is inherited in a recessive autosomal fashion; most CF patients have a CFTR Available online at www.sciencedirect.com Biochimica et Biophysica Acta 1778 (2008) 79-87 www.elsevier.com/locate/bbamem Abbreviations: CF, Cystic fibrosis; CFTR, Cystic fibrosis transmembrane conductance regulator; TM, Transmembrane; TMD, Transmembrane domain; TM3/4, Helical hairpin including residues 194-241 of CFTR; WT, Wild type; V232D-TM3/4, TM3/4 construct with mutation of Val to Asp at position 232; I231D-TM3/4, TM3/4 construct with mutation of Ile to Asp at position 231; Q207N/V232E-TM3/4, TM3/4 construct with mutation of Gln to Asn at position 207 and Val to Glu at position 232; PFO, Perfluorooctanoate; DPC, Dodecylphosphocholine; SDS, Sodium dodecylsulfate; CD, Circular dichroism; H-bond, Hydrogen bond; HSQC, Heteronuclear single quantum coherence ⁎ Corresponding author. Login to comment
35 Previous studies we performed on CFTR helix-loop-helix constructs using gel shift analysis, fluorescence measurements, and molecular modeling suggested that hairpin folding is likely stabilized by folding due to formation of a non-native side chain-side chain hydrogen bond between 'polar partners` in the interacting helices (viz., Q207 in TM3, and I231D or V232D in TM4) [20]. Login to comment
43 We then use solution NMR experiments to demonstrate the conformational variability of TM3/4 hairpin mutants (including the CF-phenotypic mutant V232D; I231D; and Q207N/V232E) vs. the wild type hairpin in micellar environments. Login to comment
46 Expression and purification of wild type and mutant TM3/4 constructs from the CFTR membrane domain The 15 N and 15 N/13 C enriched forms of the TM3/4 helical hairpin constructs (WT-, V232D-, I231D-, Q207N/V232E) were expressed and purified as previously described [19]. Login to comment
141 (a) WT; (b) V232D; (c) I231D; and (d) Q207N/V232E. Login to comment
142 Spectra were recorded in 115 mM PFO at 45 °C. Gly cross-peaks (shown in (b)) were assigned from the V232D spectrum (Fig. 2); other assignments shown were made where possible by comparison. Login to comment
149 In order to further investigate the conformational changes of TM3/4 upon introduction of a polar residue into TM4, we next performed a comparative analysis of amide 1 H/15 N chemical shifts in the 1 H-15 N HSQC spectra of WT-TM3/4 vs. I231D-TM3/4 which contains a polar mutation at the position preceding the CF-phenotypic mutation V232D-TM3/4. Login to comment
150 Note that this mutant exhibited a faster migration rate than both WT-TM3/4 and V232D-TM3/4 on SDS-PAGE [22] (Fig. 1b). Login to comment
192 To address the proposition that the formation of a given helix-helix interface may be dominated by a positional dependence of a polar residue in TM4, the 1 H-15 N HSQC spectrum of I231D-TM3/4 in PFO micelles was acquired under identical conditions as WT-TM3/4 and V232D-TM3/4. Login to comment
194 The full amide chemical shift analysis of the 1 H-15 N HSQC shows that similarly to the V232D mutant, all TM4 amides - along with residues from W216 through A221 (C-terminus of TM3) and from S222 to G226 (turn) - are highly affected by the I231D mutation (not shown), suggesting that this mutation introduces significant changes in packing relative to WT protein. Login to comment
195 In contrast, the full 1 H-15 N HSQC spectra for V232D- and Q207N/V232E-TM3/4 exhibit striking similarities (not shown, but exemplified by the 'Gly box` comparison between Fig. 4 b and d), suggesting that these two mutants adopt similar conformations in the PFO micelle environment. Login to comment
196 The 1 H-15 N HSQC spectrum of this double mutant shows that similar to V232D-TM3/4 and I231D-TM3/4 mutants, the residues in TM4 along with some in TM3 and the turn are all highly affected vs. WT upon these mutations. Login to comment
199 Molecular dynamics simulations of WT TM3/4 vs. I231D-TM3/4 further support the view that a folded two-helix hairpin forms in a micellar environment, and that a side chain-side chain Q207-D231 H-bond provides additional stabilization for the folded state [64]. Login to comment
200 Although the NMR data reported here do not provide specific evidence for H-bond formation nor are sufficient for detailed structural characterization, one can speculate that the similarities in spectra of V232D and Q207N/V232E are the result of contacts between similar residues in the V232D mutant that can effectively be substituted by N207 and E232. Login to comment
