N -methyl-D-aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors, which mediate most excitatory synaptic transmission in mammalian brains. Calcium permeation triggered by activation of NMDA receptors is the pivotal event for initiation of neuronal plasticity. Here we show the crystal structure of the intact heterotetrameric GluN1/GluN2B NMDA receptor ion channel at 4 Å. The NMDA receptors are arranged as a dimer of GluN1-GluN2B heterodimers with the two-fold symmetry axis running through the entire molecule composed of an amino terminal domain (ATD), a ligand-binding domain (LBD), and a transmembrane domain (TMD). The ATD and LBD are much more highly packed in the NMDA receptors than non-NMDA receptors, which may explain why ATD regulates ion channel activity in NMDA receptors but not in non-NMDA receptors.

Summary Since it was unexpectedly discovered that the anti-hypertensive agent, ifenprodil, has neuroprotective activity through effects to N-methyl-D-aspartate (NMDA) receptors1, enormous efforts have been made to understand the mechanism of action and to develop improved therapeutic compounds based on this knowledge2–4. Neurotransmission mediated by NMDA receptors is essential for basic brain development and function5. These receptors form heteromeric ion channels and become activated upon concurrent binding of glycine and glutamate to the GluN1 and GluN2 subunits, respectively. A functional hallmark of NMDA receptors is that their ion channel activity is allosterically regulated by binding of small compounds to the amino terminal domain (ATD) in a subtype specific manner. Ifenprodil and related phenylethanolamine compounds, which specifically inhibit GluN1/GluN2B NMDA receptors6,7, have been intensely studied for their potential use in treatment of various neurological disorders and diseases including depression, Alzheimer’s disease and Parkinson’s disease2,4. Despite great enthusiasm, mechanisms underlying recognition of phenylethanolamines and the ATD-mediated allosteric inhibition remain limited due to lack of structural information. Here we report that the GluN1 and GluN2B ATDs form heterodimer and that phenylethanolamine binds at the GluN1-GluN2B subunit interface rather than within the GluN2B cleft. The crystal structure of the GluN1b/GluN2B ATD heterodimer shows a highly distinct pattern of subunit arrangement that is different from those observed in homodimeric non-NMDA receptors and reveals the molecular determinants for phenylethanolamine binding. Restriction of domain movement in the bi-lobed structures of the GluN2B ATD by engineering an inter-subunit disulfide bond dramatically decreases ifenprodil-sensitivity indicating that conformational freedom in the GluN2B ATD is essential for ifenprodil-mediated allosteric inhibition in NMDA receptors.

SUMMARY The physiology of N-Methyl-D-aspartate (NMDA) receptors in mammals is fundamental to brain development and function. NMDA receptors are ionotropic glutamate receptors that function as heterotetramers composed mainly of GluN1 and GluN2 subunits. Activation of NMDA receptors requires binding of neurotransmitter agonists to a ligand-binding domain (LBD) and structural rearrangement of an amino terminal domain (ATD). Recent crystal structures of GluN1/GluN2B NMDA receptors in the presence of agonists and an allosteric inhibitor, ifenprodil, represent the allosterically inhibited state. However, how the ATD and LBD move to activate the NMDA receptor ion channel remains unclear. Here, we combine x-ray crystallography, single-particle electron cryomicroscopy, and electrophysiology to show that, in the absence of ifenprodil, the bi-lobed structure of GluN2 ATD adopts an open-conformation accompanied by rearrangement of the GluN1-GluN2 ATD heterodimeric interface, altering subunit orientation in the ATD and LBD and forming an active receptor conformation that gates the ion channel.

N-methyl-D-aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors (iGluRs) that mediate the majority of fast excitatory synaptic transmission in the mammalian brain. One of the hallmarks for the function of NMDA receptors is that their ion channel activity is allosterically regulated by binding of modulator compounds to the extracellular amino-terminal domain (ATD) distinct from the L-glutamate-binding domain. The molecular basis for the ATD-mediated allosteric regulation has been enigmatic because of a complete lack of structural information on NMDA receptor ATDs. Here, we report the crystal structures of ATD from the NR2B NMDA receptor subunit in the zinc-free and zinc-bound states. The structures reveal the overall clamshell-like architecture distinct from the non-NMDA receptor ATDs and molecular determinants for the zinc-binding site, ion-binding sites, and the architecture of the putative phenylethanolamine-binding site.

Sulfite oxidase deficiency is a lethal genetic disease that results from defects either in the genes encoding proteins involved in molybdenum cofactor biosynthesis or in the sulfite oxidase gene itself. Several point mutations in the sulfite oxidase gene have been identified from patients suffering from this disease worldwide. Although detailed biochemical analyses have been carried out on these mutations, no structural data could be obtained because of problems in crystallizing recombinant human and rat sulfite oxidases and the failure to clone the chicken sulfite oxidase gene. We synthesized the gene for chicken sulfite oxidase de novo, working backward from the amino acid sequence of the native chicken liver enzyme by PCR amplification of a series of 72 overlapping primers. The recombinant protein displayed the characteristic absorption spectrum of sulfite oxidase and exhibited steady state and rapid kinetic parameters comparable with those of the tissue-derived enzyme. We solved the crystal structures of the wild type and the sulfite oxidase deficiency-causing R138Q (R160Q in humans) variant of recombinant chicken sulfite oxidase in the resting and sulfate-bound forms. Significant alterations in the substrate-binding pocket were detected in the structure of the mutant, and a comparison between the wild type and mutant protein revealed that the active site residue Arg-450 adopts different conformations in the presence and absence of bound sulfate. The size of the binding pocket is thereby considerably reduced, and its position relative to the cofactor is shifted, causing an increase in the distance of the sulfur atom of the bound sulfate to the molybdenum.Sulfite oxidase (SO), 3 an enzyme containing the molybdenum cofactor (Moco), catalyzes the oxidation of sulfite to sulfate, the final step in the degradation of sulfur-containing amino acids. It resides in the intermembrane space of mitochondria, where it exists as a homodimer. The crystal structure of chicken sulfite oxidase (CLSO), purified from chicken liver, showed that each subunit contains three domains. A small heme-containing N-terminal cytochrome b 5 domain (residues 3-84) is connected to the rest of the protein via a flexible 10-residue-long loop region. The central domain (residues 92-323) contains the active site of the enzyme. Finally, the C-terminal dimerization domain (residues 324 -466) displays the same topology as the C2 subtype of the immunoglobulin superfamily (1).Upon oxidation of sulfite, Mo(VI) is reduced to Mo(IV) by two electrons. The electrons are subsequently transferred to the heme Fe(III) in the cytochrome b 5 domain in a two-step reaction, which is followed by transfer of the electrons from Fe(II) to cytochrome c (2). The distance between the two metals, molybdenum and iron, in the crystal structure of chicken liver sulfite oxidase (CLSO) is 32 Å, which is much longer than expected for the electron transfer rate observed (1). Two mechanisms were suggested to explain these results; a very efficient electron transfer through main ...