title: Discerning the Role of the Hydroxyproline Residue in the Structure of Conantokin Rl-B and Its Role in GluN2B Subunit-Selective Antagonistic Activity toward N-Methyl-d-Aspartate Receptors.
abstract: Conantokins (con) are short γ-carboxyglutamate (Gla)-containing polypeptides expressed by marine snails that function as antagonists of N-methyl-d-aspartate receptor (NMDAR) ion channels. The Gla residues govern structural conformations and antagonistic activities of the conantokins. In addition to Gla, some conantokins, e.g., conRl-B, also contain a hydroxyproline (HyP or O) residue, which in this case is centrally located in the peptide at position 10. Because conRl-B specifically inhibits ion channels of GluN2B subunit-containing heterotetrameric NMDARs, we evaluated the unusual role of HyP10in this effect. To accomplish this goal, we examined synthetic variants of conRl-B in which HyP10was either deleted (conRl-B[ΔO10]) or replaced with alanine (conRl-B[O10A]) or proline (conRl-B[O10P]). The solution structures of these variants were determined by nuclear magnetic resonance spectroscopy. Deletion of HyP10, or replacement of HyP10with Ala10, attenuated the distortion in the central region of the apo-conRl-B helix and allowed Mg2+-complexed end-to-end α-helix formation. The inhibitory properties of these variants were assessed by measuring NMDA/Gly-stimulated intracellular Ca2+influx in mice neurons. ConRl-B[O10P] retained its NMDAR ion channel inhibitory activity in wild-type (WT) neurons but lost its GluN2B specificity, whereas conRl-B[ΔO10] showed overall diminished inhibitory function. ConRl-B[O10A] showed attenuated inhibitory function but retained its GluN2B specificity. Thus, HyP10plays a critical role in maintaining the structural integrity of conRl-B, which can be correlated with its GluN2B subunit-selective inhibition. Weakened inhibition by conRl-B was also observed in neurons lacking either the GluN2C or GluN2D subunit, compared to WT neurons. This suggests that GluN2C and GluN2D are also required for inhibition by conRl-B.
authors.affiliation: W. M. Keck Center for Transgene Research and ‡Department of Chemistry and Biochemistry, University of Notre Dame , Notre Dame, Indiana 46556, United States.
authors.forename: Yue
authors.initials: Y
authors.lastname: Yuan
authors.affiliation: W. M. Keck Center for Transgene Research and ‡Department of Chemistry and Biochemistry, University of Notre Dame , Notre Dame, Indiana 46556, United States.
authors.forename: Rashna D
authors.identifier: http://orcid.org/0000-0002-2291-5020
authors.initials: RD
authors.lastname: Balsara
authors.affiliation: W. M. Keck Center for Transgene Research and ‡Department of Chemistry and Biochemistry, University of Notre Dame , Notre Dame, Indiana 46556, United States.
authors.forename: Jaroslav
authors.initials: J
authors.lastname: Zajicek
authors.affiliation: W. M. Keck Center for Transgene Research and ‡Department of Chemistry and Biochemistry, University of Notre Dame , Notre Dame, Indiana 46556, United States.
authors.forename: Shailaja
authors.initials: S
authors.lastname: Kunda
authors.affiliation: W. M. Keck Center for Transgene Research and ‡Department of Chemistry and Biochemistry, University of Notre Dame , Notre Dame, Indiana 46556, United States.
authors.forename: Francis J
authors.initials: FJ
authors.lastname: Castellino
mesh_terms: D015055:1-Carboxyglutamic Acid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000409:Alanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D052078:Conus Snail / Q000737:chemistry; D006909:Hydroxyproline / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D018345:Mice, Knockout; D008958:Models, Molecular; D009154:Mutation; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D011392:Proline / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D012996:Solutions
chemical_list: C121001:NR2B NMDA receptor; D010455:Peptides; D016194:Receptors, N-Methyl-D-Aspartate; D012996:Solutions; D015055:1-Carboxyglutamic Acid; D011392:Proline; D000409:Alanine; D006909:Hydroxyproline; D002118:Calcium