202 Conversely, this model would predict that in the case of I231D, there must be a relative reorientation by 100° of one helix with respect to the other for participation of I231D in interhelical interactions vs. either V232D or Q207N/V232E. Login to comment
209 Although hairpin constructs have degrees of conformational freedom in SDS or PFO micellar environments that would not be available to the corresponding helices embedded in an intact CFTR TM domain, our results suggest that hairpin interfaces - and possibly helix boundaries - may vary as a function of the position of a non-native polar mutation (i.e., V232D vs. I231D in TM4), and thereby indicate the susceptibility of the native protein structure to a corresponding destiny. Login to comment
6 Comparative analysis of 15 N and 1 H chemical shift variations among HSQC spectra of WT-, V232D-, I231D- and Q207N/V232E-TM3/4 indicated that hairpin conformations vary with the position of a polar mutation (i.e., V232D and I231D vs. WT), but remain similar when hairpins with identically-positioned polar partners are compared (i.e., V232D vs. Q207N-V232E). Login to comment
14 CF is inherited in a recessive autosomal fashion; most CF patients have a CFTR Available online at www.sciencedirect.com Biochimica et Biophysica Acta 1778 (2008) 79-87 www.elsevier.com/locate/bbamem Abbreviations: CF, Cystic fibrosis; CFTR, Cystic fibrosis transmembrane conductance regulator; TM, Transmembrane; TMD, Transmembrane domain; TM3/4, Helical hairpin including residues 194-241 of CFTR; WT, Wild type; V232D-TM3/4, TM3/4 construct with mutation of Val to Asp at position 232; I231D-TM3/4, TM3/4 construct with mutation of Ile to Asp at position 231; Q207N/V232E-TM3/4, TM3/4 construct with mutation of Gln to Asn at position 207 and Val to Glu at position 232; PFO, Perfluorooctanoate; DPC, Dodecylphosphocholine; SDS, Sodium dodecylsulfate; CD, Circular dichroism; H-bond, Hydrogen bond; HSQC, Heteronuclear single quantum coherence Ìe; Corresponding author. Login to comment
36 Previous studies we performed on CFTR helix-loop-helix constructs using gel shift analysis, fluorescence measurements, and molecular modeling suggested that hairpin folding is likely stabilized by folding due to formation of a non-native side chain-side chain hydrogen bond between 'polar partners` in the interacting helices (viz., Q207 in TM3, and I231D or V232D in TM4) [20]. Login to comment
44 We then use solution NMR experiments to demonstrate the conformational variability of TM3/4 hairpin mutants (including the CF-phenotypic mutant V232D; I231D; and Q207N/V232E) vs. the wild type hairpin in micellar environments. Login to comment
47 Expression and purification of wild type and mutant TM3/4 constructs from the CFTR membrane domain The 15 N and 15 N/13 C enriched forms of the TM3/4 helical hairpin constructs (WT-, V232D-, I231D-, Q207N/V232E) were expressed and purified as previously described [19]. Login to comment
193 To address the proposition that the formation of a given helix-helix interface may be dominated by a positional dependence of a polar residue in TM4, the 1 H-15 N HSQC spectrum of I231D-TM3/4 in PFO micelles was acquired under identical conditions as WT-TM3/4 and V232D-TM3/4. Login to comment
197 The 1 H-15 N HSQC spectrum of this double mutant shows that similar to V232D-TM3/4 and I231D-TM3/4 mutants, the residues in TM4 along with some in TM3 and the turn are all highly affected vs. WT upon these mutations. Login to comment
203 Conversely, this model would predict that in the case of I231D, there must be a relative reorientation by 100&#b0; of one helix with respect to the other for participation of I231D in interhelical interactions vs. either V232D or Q207N/V232E. Login to comment
210 Although hairpin constructs have degrees of conformational freedom in SDS or PFO micellar environments that would not be available to the corresponding helices embedded in an intact CFTR TM domain, our results suggest that hairpin interfaces - and possibly helix boundaries - may vary as a function of the position of a non-native polar mutation (i.e., V232D vs. I231D in TM4), and thereby indicate the susceptibility of the native protein structure to a corresponding destiny. Login to comment

[hide] Coarse-grained molecular dynamics simulations of m... J Struct Biol. 2007 Mar;157(3):593-605. Epub 2006 Oct 20. Bond PJ, Holyoake J, Ivetac A, Khalid S, Sansom MS
Coarse-grained molecular dynamics simulations of membrane proteins and peptides.
