Electron Crystallography of Euglenoid Four-Transmembrane Protein Revealed the Linear Polymerization by a Combination of Three-Ways of Intermolecular Interaction

In Euglena gracilis, cell membranes on the ridge regions of the striped surface structure are covered with paracrystalline arrays, mainly composed of the integral membrane protein, called IP39. It has recently been cloned as a putative four-membrane-spanning protein with a conserved sequence motif of PMP-22/EMP/MP20/Claudin superfamily. Here, we report the three-dimensional structure of Euglena IP39 at 7 A resolution determined by image-based electron crystallography of two-dimensional (2D) crystals. The 2D crystal array of IP39 appears to be a striated pattern of the antiparallel double-rows, in which the trimeric units of IP39 are longitudinally polymerized, resulting in continuously extending zigzag lines. Each protomer shows an overall cylindrical density (approximately 20 A in diameter) with ‘nock’ and ‘pointed’ shapes at the respective sides of the membrane. Structural analysis of another 2D crystal bound with anti-phosphotyrosine Fab fragments reveals that the ‘nock’-like protruded structure is facing to the cytoplasmic side. A four-helical bundle model is consistently fitted to the EM density map and shows that the transmembrane regions are mainly involved in the intermolecular interactions to form the linear strands. The polymerization pattern of IP39 in the 2D crystal, however, exhibits unexpected interactions between respective protomers. In the trimeric unit of the single strand, one of the three protomers is likely to be rotated at approximately 180 degree in the opposite direction to the others, indicating that there are at least three different ways of possible intermolecular interactions in the transmembrane regions between neighboring protomers. A combination of such multiple interactions would be important for the continuous linear polymerization, thus providing important implications in the strand formation of other four-transmembrane proteins of this family in the lipid environment.