Connexons Coupling to Gap Junction Channel: Potential Role for Extracellular Protein Stabilization Centers.

Ágnes Simon,Zsolt Szabó,László Héja,Julianna Kardos

doi:10.3390/biom12010049

Ágnes Simon, Zsolt Szabó + Show 2 more

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https://doi.org/10.3390/biom12010049

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Abstract

Connexin (Cx) proteins establish intercellular gap junction channels (Cx GJCs) through coupling of two apposed hexameric Cx hemichannels (Cx HCs, connexons). Pre- and post-GJ interfaces consist of extracellular EL1 and EL2 loops, each with three conserved cysteines. Previously, we reported that known peptide inhibitors, mimicking a variety of Cx43 sequences, appear non-selective when binding to homomeric Cx43 vs. Cx36 GJC homology model subtypes. In pursuit of finding potentially Cx subtype-specific inhibitors of connexon-connexon coupling, we aimed at to understand better how the GJ interface is formed. Here we report on the discovery of Cx GJC subtype-specific protein stabilization centers (SCs) featuring GJ interface architecture. First, the Cx43 GJC homology model, embedded in two opposed membrane bilayers, has been devised. Next, we endorsed the fluctuation dynamics of SCs of the interface domain of Cx43 GJC by applying standard molecular dynamics under open and closed cystine disulfide bond (CS-SC) preconditions. The simulations confirmed the major role of the unique trans-GJ SC pattern comprising conserved (55N, 56T) and non-conserved (57Q) residues of the apposed EL1 loops in the stabilization of the GJC complex. Importantly, clusters of SC patterns residing close to the GJ interface domain appear to orient the interface formation via the numerous SCs between EL1 and EL2. These include central 54CS-S198C or 61CS-S192C contacts with residues 53R, 54C, 55N, 197D, 199F or 64V, 191P, respectively. In addition, we revealed that GJC interface formation is favoured when the psi dihedral angle of the nearby 193P residue is stable around 180° and the interface SCs disappear when this angle moves to the 0° to −45° range. The potential of the association of non-conserved residues with SC motifs in connexon-connexon coupling makes the development of Cx subtype-specific inhibitors viable.

Highlights

Vertebrate cells perform dominant remodelling of intercellular adhesion and communication via specific subunit combinations of hexameric connexin (Cx) hemichannels (CxHCs, connexons) that couple to form dodecameric gap junction channels (Cx GJCs) [1,2,3,4,5,6,7,8].Canonical Cx proteins present three conserved cysteines in their extracellular loopsEL1 and EL2, except Cx23 subtype identified in mammals and zebrafish
Certain Cys residues seem to form protein stabilization centers (SCs), meaning that they are far in sequence, at least one of their atoms are closer than VWR(1) + VWR(2) + 1 Å, where VWR designates van der Waals radii (VWR) and residues in their vicinity intensively interact with each other [29]
Taking cysteine-rich domains as major players of intercellular communication, we Taking cysteine-rich domains as major players of intercellular communication, we could refer to the cytoplasmic carboxy terminus securing Cx GJCs in plaques [60]

Summary

Introduction

Vertebrate cells perform dominant remodelling of intercellular adhesion and communication via specific subunit combinations of hexameric connexin (Cx) hemichannels (CxHCs, connexons) that couple to form dodecameric gap junction channels (Cx GJCs) [1,2,3,4,5,6,7,8].Canonical Cx proteins present three conserved cysteines in their extracellular loopsEL1 and EL2, except Cx23 subtype identified in mammals and zebrafish. The Cx23a isoform has only two conserved cysteines in its extracellular EL1 and EL2 loops and is characterized by less efficient GJC coupling [9]. Pannexin [10] and innexin [11] channels, releasing small molecules and ions contain two conserved cysteines in EL1 and EL2 loops and they hardly serve GJC functions either. We may conjecture the implementation of the GJC function with the higher number of extracellular cysteines in EL1 and EL2 loops. An exception to this rule is the Cx31.3 isoform that does not form functional GJC [5] despite the Biomolecules 2022, 12, 49.

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