Crosslinking constraints and computational models as complementary tools in modeling the extracellular domain of the glycine receptor.

Zhenyu Liu,Maria Kurnikova,Michael Yonkunas,Agnieszka Szarecka,Michael Cascio,Kirill Speranskiy,Panayiotis V Benos

doi:10.1371/journal.pone.0102571

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.

Highlights

Anionic–selective glycine receptors (GlyRs5) play critical roles in fast neuronal communication and in neural development
This functional coupling of the extracellular domain (ECD) and TMD in GlyR is supported by the presence of point mutations in the interfacial loops linking these two domains in GlyR sequences of some individuals with hyperekplexia, a neurological disease characterized by an excessive startle response wherein channel gating is effectively uncoupled from ligand binding [8]
The relative structural drift measured as the root mean square deviation (RMSD) of Ca atoms from the initial structure was,3.5 A, a relatively small value considering that the starting structure was a homology model

Summary

Introduction

Anionic–selective glycine receptors (GlyRs5) play critical roles in fast neuronal communication and in neural development. The relative structural drift measured as the root mean square deviation (RMSD) of Ca atoms from the initial structure was ,3.5 A , a relatively small value considering that the starting structure was a homology model This stability indicates that AChBP, despite low sequence identity with pLGICs, is a very good template for its ligand binding ECD. Crosslinking/MS studies are potentially capable of providing a network of intra- and intersubunit constraints to critically evaluate and refine structural models of GlyR and other pLGICs

Experimental Procedures

Results

Discussion