J Struct Biol. 2007 Mar;157(3):593-605. Epub 2006 Oct 20., [PMID:17116404]

Abstract [show]
Molecular dynamics (MD) simulations provide a valuable approach to the dynamics, structure, and stability of membrane-protein systems. Coarse-grained (CG) models, in which small groups of atoms are treated as single particles, enable extended (>100 ns) timescales to be addressed. In this study, we explore how CG-MD methods that have been developed for detergents and lipids may be extended to membrane proteins. In particular, CG-MD simulations of a number of membrane peptides and proteins are used to characterize their interactions with lipid bilayers. CG-MD is used to simulate the insertion of synthetic model membrane peptides (WALPs and LS3) into a lipid (PC) bilayer. WALP peptides insert in a transmembrane orientation, whilst the LS3 peptide adopts an interfacial location, both in agreement with experimental biophysical data. This approach is extended to a transmembrane fragment of the Vpu protein from HIV-1, and to the coat protein from fd phage. Again, simulated protein/membrane interactions are in good agreement with solid state NMR data for these proteins. CG-MD has also been applied to an M3-M4 fragment from the CFTR protein. Simulations of CFTR M3-M4 in a detergent micelle reveal formation of an alpha-helical hairpin, consistent with a variety of biophysical data. In an I231D mutant, the M3-M4 hairpin is additionally stabilized via an inter-helix Q207/D231 interaction. Finally, CG-MD simulations are extended to a more complex membrane protein, the bacterial sugar transporter LacY. Comparison of a 200 ns CG-MD simulation of LacY in a DPPC bilayer with a 50 ns atomistic simulation of the same protein in a DMPC bilayer shows that the two methods yield comparable predictions of lipid-protein interactions. Taken together, these results demonstrate the utility of CG-MD simulations for studies of membrane/protein interactions.

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11 In an I231D mutant, the M3-M4 hairpin is additionally stabilized via an inter-helix Q207/D231 interaction. Login to comment
94 Simulation Duration (ns) Number of lipids/waters Approximate bilayer/micelle formation time (ns) WALP16 3 £ 200 256 DPPC + 3,187 W 10, 8, 26 WALP19 3 £ 200 256 DPPC + 3,228 W 144, 49, 41 WALP23 3 £ 200 256 DPPC + 3,133 W 8, 40, 33 LS3 3 £ 200 256 DPPC + 3,333 W 20, 80, 40 Vpu 3 £ 200 256 DPPC + 3,117 W 7, 14, 20 Fd 3 £ 200 256 DPPC + 3,150 W 60, 16, 92 CFTR M3-M4 WT 3 £ 500 64 DPC + 15,551 W 60, 90, 100 CFTR M3-M4 I231D 1 £ 500, 64 DPC + 15,546 W 200 2 £ 200 120, 100 LacY CG 1 £ 200 217 DPPC + 2,504 W 20 LacY AT 1 £ 50 173 DMPC + 14,845 W - lipid bilayers (Im and Brooks, 2005;Nymeyer et al., 2005;Petrache et al., 2002). Login to comment
148 (B) Start and end snapshots for the CFTR M3-M4 I231D simulation. Login to comment
149 The initial structure is the starting model of the CFTR M3-M4 I231D mutant, modelled (based on secondary structure predictions) as two -helices connected by an extended loop. Login to comment
220 It is thought that the Q207 sidechain in M3 may form an interhelical H-bond with a CF-phenotypic mutant V232D in M4 (Therien et al., 2001). Login to comment
221 Asp-scanning mutagenesis, in which Asp was placed in M4 successively at residues 221-241, combined with gel shift assays to assess H-bonding suggested that the I231D formed the strongest predicted hydrogen bond with Q207 (Choi et al., 2004). Login to comment
222 To explore the nature of -helical hairpin formation in CFTR M3-M4 we therefore performed two simulations, of CFTR M3-M4 WT and of the I231D mutant. Login to comment
233 Interestingly, the average time for formation of a stable micelle was »80 ns for the WT, but »140ns for the I231D mutant. Login to comment
242 (A) Distributions of distance between the sidechain particles for residues 207 and 231 for simulations CFTR M3-M4 WT (black line) and M3-M4 I231D (grey line). Login to comment
244 For the WT snapshot the two spheres represent the Q207 and I231 sidechain particles, for the I231D snapshot they correspond to Q207 and D231. Login to comment
147 (B) Start and end snapshots for the CFTR M3-M4 I231D simulation. Login to comment
232 Interestingly, the average time for formation of a stable micelle was &#bb;80 ns for the WT, but &#bb;140ns for the I231D mutant. Login to comment
241 (A) Distributions of distance between the sidechain particles for residues 207 and 231 for simulations CFTR M3-M4 WT (black line) and M3-M4 I231D (grey line). Login to comment
243 For the WT snapshot the two spheres represent the Q207 and I231 sidechain particles, for the I231D snapshot they correspond to Q207 and D231. Login to comment

[hide] Non-native interhelical hydrogen bonds in the cyst... Biochemistry. 2004 Jun 29;43(25):8077-83. Choi MY, Cardarelli L, Therien AG, Deber CM
Non-native interhelical hydrogen bonds in the cystic fibrosis transmembrane conductance regulator domain modulated by polar mutations.
