title: P2X5 subunit assembly requires scaffolding by the second transmembrane domain and a conserved aspartate.
abstract: Functional homomeric and heteromeric ATP-gated P2X receptor channels have been shown to display a characteristic trimeric architecture. Of the seven different isoforms (designated P2X(1)-P2X(7)), P2X(5) occurs in humans primarily as a non-functional variant lacking the C-terminal end of the ectodomain and the outer half of the second transmembrane domain. We show that this truncated variant, which results from the splice-skipping of exon 10, is prone to subunit aggregation because the residual transmembrane domain 2 is too short to insert into the membrane. Alleviation of the negative hydrophobic mismatch by the addition of a stretch of moderately hydrophobic residues enabled formation of a second membrane-spanning domain and strictly parallel homotrimerization. Systematic mutagenesis identified only one transmembrane domain 2 residue, Asp(355), which supported homotrimerization in a side chain-specific manner. Our results indicate that transmembrane domain 2 formation contributes 2-fold to hP2X(5) homotrimerization by tethering the end of the ectodomain to the membrane, thereby topologically restricting conformational mobility, and by intramembrane positioning of Asp(355). While transmembrane domain 2 appears to favor assembly by enabling productive subunit interactions in the ectodomain, Asp(355) seems to assist by simultaneously driving intramembrane helix interactions. Overall, these results indicate a complex interplay between topology, helix-helix interactions, and oligomerization to achieve a correctly folded structure.
authors.affiliation: Department of Molecular Pharmacology, Rheinisch-Westfaelische Technische Hochschule (RWTH) Aachen, Wendlingweg 2, D-52074 Aachen, Germany.
authors.forename: Wiebke
authors.initials: W
authors.lastname: Duckwitz
authors.forename: Ralf
authors.initials: R
authors.lastname: Hausmann
authors.forename: Armaz
authors.initials: A
authors.lastname: Aschrafi
authors.forename: Günther
authors.initials: G
authors.lastname: Schmalzing
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry*; D005091:Exons; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009865:Oocytes / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000737:chemistry / Q000502:physiology*; D058485:Receptors, Purinergic P2X5; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis
chemical_list: C546465:P2RX5 protein, human; D018048:Receptors, Purinergic P2; D058485:Receptors, Purinergic P2X5; D001224:Aspartic Acid