Large-scale expression and thermodynamic characterization of a glutamate receptor agonist-binding domain

Abstract

The ionotropic glutamate receptors (GluR) are the primary mediators of excitatory synaptic transmission in the brain. GluR agonist binding has been localized to an extracellular domain whose core is homologous to the bacterial periplasmic binding proteins (PBP). We have established routine, baculovirus-mediated expression of a complete ligand-binding domain construct at the 10-L scale, yielding 10±40 milligrams of purified protein. This construct contains peptides that lie outside the PBP-homologous core and that connect the domain core to the transmembrane domains of the channel and to the N-terminal`X'-domain. These linker peptides have been implicated in modulating channel physiology. Such extended constructs have proven difficult to express in bacteria, but the protein described here is stable and monomeric. Isothermal titration calorimetry reveals that glutamate binding to the domain involves a substantial heat capacity change and that at physiological temperatures, the reaction is both entropically and enthalpically favorable.Keywords: glutamate receptor ion channels; calorimetry; baculovirus/insect-cell expression; protein purification; ligand-binding thermodynamics.Glutamate receptor ion channels (GluR) are responsible for most excitatory synaptic communication in the central nervous system. They play an important role in the regulation of synaptic strength and are involved in a number of neuropathological processes, including post-traumatic neuronal damage, epilepsy and neurodegenerative diseases. GluR can be divided into three subclasses based on their pharmacological selectivity for the agonists a-amino-5-methyl-3-hydroxy-4-isoxazole propionate (AMPA), kainate and N-methyl-d-aspartate (NMDA) (reviewed in [1]). Like many ion channels, the GluR exhibit desensitization [2], the kinetics and extent of which depend on the agonist used.The GluR ligand-binding domain (`S1S2') is formed by two extracellular peptide sequences, known as S1 and S2, each approximately 150 amino acids long [3±5]. S1 is located N-terminal to the first transmembrane domain, and S2 is between the second and third transmembrane domains. The core of the ligand-binding domain, consisting of 80±85% of S1S2, exhibits homology to the prokaryotic periplasmic binding proteins (PBP). The PBP bind their ligands between two lobes, trapping them by a`Venus flytrap'-style cleft closure[6] (reviewed in [7]), although more restricted conformational changes are also observed, particularly among oligomeric members of the PBP family [8,9]. Sequences lacking PBP homology are located at both ends of the S1 and S2 peptides and connect the core of the domain to the`X'-domain located at the N-terminus and to the three transmembrane domains. These peptides have been implicated in modulating the electrophysiological characteristics of the channel and include the`flip/flop' alternative splice sequence important for desensitization kinetics [10±12].A fusion protein, in which the complete S1 and S2 sequences of the AMPA receptor GluRD are linked, reproduces the...

Flexibility of a Glutamate-Binding Domain

“…The idea that binding first occurs to lobe 1 (docking) followed by closure of lobes and interaction with lobe 2 (locking) has been proposed previously (Abele et al, 2000), but these simulations present a plausible pathway leading from the open state through two intermediates to a closed and fully bound state. Furthermore, the energetics of the process (stabilization of 9-12 kcal/mol) is similar to that obtained with isothermal titration calorimetry (approximately 8 kcal/mol, Madden et al, 2000).…”

First Images of a Glutamate Receptor Ion Channel: Oligomeric State and Molecular Dimensions of GluRB Homomers

“…We next compared expression in Sf21 cells with that in High Five insect cells, which are suitable for the expression of a variety of membrane proteins (22) and for large-scale production (14). Equal volumes of Sf21 and High Five cells were infected with v506-2 at MOI ) 5 and harvested 90 h later.…”

“…Large-scale cell culture was performed in shaker flasks. Protein expression was performed in Sf21 or Trichoplusia ni High Five insect cells infected with multiplicity of infection (MOI) g 4 in Ex-cell 400 medium (JRH Biosciences), and viral expansion was performed in Sf9 cells in TNM-FH medium supplemented as described (14). Cell viability was assayed 86 h after infection by staining with trypan blue (Gibco).…”

Stereochemistry of Quinoxaline Antagonist Binding to a Glutamate Receptor Investigated by Fourier Transform Infrared Spectroscopy

“…Protein Preparation and Characterization-The GluR4-S1S2 protein was expressed, purified, and characterized as described (9). In brief, S1S2 was expressed as a secreted construct in the baculovirus system.…”