Ligand-Induced Conformational Changes in Tetrameric IP3R1 Revealed by Single-Particle Cryo-EM

Abstract

A functional hallmark of inositol 1,4,5-trisphosphate receptors (IP3Rs), the main Ca2+ release channels in the endoplasmic reticulum of virtually all eukaryotic cells, is the coupled interplay between the binding of primary ligands, IP3 and Ca2+, and channel gating. IP3Rs are exceptionally large integral membrane proteins, comprising four subunits of over 300 kDa each. A central mechanistic question of IP3R function is how IP3 binding in the N-terminal sequence of the channel protein is communicated to the ion-conduction pore, which is formed close to the C-terminus. Using single-particle cryo-EM, we have performed structural analysis of purified tetrameric IP3R1 vitrified in the presence of micromolar Ca2+ and saturating concentration of adenophostin A (AdA), a structural mimetic of IP3, that is a high-affinity, full agonist of IP3Rs. Cryo-EM images of IP3R1 were recorded on a Gatan 4k x 4k CCD camera in the JEM2010F cryomicroscope operated at low-dose conditions. Using EMAN2, ∼40,000 particle images were merged to generate a preliminary 3D map of ligand-bound IP3R1 at ∼15 A resolution. A comparison with our recent structure of IP3R1, determined without the addition of any channel ligands (Ludtke et al., 2010: Structure 19, 1192-99), reveals structural rearrangements in the cytoplasmic domains of the ligand-bound IP3R1 channel. Furthermore, we have performed docking studies of available crystal structures of the ligand-binding domains into reconstructed cryo-EM density maps of the entire IP3R1. All together our studies suggest that AdA/Ca2+ binding induces conformational changes in the quaternary structure of tetrameric IP3R1 that might underlie the long-range allosteric mechanism of agonist-mediated activation of the ion-conducting pore of IP3R. Supported by grants from NIH (R21AR063255, R01GM072804, R01GM080139, P41GM103832) and by AHA (12GRNT10510002).