ABCMdb

PMID: 11888281

Partridge AW, Melnyk RA, Deber CM
Polar residues in membrane domains of proteins: molecular basis for helix-helix association in a mutant CFTR transmembrane segment.
Biochemistry. 2002 Mar 19;41(11):3647-53., 2002-03-19 [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC7 p.Val232Asp To systematically explore the structural consequences of H-bonds between TM helices, we focused on TM4 of the cystic fibrosis conductance regulator (CFTR) and its cystic fibrosis- (CF-) phenotypic mutation, V232D, as a model system. Login to comment 2 ABCC7 p.Val232Asp Synthetic peptides corresponding to wild-type (TM4-wt) (residues 219-242: LQASAFCGLGFLIVLALFQAGLGR) and mutant (TM4-V232D) sequences both adopt helical structures in SDS micelles and display dimer bands on SDS-PAGE arising from disulfide bond formation via wild-type residue Cys-225. Login to comment 3 ABCC7 p.Val232Asp However, the TM4-V232D peptide additionally forms a ladder of noncovalent oligomers, including tetramers, hexamers, and octamers, mediated by a hydrogen bond network involving Asp-Gln side chain-side chain interactions. Login to comment 19 ABCC7 p.Val232Asp To investigate the roles of polar side chains in TM helices, we synthesized peptides corresponding to TM4 from CFTR either with or without the CF V232D mutation. Login to comment 20 ABCC7 p.Val232Asp We found that whereas the wild-type TM4 peptide failed to form any noncovalent helical associations, the TM4 peptide containing the V232D mutation generates a well-defined oligomeric ladder of TM4 helices mediated through a network of interhelical side chain-side chain H-bonds between D232 † This work was supported, in part, by grants to C.M.D. from the Canadian Cystic Fibrosis Foundation, the Canadian Institutes for Health Research (CIHR), and the National Institutes of Health (NIDDK). Login to comment 65 ABCC7 p.Val232Asp The TM4 peptide was designed identically to TM4-wt except that it contained the V232D mutation. Login to comment 67 ABCC7 p.Val232Asp Effect of the V232D Mutation on Helical Packing. Login to comment 71 ABCC7 p.Val232Asp However, the TM4 peptide containing the V232D mutation displays additional bands consistent with those of a tetramer, hexamer, and octamer. Login to comment 82 ABCC7 p.Phe229Ala ABCC7 p.Gln220Ala ABCC7 p.Phe224Ala ABCC7 p.Ser222Ala For example, the point mutations Q220A, S222A, F224A, and F229A each resulted in species patterns with a more heavily populated dimer band versus the higher order oligomer bands. Login to comment 83 ABCC7 p.Gly226Ala In contrast, the G226A mutation stabilized the oligomer ladder, as evidenced by the presence of additional higher order oligomeric bands corresponding to decamers and dodecamers. Login to comment 107 ABCC7 p.Cys225Ala Spectra are TM4-VD and TM4-VD(C225A). Login to comment 122 ABCC7 p.Val232Asp In this context, we simulated H-bond formation in membrane-interactive peptides using segments featuring the CF-phenotypic mutation V232D from TM4 of the CFTR transmembrane domain. Login to comment 135 ABCC7 p.Val232Asp The V232D peptide was reduced in the lane indicated by incubating the peptide with TCEP in aqueous solution for 5 min prior to addition of Tricine sample buffer. Login to comment 163 ABCC7 p.Val232Asp However, due to FIGURE 6: Molecular models proposed for the oligomerization of the CFTR transmembrane helix 4 containing the V232D mutation (TM4-VD). Login to comment 178 ABCC7 p.Val232Asp The V232D mutation in TM4 creates a membrane-buried electrostatic locus, whether it is in the context of a TM peptide or in the full-length protein. Login to comment 182 ABCC7 p.Val232Asp For example, where the V232D mutation arises in full-length CFTR, the D232 residue on one TM4 helix could partner with the Q237 residue in the complementary TM4 helix in another CFTR molecule, to produce a functionally impaired H-bonded dimer. Login to comment 185 ABCC7 p.Val232Asp In the single-spanning TM peptides studied here, the V232D mutation results in a homophilic association of TM4 helices mediated by D232 and Q237 interhelical H-bonding. Login to comment 187 ABCC7 p.Val232Asp That the V232D mutation produces a non-wild-type H-bond in both systems suggests that a given mutant polar residue arising in the CFTR membrane domain could have multiple polar partners available in vivo. Login to comment