Erkan Karakas

Crystal structure of a heterotetrameric NMDA receptor ion channel

Intact NMDA receptor structure revealed For brains to develop and form memories, a signal must be transmitted from one neuron to the next. Glutamate is an important neurotransmitter that excites the receiving nerve cell by binding to an ion channel called an N-Methyl-d-Aspartate (NMDA) receptor. This activates the NMDA receptors, causing calcium ions to flood in, triggering signal transduction. Either under- or overactivation can result in a variety of neurological disorders and diseases. Karakas and Furukawa describe the crystal structure of an intact NMDA receptor composed of four separate subunits. Science, this issue p. 992 Specific interactions between key neuronal receptor subunits and domains are critical for functional regulation. 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 angstroms. The NMDA receptors are arranged as a dimer of GluN1-GluN2B heterodimers with the twofold 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.

Subunit Arrangement and Phenylethanolamine Binding in GluN1/GluN2B NMDA Receptors

Since it was discovered that the anti-hypertensive agent ifenprodil has neuroprotective activity through its effects on NMDA (N-methyl-D-aspartate) receptors, a determined effort has been made to understand the mechanism of action and to develop improved therapeutic compounds on the basis of this knowledge. Neurotransmission mediated by NMDA receptors is essential for basic brain development and function. These receptors form heteromeric ion channels and become activated after 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 and GluN2B NMDA receptors, have been intensely studied for their potential use in the treatment of various neurological disorders and diseases, including depression, Alzheimer’s disease and Parkinson’s disease. Despite considerable enthusiasm, mechanisms underlying the recognition of phenylethanolamines and ATD-mediated allosteric inhibition remain limited owing to a lack of structural information. Here we report that the GluN1 and GluN2B ATDs form a heterodimer and that phenylethanolamine binds at the interface between GluN1 and GluN2B, rather than within the GluN2B cleft. The crystal structure of the heterodimer formed between the GluN1b ATD from Xenopus laevis and the GluN2B ATD from Rattus norvegicus shows a highly distinct pattern of subunit arrangement that is different from the arrangements observed in homodimeric non-NMDA receptors and reveals the molecular determinants for phenylethanolamine binding. Restriction of domain movement in the bi-lobed structure of the GluN2B ATD, by engineering of an inter-subunit disulphide bond, markedly decreases sensitivity to ifenprodil, indicating that conformational freedom in the GluN2B ATD is essential for ifenprodil-mediated allosteric inhibition of NMDA receptors. These findings pave the way for improving the design of subtype-specific compounds with therapeutic value for neurological disorders and diseases.

Structure of the zinc-bound amino-terminal domain of the NMDA receptor NR2B subunit

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.

Structural basis for DNA recognition and processing by UvrB.

DNA-damage recognition in the nucleotide excision repair (NER) cascade is a complex process, operating on a wide variety of damages. UvrB is the central component in prokaryotic NER, directly involved in DNA-damage recognition and guiding the DNA through repair synthesis. We report the first structure of a UvrB–double-stranded DNA complex, providing insights into the mechanism by which UvrB binds DNA, leading to formation of the preincision complex. One DNA strand, containing a 3′ overhang, threads behind a β-hairpin motif of UvrB, indicating that this motif inserts between the strands of the double helix, thereby locking down either the damaged or undamaged strand. The nucleotide directly behind the β-hairpin is flipped out and inserted into a small, highly conserved pocket in UvrB.

Structural insights into the first incision reaction during nucleotide excision repair.

Nucleotide excision repair is a highly conserved DNA repair mechanism present in all kingdoms of life. The incision reaction is a critical step for damage removal and is accomplished by the UvrC protein in eubacteria. No structural information is so far available for the 3′ incision reaction. Here we report the crystal structure of the N‐terminal catalytic domain of UvrC at 1.5 Å resolution, which catalyzes the 3′ incision reaction and shares homology with the catalytic domain of the GIY‐YIG family of intron‐encoded homing endonucleases. The structure reveals a patch of highly conserved residues surrounding a catalytic magnesium‐water cluster, suggesting that the metal binding site is an essential feature of UvrC and all GIY‐YIG endonuclease domains. Structural and biochemical data strongly suggest that the N‐terminal endonuclease domain of UvrC utilizes a novel one‐metal mechanism to cleave the phosphodiester bond.

Activation of NMDA receptors and the mechanism of inhibition by ifenprodil

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.

