Functional evidence for a twisted conformation of the NMDA receptor GluN2A subunit N-terminal domain

Abstract

Ionotropic glutamate receptors (iGluRs) possess in their extracellular region a large N-terminal domain (NTD) that precedes the agonist-binding domain and displays a clamshell-like architecture similar to the bacterial leucine/isoleucine/valine-binding protein (LIVBP). In addition to their role in receptor assembly, in NMDA receptors (NMDARs), the NTDs of GluN2A and GluN2B subunits form a major site for subunit-specific regulation of ion channel activity, in particular through binding of allosteric modulators such as the synaptically-enriched zinc ion. A recent crystallographic study of the isolated GluN2B NTD has revealed an unexpected twisted closed-cleft conformation caused by a rotation of ∼50° in the interlobe orientation compared with all other known LIVBP-like structures (Karakas et al., 2009). By measuring currents carried by recombinant NMDARs, we now provide functional evidence, through disulfide cross-linking and the identification of a new zinc-binding residue (D283), that the GluN2A NTD of intact GluN1/GluN2A receptors adopts a similar twisted conformation in its closed-cleft state. We propose that the twisted NTD conformation is a distinct structural feature of NMDARs (at least for GluN2A and GluN2B subunits), arguing for interactions between the NTDs in the tetrameric complex that are likely to differ between NMDA and AMPA/kainate receptors.

Introduction

The vast majority of excitation in the mammalian brain and spinal cord is mediated by the amino-acid glutamate. Following neuronal activity, glutamate is released in the extracellular medium and acts on two large families of plasma membrane receptors: metabotropic glutamate receptors (mGluRs), which are G-protein coupled receptors, and iGluRs which form glutamate-gated ion channels selective for cations. The iGluR family is further divided into three subfamilies named for their selective agonists, AMPA, kainate and NMDA, with diverse functions and various genes encoding multiple receptor subunits for each subfamily (Dingledine et al., 1999).

Considerable progress has been made during the last decade regarding the molecular architecture of glutamate receptors. The combination of functional, biochemical and structural studies has established that both mGluRs and iGluRs possess in their extracellular region globular clamshell-like domains that have homology to soluble bacterial periplasmic binding proteins (PBPs) (O’Hara et al., 1993, Mayer, 2006). The extracellular region of iGluR subunits are actually made of two adjacent PBP-like domains: the N-terminal domain (NTD), which encompasses the first ∼380 amino acids and is related to LIVBP, and the agonist-binding domain (ABD) that is directly connected to the transmembrane region and that is related to the glutamine binding-protein (QBP). In the mGluR family, each subunit comprises a single extracellular clamshell-like domain that forms the glutamate-binding domain and that, similarly to the iGluR NTDs, is related to LIVBP. Crystal structures of AMPA, kainate and NMDA ABDs, (Armstrong and Gouaux, 2000, Furukawa and Gouaux, 2003, Furukawa et al., 2005, Mayer, 2005, Yao et al., 2008), and of dimeric glutamate-binding domains of mGluRs (Kunishima et al., 2000), both with and without ligands, have revealed that all these domains share with PBPs a common overall structure and mode of operation. They fold as two lobes tethered together by a hinge defining a large central interlobe cleft. The activating ligand (i.e. agonist) binds in the central cleft, promotes domain closure and stabilization of a closed-cleft conformation through a hinge-bending motion (Quiocho and Ledvina, 1996, Jingami et al., 2003, Mayer, 2006).

Structural and functional insights on iGluR NTDs were obtained more recently. These domains play a major role in subunit assembly, insuring that only subunits within a subfamily assemble with one another (Leuschner and Hoch, 1999, Meddows et al., 2001, Ayalon and Stern-Bach, 2001, Ayalon et al., 2005). As expected, the structures of AMPA and kainate NTDs, either in isolation (Kumar et al., 2009, Jin et al., 2009, Clayton et al., 2009) or in an intact tetrameric GluA2 AMPA receptor (Sobolevsky et al., 2009), show the typical LIVBP-like fold, and in agreement with their role as assembly signals, NTDs form tightly associated dimers. Intriguingly, there are no known ligands that bind the NTDs of AMPA and kainate receptors and structural constraints within the NTD dimer render conformational motions of the hinge-bending type unlikely (Kumar et al., 2009, Jin et al., 2009). The situation strikingly differs for NMDARs. Indeed, in this receptor family, the NTDs are capable of functional regulation of channel activity. By spontaneously oscillating between an open-cleft and a closed-cleft conformation, the GluN2 NTDs tune channel open probability in a subunit-specific fashion (Gielen et al., 2009; see also Yuan et al., 2009). GluN2 NTDs are also a major determinant of NMDAR pharmacology providing binding sites for small ligands that allosterically modulate ion channel activity. These ligands include ifenprodil and derivatives, a large family of synthetic compounds that selectively antagonize NMDARs containing the GluN2B subunit (Masuko et al., 1999, Perin-Dureau et al., 2002, Marinelli et al., 2007, Mony et al., 2009a, Mony et al., 2009b). The zinc ion, accumulated at many excitatory synapses in the brain (Paoletti et al., 2009), is another ligand that produce potent inhibition of NMDARs by targeting the NTDs. Zinc binds the NTD of both GluN2A and GluN2B subunits, and it is believed that zinc binding in the central cleft triggers domain closure, producing ligand-induced conformational changes similar to those observed in other LIVBP-like proteins (Paoletti et al., 2000, Rachline et al., 2005, Gielen et al., 2008).

The first NTD structure of an NMDAR, that of the GluN2B subunit, has recently been solved, providing a detailed molecular map of this important regulatory domain (Karakas et al., 2009). Unexpectedly, this domain, captured in a closed-cleft conformation, shows a distinct clamshell-like architecture, with a lobe 1/lobe 2 orientation twisted by ∼50° compared to all other LIVBP-like structures released to date. Interestingly, this twisted conformation maps previously unpredicted residues in the zinc and ifenprodil binding sites (Karakas et al., 2009). We wondered if this novel NTD twisted conformation is a peculiar property of the GluN2B NTD or if it is transferable to other NMDAR subunits, in particular to the GluN2A NTD, a domain also capable of experiencing cleft closures and openings. By engineering disulfide bridges and identifying a new residue critical for high-affinity GluN2A-specific NMDAR zinc inhibition, we provide functional evidence that the twisted closed-cleft conformation is not unique to GluN2B NTD but also applies to the GluN2A NTD.