Julie Briot, Nazzareno D’Avanzo, Jurgen Sygusch and Lucie Parent
L-type calcium channels (LTCC) are responsible for Ca2+ influx into muscle and neurons. These macromolecular complexes minimally comprise the main poreforming CaVα1 and auxiliary subunits CaVβ and CaVα2δ1. The ultrastructure of the oligomeric LTCC complexes from heart and skeletal muscle has been reported previously at ≈ 20 Å, a resolution that prevent identification of structural domains. Recent improvements in cryo-electronic microscopy (EM) methods made it possible to obtain a three-dimensional structure of the rabbit skeletal muscle LTCC CaV1.1 complex at a resolution of 4.2 Å and recently at 3.6 Å. This technique requires only nanograms of purified proteins and circumvents crystallization as a means for structure determination. The high resolution cryo-EM structure shows the molecular architecture of the subunits comprising the oligomeric complex and for the first time, a high-resolution glance of the largely extracellular CaVα2δ1 protein with its extracellular domains (Cache1, VWA, and Cache2). Although the CaVα2δ1 protein is a single-pass transmembrane protein, the complex topology of its extracellular domain represents a technical challenge for structure determination using conventional purification approaches. Herein we show the merits of a strategy based upon the purification of small structural domains that can be elucidated individually before these domains are reassembled into the quaternary structure. A structural model was derived using ab initio structure prediction constrained by small angle X-ray scattering profile of the refolded Cache2 domain. The excellent agreement between the predicted structure and the available cryo-EM structure suggests a novel and rapid procedure to discover structural information of protein domains.
