Structural studies of N-terminal mutants of connexin 32 using (1)H NMR spectroscopy.
The amino terminus of gap junction proteins, connexins, plays a fundamental role in voltage gating and ion permeation. We have previously shown with (1)H NMR that the structure of the N-terminus of functional connexin molecules contains a flexible turn around G12 (Arch. Biochem. Biophys.490:9,2009) allowing the N-terminus to form a portion of the channel pore near the cytoplasmic entrance. The mutants of nonfunctional connexin molecules G12S and G12Y were found to prevent this turn. Previous functional studies of loci at which Cx32 mutations cause a peripheral neuropathy, Charcot-Marie-Tooth disease, have shown that G12S is not plasma membrane inserted. Presently, we solve the structure of nonfunctional Connexin 32 mutants W3D and Y7D which do not appear to be membrane inserted. Using 2D (1)H NMR, we report that similar to G12S and G12Y, alterations in hydrophobic sidechain interactions disrupt (Y7D) or constrain (W3D) the flexible turn around G12. The alteration in the open turn around residue 12, observed in all nonfunctional mutants G12S, G12Y, W3D and Y7D correlates with loss of function. We propose that loss of the open turn causes the N-terminus to extend out of the channel pore and this misfolding may target mutants for destruction in the endoplasmic reticulum.
Department of Chemistry, Brooklyn College of the City University of New York, Brooklyn, NY 11210, United States.
B D
BD
Kalmatsky
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Y
Batir
T A
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Bargiello
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Dowd
D000595:Amino Acid Sequence; D017630:Connexins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D012996:Solutions; D000097002:Gap Junction beta-1 Protein
D017630:Connexins; D050505:Mutant Proteins; D010446:Peptide Fragments; D012996:Solutions; D000097002:Gap Junction beta-1 Protein