Structural studies of N-terminal mutants of Connexin 32 using 1H NMR spectroscopy

Abstract

The amino terminus of gap junction proteins, connexins, plays a fundamental role in voltage gating and ion permeation. We have previously shown with 1H 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 1H 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.

Voltage Regulation of Connexin Channel Conductance

“…These studies demonstrated the presence of a flexible open turn initiated by the 12th residue (glycine in both Cx26 and Cx32) that would allow the n-terminal half of the N-terminus to be positioned within the channel pore. 53 , 54 , 55 Demonstration that cysteine substitutions of the 4th, 5th, and 8th position can be modified by a MTS reagents strongly support the proposed structure 14 and Oh and Bargiello (unpublished observations). Significantly, the NMR derived structure agrees well with that of the N-terminus in the crystal structure of Cx26 gap junctions.…”

Structural studies of N-terminal mutants of Connexin 26 and Connexin 32 using 1H NMR spectroscopy

“…N-terminal peptides with mutations that fail to form a flexible turn do not form functional channels [25, 26]. The solution structure of the Cx26 N-terminal G12R peptide indicates formation of a highly flexible open turn, whereas that of Cx32G12R forms a highly structured more constrained turn.…”

“…Our past studies have shown that formation of an open flexible turn in Cx32 N-terminal peptides correlates with channel expression. Cx32 peptides containing substitutions G12S, Y7D and W3D do not form the open turn initiated by G12 and these mutations fail to produce functional channels [25, 26, 54]. In the case of G12S, the loss of expression is a consequence of a trafficking/assembly defect [19], and it is likely that the same is true for Y7D and W3D.…”

“…We solve the structure of the Cx32G12R N-terminal peptide to compare it to the Cx26G12R N-terminus as well as with that of the wild-type Cx32 N-terminus. All structures are compared with previously solved Connexin 32 loss of function mutation structures to understand structure-function relationships of Connexin molecules in health and disease [25, 26]. …”

Proteins and Mechanisms Regulating Gap-Junction Assembly, Internalization, and Degradation

“…The short NH 2 -terminal domains of the six Cx subunits form the funnel-shaped channel wall (FIGURE 2C), which is stabilized and held in place by a circular network of hydrogen bonds formed between the D2 residue on one Cx subunit and the backbone amide of T5 on another Cx subunit, as well as between W3 and M32 in TM1 within the same Cx subunit; yet the crystallographic data indicate that the NH 2 termini are the most mobile domains in the Cx26 channel structure. Indeed, the secondary structure of the NH 2 terminus can be broken down into two regions: an NH 2 -terminal ␣-helix or hydrophobic domain followed by an open turn (FIGURE 2A, (81), NH 2 -terminal FPtagged GJ channels are nonfunctional based on their inability to transfer the GJ-permeable dye lucifer yellow (99). Thus, although NH 2 -terminal GFP-tagged Cxs may be useful to address certain questions (11,23,27,60), their inability to form functional channels may be as destructive as the inability of COOH-terminal GFP-tagged Cxs to bind to the scaffolding protein ZO-1 (73) (see Cx43/ ZO-1 Binding for more detail), and this needs to be taken into close consideration if meaningful results are to be obtained.…”