X-Ray Structure of the Cx26 Gap Junction Channel and Comparison with the Cryo-EM Structure of Cx43

Abstract

Intercellular gap junction channels are formed by the end-to-end docking of connexin (Cx) hexamers that traverse apposing cell membranes. Each Cx subunit has four transmembrane (TM) α-helices and two extracellular loops (E1 and E2). Three-dimensional crystals of recombinant, purified Cx26 were grown using a new class of detergents designated facial amphiphiles (FAs), which have a cholate backbone with polar groups extending from one face and a short alkyl chain extending from the opposite face. FA-solubilized Cx26 crystallized into the H32 space group with two monomers in the asymmetric unit, and the crystals diffracted isotropically to 3.3-A resolution. Using a molecular replacement search model based on a cryoEM map of Cx43 at 5.7-A resolution [Fleishman et al., Mol. Cell 15: 879-888 (2004)], we solved the structure independently from a previously reported 3.5-A resolution X-ray structure [2ZW3, Maeda et al., Nature 458: 597-602 (2009)]. The overall R/Rfree values and completeness were 0.311/0.328 and 98.9%, respectively, and the Molprobity score was 2.07 (100th percentile). The RMS differences between 2ZW3 and our structure were 0.9 and 1.7-A for the main-chain and side-chain atoms in the TM helices and 1.3 and 1.9-A for the main-chain and side-chain atoms in E1 and E2. Although the topology and fold recapitulated 2ZW3, the maximum differences were significant: 2.7 and 5.9-A for the main chain and side chain atoms in the TM helices and 3.9 and 7.6-A for the main-chain and side-chain atoms in E1 and E2. We generated an electron density map at a resolution comparable to the cryoEM structure of the authentic Cx43 channel. The similarity of the maps suggests that detergent-solubilized Cx26 that crystallized as a dodecamer represents the authentic gap junction channel.