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. Author manuscript; available in PMC: 2022 Jul 1.
Published in final edited form as: J Physiol. 2021 May 13;599(13):3313–3335. doi: 10.1113/JP281339

Fig. 2. Arginine-9 of Cx46 and designed variants adopts a dynamic ensemble of stable conformational states that prevent pore constriction.

Fig. 2

a. Cryo-EM based structure of Cx46 (PDB 6MHQ), viewed along the pore axis with residues R9 and E12 displayed in stick representation. R9 is oriented toward the center of the pore permeation pathway, and forming a primary constriction site. b. Representative snap-shot of Cx46 following MD equilibration, viewed as in panel a. R9 adopts alternative conformational states that reorient this sidechain away from the pore permeation pathway, that appear to be stabilized by salt bridge interaction with a neighboring E12 (asterisk, *) and/or π-π interactions with R9 from a neighboring subunit (dagger, †). c. Zoom view (boxed in panel b) of a representative R9 salt bridge interaction with a neighboring E12 residue. d. Zoom view (boxed in panel b) of a representative π-π interaction between two R9 residues of neighboring subunits. Distances displayed in panels c,d are between nearest heavy atoms. e. Probability distribution of the distance between position-9 to position-12 in the neighboring subunit obtained from MD simulation for Cx46, Cx50-N9R and Cx50-46NT (blue traces, represent R9-E12(i+1) distances) and Cx50, Cx46-R9N and Cx46-50NT (red traces, represent N9-E12(i+1) distances). f. Probability distribution of the distance of position-9 to position-9 in the neighboring subunit obtained from MD simulation for Cx46, Cx50-N9R and Cx50-46NT (blue traces, represent R9-R9(i+1) distances) and Cx50, Cx46-R9N and Cx46-50NT (red traces, represent N9-N9(i+1) distances). Distances displayed in panels e,f were measured between R9 Cζ or N9 Cδ and E12 Cγ to capture equivalent rotameric states, and are therefore greater than those displayed in panels c,d.