Structure of the C-terminal half of UvrC reveals an RNase H endonuclease domain with an Argonaute-like catalytic triad

Removal and repair of DNA damage by the nucleotide excision repair pathway requires two sequential incision reactions, which are achieved by the endonuclease UvrC in eubacteria. Here, we describe the crystal structure of the C‐terminal half of UvrC, which contains the catalytic domain responsible for 5′ incision and a helix–hairpin–helix–domain that is implicated in DNA binding. Surprisingly, the 5′ catalytic domain shares structural homology with RNase H despite the lack of sequence homology and contains an uncommon DDH triad. The structure also reveals two highly conserved patches on the surface of the protein, which are not related to the active site. Mutations of residues in one of these patches led to the inability of the enzyme to bind DNA and severely compromised both incision reactions. Based on our results, we suggest a model of how UvrC forms a productive protein–DNA complex to excise the damage from DNA.

Emerging structural insights into the function of ionotropic glutamate receptors

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate excitatory neurotransmission crucial for brain development and function, including learning and memory formation. Recently a wealth of structural studies on iGluRs including AMPA receptors (AMPARs), kainate receptors, and NMDA receptors (NMDARs) became available. These studies showed structures of non-NMDARs including AMPAR and kainate receptor in various functional states, thereby providing the first visual sense of how non-NMDAR iGluRs may function in the context of homotetramers. Furthermore, they provided the first view of heterotetrameric NMDAR ion channels, and this illuminated the similarities with and differences from non-NMDARs, thus raising a mechanistic distinction between the two groups of iGluRs. We review mechanistic insights into iGluR functions gained through structural studies of multiple groups.

Structural Determinants and Mechanism of Action of a GluN2C-selective NMDA Receptor Positive Allosteric Modulator

NMDA receptors are tetrameric complexes of GluN1, GluN2A-D, and GluN3A-B subunits and are involved in normal brain function and neurologic disorders. We identified a novel class of stereoselective pyrrolidinone (PYD) positive allosteric modulators for GluN2C-containing NMDA receptors, exemplified by methyl 4-(3-acetyl-4-hydroxy-1-[2-(2-methyl-1H-indol-3-yl)ethyl]-5-oxo-2,5-dihydro-1H-pyrrol-2-yl)benzoate. Here we explore the site and mechanism of action of a prototypical analog, PYD-106, which at 30 μM does not alter responses of NMDA receptors containing GluN2A, GluN2B, and GluN2D and has no effect on AMPA [α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid] and kainate receptors. Coapplication of 50 μM PYD-106 with a maximally effective concentration of glutamate and glycine increases the response of GluN1/GluN2C NMDA receptors in HEK-293 cells to 221% of that obtained in the absence of PYD (taken as 100%). Evaluation of the concentration dependence of this enhancement revealed an EC50 value for PYD of 13 μM. PYD-106 increased opening frequency and open time of single channel currents activated by maximally effective concentrations of agonist but only had modest effects on glutamate and glycine EC50. PYD-106 selectively enhanced the responses of diheteromeric GluN1/GluN2C receptors but not triheteromeric GluN1/GluN2A/GluN2C receptors. Inclusion of residues encoded by GluN1-exon 5 attenuated the effects of PYD. Three GluN2C residues (Arg194, Ser470, Lys470), at which mutagenesis virtually eliminated PYD function, line a cavity at the interface of the ligand binding and the amino terminal domains in a homology model of GluN1/GluN2C built from crystallographic data on GluN1/GluN2B. We propose that this domain interface constitutes a new allosteric modulatory site on the NMDA receptor.

Mapping the Binding of GluN2B-Selective N-Methyl-d-aspartate Receptor Negative Allosteric Modulators

We have used recent structural advances in our understanding of the N-methyl-d-aspartate (NMDA) receptor amino terminal domain to explore the binding mode of multiple diaryl GluN2B-selective negative allosteric modulators at the interface between the GluN1 and GluN2B amino-terminal domains. We found that interaction of the A ring within the binding pocket seems largely invariant for a variety of structurally distinct ligands. In addition, a range of structurally diverse linkers between the two aryl rings can be accommodated by the binding site, providing a potential opportunity to tune interactions with the ligand binding pocket via changes in hydrogen bond donors, acceptors, as well as stereochemistry. The most diversity in atomic interactions between protein and ligand occur in the B ring, with functional groups that contain electron donors and acceptors providing additional atomic contacts within the pocket. A cluster of residues distant to the binding site also control ligand potency, the degree of inhibition, and show ligand-induced increases in motion during molecular dynamics simulations. Mutations at some of these residues seem to distinguish between structurally distinct ligands and raise the possibility that GluN2B-selective ligands can be divided into multiple classes. These results should help facilitate the development of well tolerated GluN2B subunit-selective antagonists.