Autosomal dominant polycystic kidney disease (ADPKD) is one of the most prevalent human genetic diseases, afflicting approximately 0.2% of the world population.It is caused by mutations in genes encoding TRPP2 and PKD1, which form an ion channel/receptor complex on the cell surface.PKD1 is a member of the polycystin family whereas TRPP2 is a member of the transient receptor potential (TRP) ion channel family.Using a combination of biochemistry, X-ray crystallography and a single molecule method to determine the subunit composition of proteins in the plasma membrane of live cells, we find that the TRPP2/PKD1 complex contains 3 TRPP2 and 1 PKD1.A newly identified coiled-coil domain in the C terminus of TRPP2 is critical for the formation of this complex.This coiled-coil domain forms a homotrimer, both in solution and in crystal structure, and binds to a single coiled-coil domain in the C terminus ofPKD1.To gain molecular and functional insights into this heterotetrameric coiled-coil complex, we computationally constructed a structural model by combining a peptide docking method and molecular dynamics simulations.The model shows that this tetrameric complex has a novel "di-trimer" configuration: an upstream trimer made of three TRPP2 helices and a downstream trimer made of two TRPP2 helices and one PKD1 helix.Mutagenesis and biochemical analysis derived from this model identify critical TRPP2/PKD1 interface contacts essential for the heteromeric coiled-coil complex.Mutation of these interface positions in the full-length proteins showed that these interactions were critical for the assembly of the full-length complex in cells.Our results provide a means to specifically weaken the TRPP2 and PKD1 association, thus facilitating future in vitro and in vivo studies on the functional importance of this association.Our results also have significant implications for the assembly, regulation and function of the TRPP2/PKD1 complex, and for the pathogenic mechanism of ADPKD-causing mutations that disrupt the TRPP2/PKD 1 coiled-coil complex.