Biochemistry. 2004 Jun 29;43(25):8077-83., [PMID:15209503]

Abstract [show]
Polar residues comprise about 15% of the transmembrane (TM) domains of proteins, where they can stabilize structure via native side chain-side chain interhelical hydrogen bonds between TM helices. However, non-native H-bonds may be implicated in disease states, through limiting protein dynamics during transport and/or misfolding the protein by inducing non-native rotational positions about TM helical axes. Here we have undertaken an investigation of the presence and strength of H-bond interactions within a series of helix-loop-helix ("hairpin") constructs derived from TM helices 3 and 4 (italic) of the cystic fibrosis transmembrane conductance regulator (CFTR) (prototypic sequence G(194)LALAHFVWIAPLQ(207)VALLMGLIWELLQASAFAGLGFLIV(232)LALFQ(237)AGLG(241)) in which wild-type Q207 in TM3 forms an interhelical H-bond with CF-phenotypic mutant V232D in TM4 [Therien, A. G., Grant, F. E., and Deber, C. M. (2001) Nat. Struct. Biol 8, 597-601]. In the present work, a library of 21 TM3/4 constructs was prepared, where Asp residues were placed individually at TM4 positions 221-241. Using gel shift assays-in which H-bond-linked hairpins (closed conformation) migrate faster than the elongated forms (open conformation)-we found that Q207 in TM3 is able to "capture" all 21 TM4 D mutations into measurable populations of interhelical H-bonds. A similar library of TM4 D mutants-but also containing Q207L-reverted to wild-type migration rates, confirming Q207 as the polar partner for TM4 D residues. In view of the broad capture range of Q207, these results emphasize the potential consequences to folding and dynamics of introducing polar mutations into the TM domains of membrane proteins in the vicinity of a native polar TM residue.

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87 When these results are quantitated for the full TM4 D mutant library (Figure 3b), we find that the I231D construct has the largest negative percentage molecular weight decrease relative to the wt (-11.65% ( 0.61%), and hence migrated the furthest among all the mutants in TM4 on SDS-PAGE. Login to comment
89 Interestingly, mutants G228D through I231D, and L233D through F236D, have a more negative value of the percent of MW decrease relative to the wt than the CF-phenotypic mutant V232D (Figure 3b) (see the Discussion). Login to comment
131 Molecular Modeling Studies on the I231D Construct. Login to comment
162 Thus, for example, if one assumes that I231D in TM4 forms the closest contacts with Q207 in TM3, there are 10 residues ()2.7 turns of helix) from I231D for G241D. Login to comment
170 The present model TM3/4 systems also assume that sequentially consecutive helices will be adjacent and aligned in the native proteins FIGURE 6: Energy-minimized structural models of antiparallel CFTR transmembrane helical segments 3 and 4 in the I231D mutant. Login to comment
171 Space-filling model of the I231D helical hairpin (intervening loop omitted). Login to comment
173 (a) View of the I231D model showing the side chain carboxamide from Q207 in TM3 interacting with the side chain carboxylate of D231 in TM4. Login to comment
185 In fact, the I231D construct has the greatest percentage molecular weight decrease relative to the wt, and ostensibly forms the tightest H-bond with Q207 (illustrated with energy-minimized helical dimers in Figure 6). Login to comment