title: Crosslinking constraints and computational models as complementary tools in modeling the extracellular domain of the glycine receptor.
abstract: The glycine receptor (GlyR), a member of the pentameric ligand-gated ion channel superfamily, is the major inhibitory neurotransmitter-gated receptor in the spinal cord and brainstem. In these receptors, the extracellular domain binds agonists, antagonists and various other modulatory ligands that act allosterically to modulate receptor function. The structures of homologous receptors and binding proteins provide templates for modeling of the ligand-binding domain of GlyR, but limitations in sequence homology and structure resolution impact on modeling studies. The determination of distance constraints via chemical crosslinking studies coupled with mass spectrometry can provide additional structural information to aid in model refinement, however it is critical to be able to distinguish between intra- and inter-subunit constraints. In this report we model the structure of GlyBP, a structural and functional homolog of the extracellular domain of human homomeric α1 GlyR. We then show that intra- and intersubunit Lys-Lys crosslinks in trypsinized samples of purified monomeric and oligomeric protein bands from SDS-polyacrylamide gels may be identified and differentiated by MALDI-TOF MS studies of limited resolution. Thus, broadly available MS platforms are capable of providing distance constraints that may be utilized in characterizing large complexes that may be less amenable to NMR and crystallographic studies. Systematic studies of state-dependent chemical crosslinking and mass spectrometric identification of crosslinked sites has the potential to complement computational modeling efforts by providing constraints that can validate and refine allosteric models.
authors.affiliation: Center for Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States of America; Department of Cell and Molecular Biology, Grand Valley State University, Allendale, Michigan, United States of America.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Szarecka
authors.affiliation: Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Michael
authors.initials: M
authors.lastname: Yonkunas
authors.affiliation: Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Kirill
authors.initials: K
authors.lastname: Speranskiy
authors.affiliation: Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Maria
authors.initials: M
authors.lastname: Kurnikova
authors.affiliation: Center for Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America; Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Michael
authors.initials: M
authors.lastname: Cascio
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry; D004120:Dimethyl Suberimidate / Q000737:chemistry; D006860:Hydrogen Bonding; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D021122:Protein Subunits / Q000737:chemistry; D018009:Receptors, Glycine / Q000737:chemistry*; D061987:Sf9 Cells; D018411:Spodoptera; D040681:Structural Homology, Protein
chemical_list: D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D021122:Protein Subunits; D018009:Receptors, Glycine; D004120:Dimethyl Suberimidate