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Purification and Crystallization of the C‐terminal Interaction Domains of TRPP Ion Channel Proteins
Author(s) -
Martin Hannah,
Agbaje Zainab,
Yu Yong
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.772.7
Subject(s) - transient receptor potential channel , chemistry , polycystic kidney disease , trpc1 , microbiology and biotechnology , autosomal dominant polycystic kidney disease , trpv , coiled coil , biochemistry , receptor , biophysics , biology , endocrinology , kidney , trpv1
Members of the transient receptor potential (TRP) protein family of ion channels are essential for various cell signaling pathways throughout the body. Proteins in the polycystin subfamily of TRP channels (TRPP) form receptor‐ion channel complexes with members of the polycystic kidney disease (PKD) protein family in 3:1 stoichiometry (3TRPP:1PKD). TRPP2 forms a flow‐sensing complex in primary cilia with its PKD partners that determines left‐right asymmetry in the embryonic node and proper morphology of kidney tubules. Mutations in TRPP2 cause autosomal dominant polycystic kidney disease (ADPKD), which affects over 400,000 Americans and often leads to end stage renal failure. TRPP3, with its binding partner PKD1L3, is a sour taste receptor candidate found in taste cells in the tongue. The C‐terminus of the TRPP channel has been shown to be essential for channel assembly and function. In both TRPP2 and TRPP3, the C‐terminal coiled‐coil domains form a homotrimer and play a key role in the assembly of the TRPP/PKD complexes. Another C1 domain was found in TRPP3 that helps direct homomeric assembly. TRPP2 also has a calcium binding EF‐hand motif involved in regulation of channel activity, and an ER‐retention motif that is important for its trafficking from the ER to the cell membrane. Although the structures of the coiled‐coil domains of both proteins and the EF‐hand motif of TRPP2 have been solved separately, we still don't have an overall picture of how the different domains and motifs interact and assemble with each other. Recently, a cryo‐EM structure of TRPP2 has been reported. However, it lacks the C‐terminus. Clearly, the assembly of these proteins is multi‐faceted and significant for their function, and resolving the structure of that assembly is a necessary step towards understanding the function of the protein in vivo . Our preliminary data show oligomer formation of these proteins. In this structural study, we selected several C‐terminal fragments of all three TRPP proteins based on secondary structure prediction and functional importance, and expressed and purified them using E. coli . Our goal is to crystallize select members of the TRPP family and determine their structures through X‐ray diffraction. This study will elucidate the interaction and assembly of the C‐terminal domains of TRPP proteins and the relevance of key amino acid residues to channel function. Support or Funding Information This work was supported by NIH Grant DK102092.