NMR and Structural Data for Connexin 32 and Connexin 26 N-terminal Peptides

In this article we present 1H and 13C chemical shift assignments, secondary structural propensity data and normalized temperature coefficient data for N-terminal peptides of Connexin 26(Cx26), Cx26G12R and Cx32G12R mutants seen in syndromic deafness and Charcot Marie Tooth Disease respectively, published in “Structural Studies of N-Terminal Mutants of Connexin 26 and Connexin 32 Using 1HNMR Spectroscopy” (Y.Batir,T.A.Bargiello,T.L.Dowd, 2016)[1]. The mutation G12R affects thestructure of both Cx26 and Cx32 peptides differently. We present data from secondary structure propensity chemical shift analysis which calculates a secondary structurepropensity (SSP) score for both disordered or folded peptides and proteins using the difference between the 13C secondary chemical shifts of the Cα and Cβ protons.This data supplements the calculated NMR structures from NOESY data[1]. We present and compare the SSP data for the Cx26 vs Cx26G12R peptides and the Cx32 and Cx32G12R peptides. In addition,we present plots of temperature coefficients obtained for Cx26, Cx26G12R and Cx32G12R peptides collected previously [1] and normalized to their random coil temperature coefficients, “Random coil 1H chemical shifts obtained as a function of temperature and trifluoroethanol concentration for the peptide series GGXGG” (G. Merutka, H.J.Dyson, P.E.Wright,1995) [2]. Reductions in these normalized temperature coefficients are directly observable for residues in different segments of the peptide and this data informs on solvent accessibility of the NH protons and NH protons which may be more constrained due to the formation of H bonds

Conformational changes in a pore-forming region underlie voltage-dependent “loop gating” of an unapposed connexin hemichannel

The structure of the pore is critical to understanding the molecular mechanisms underlying selective permeation and voltage-dependent gating of channels formed by the connexin gene family. Here, we describe a portion of the pore structure of unapposed hemichannels formed by a Cx32 chimera, Cx32*Cx43E1, in which the first extracellular loop (E1) of Cx32 is replaced with the E1 of Cx43. Cysteine substitutions of two residues, V38 and G45, located in the vicinity of the border of the first transmembrane (TM) domain (TM1) and E1 are shown to react with the thiol modification reagent, MTSEA–biotin-X, when the channel resides in the open state. Cysteine substitutions of flanking residues A40 and A43 do not react with MTSEA–biotin-X when the channel resides in the open state, but they react with dibromobimane when the unapposed hemichannels are closed by the voltage-dependent “loop-gating” mechanism. Cysteine substitutions of residues V37 and A39 do not appear to be modified in either state. Furthermore, we demonstrate that A43C channels form a high affinity Cd2+ site that locks the channel in the loop-gated closed state. Biochemical assays demonstrate that A43C can also form disulfide bonds when oocytes are cultured under conditions that favor channel closure. A40C channels are also sensitive to micromolar Cd2+ concentrations when closed by loop gating, but with substantially lower affinity than A43C. We propose that the voltage-dependent loop-gating mechanism for Cx32*Cx43E1 unapposed hemichannels involves a conformational change in the TM1/E1 region that involves a rotation of TM1 and an inward tilt of either each of the six connexin subunits or TM1 domains

Molecular dynamics simulations of the Cx26 hemichannel: Evaluation of structural models with Brownian dynamics

The recently published crystal structure of the Cx26 gap junction channel provides a unique opportunity for elucidation of the structure of the conductive connexin pore and the molecular determinants of its ion permeation properties (conductance, current–voltage [I-V] relations, and charge selectivity). However, the crystal structure was incomplete, most notably lacking the coordinates of the N-terminal methionine residue, which resides within the pore, and also lacking two cytosolic domains. To allow computational studies for comparison with the known channel properties, we completed the structure. Grand canonical Monte Carlo Brownian dynamics (GCMC/BD) simulations of the completed and the published Cx26 hemichannel crystal structure indicate that the pore is too narrow to permit significant ion flux. The GCMC/BD simulations predict marked inward current rectification and almost perfect anion selectivity, both inconsistent with known channel properties. The completed structure was refined by all-atom molecular dynamics (MD) simulations (220 ns total) in an explicit solvent and POPC membrane system. These MD simulations produced an equilibrated structure with a larger minimal pore diameter, which decreased the height of the permeation barrier formed by the N terminus. GCMC/BD simulations of the MD-equilibrated structure yielded more appropriate single-channel conductance and less anion/cation selectivity. However, the simulations much more closely matched experimentally determined I-V relations when the charge effects of specific co- and posttranslational modifications of Cx26 previously identified by mass spectrometry were incorporated. We conclude that the average equilibrated structure obtained after MD simulations more closely represents the open Cx26 hemichannel structure than does the crystal structure, and that co- and posttranslational modifications of Cx26 hemichannels are likely to play an important physiological role by defining the conductance and ion selectivity of Cx26 channels. Furthermore, the simulations and data suggest that experimentally observed heterogeneity in Cx26 I-V relations can be accounted for by variation in co- and posttranslational modifications

Determinants of Gating Polarity of a Connexin 32 Hemichannel

There is good evidence supporting the view that the transjunctional voltage sensor (V(j)-sensor) of Cx32 and other Group 1 connexins is contained within a segment of the N-terminus that contributes to the formation of the channel pore. We have shown that the addition of negatively charged amino acid residues at several positions within the first 10 amino acid residues reverses the polarity of V(j)-gating and proposed that channel closure is initiated by the inward movement of this region. Here, we report that positive charge substitutions of the 2nd, 5th, and 8th residues maintain the negative polarity of V(j)-gating. These data are consistent with the original gating model. Surprisingly, some channels containing combinations of positive and/or negative charges at the 2nd and 5th positions display bipolar V(j)-gating. The appearance of bipolar gating does not correlate with relative orientation of charges at this position. However, the voltage sensitivity of bipolar channels correlates with the sign of the charge at the 2nd residue, suggesting that charges at this position may have a larger role in determining gating polarity. Taken together with previous findings, the results suggest that the polarity V(j)-gating is not determined by the sign of the charge lying closest to the cytoplasmic entry of the channel, nor is it likely to result from the reorientation of an electrical dipole contained in the N-terminus. We further explore the mechanism of polarity determination by utilizing the one-dimensional Poisson-Nernst-Plank model to determine the voltage profile of simple model channels containing regions of permanent charge within the channel pore. These considerations demonstrate how local variations in the electric field may influence the polarity and sensitivity of V(j)-gating but are unlikely to account for the appearance of bipolar V(j)-gating