The PKD1 and PKD2 genes are the genes that are mutated in patients suffering from autosomal dominant polycystic kidney disease. The human PKD2 gene codes for a 968-amino acid long membrane protein called polycystin-2 that represents a cation channel whose activity can be regulated by Ca 2؉ ions. By CD, fluorescence, and NMR spectroscopy, we have studied a 117-amino acid-long fragment of the cytoplasmic domain of polycystin-2, polycystin-2-(680 -796) that was proposed to contain a Ca The PKD2 gene is one of the two genes mutated in patients with autosomal-dominant polycystic kidney disease (1). It encodes polycystin-2, a 968-amino acid-long protein with 6 putative membrane-spanning domains. According to structural predictions, both its N and C termini extend into the cytoplasm; furthermore, a pore-forming region has been postulated between the fifth and sixth membrane-spanning domains. By sequence comparison a coiled-coil domain and a Ca 2ϩ -binding EF-hand have been predicted in the C terminus of polycystin-2 (1) and a homology model has been published recently (2). However, so far only indirect evidence for the presence of these motifs has been presented. Electrophysiological measurements by several groups have demonstrated that polycystin-2 is a non-selective cation channel and that its channel activity is regulated by calcium. One report has shown the activation of polycystin-2 by the addition of 1 M Ca 2ϩ to the bath solution and its inhibition by millimolar concentrations of Ca 2ϩ (3). An R742X mutant protein, however, did not respond to the addition of Ca 2ϩ . These initial observations were confirmed by other publications that reported that Ca 2ϩ concentrations of up to 1.26 mM increase the probability for the open state of full-length polycystin-2 (4), whereas higher concentrations are inhibitory (4, 5). Again, the activity of a truncated protein, this time a 703-amino acid mutant, is not modulated by calcium (4).From the above it seems obvious that the C terminus via binding of Ca 2ϩ exerts a modulatory activity on the channel activity of polycystin-2. This part of the protein, however, also is of particular interest because it interacts with a wide variety of other proteins, among them polycystin-1, ion channels (inositol 1,4,5-trisphosphate receptor, polycystin-2 itself, and TRPC1), cytoskeletal proteins (␣-actinin, CD2AP, mDia1, troponin I, and tropomyosin-1), intracellular trafficking proteins (PACS-1, PACS-2, and PIGEA-14) and even a transcription factor (Id2) (6). Whether all of these interactions are direct or require additional cofactors is not clear at present; furthermore, it is not known whether any of them are cooperative or exclusive. We have therefore decided to subject the C terminus of polycystin-2 to an extensive biochemical and structural analysis. 3 The abbreviations used are: DSS, 2,2-dimethyl-2-silapentane-5-sulfonic acid; CDPK-␣, calmodulin-like domain of the calcium dependent soybean protein kinase ␣; HSQC, heteronuclear single quantum coherence; ISIC, intelligent structural infor...