Chemical shift assignments of calmodulin bound to the GluN1 C0 domain (residues 841–865) of the NMDA receptor

Neuroplasticity and synaptic transmission in the brain are regulated by N-methyl-D-aspartate receptors (NMDARs) that consist of hetero-tetrameric combinations of the glycine-binding GluN1 and glutamate-binding GluN2 subunits. Calmodulin (CaM) binds to the cytosolic C0 domain of GluN1 (residues 841–865) that may play a role in the Ca2+-dependent inactivation (CDI) of NMDAR channel activity. Dysregulation of NMDARs are linked to various neurological disorders, including Alzheimer’s disease, depression, stroke, epilepsy, and schizophrenia. Here, we report complete NMR chemical shift assignments of Ca2+-saturated CaM bound to the GluN1 C0 domain of the human NMDAR (BMRB no. 51715).


Biological context
N-methyl-D-aspartate receptors (NMDARs) in the brain are localized at the post-synaptic membrane where they regulate neuronal excitability and confer synaptic plasticity (Traynelis et al. 2010). NMDARs contain two copies each of GluN1 and GluN2 subunits, which activate upon binding to the co-agonist glycine and neurotransmitter agonist glutamate, respectively (Benveniste and Mayer 1991;Clements and Westbrook 1991). Under resting basal conditions, the intracellular Ca 2+ concentration is kept below 100 nM due to the powerful action of Ca 2+ pumps and exchangers (Berridge et al. 2003;Clapham 2007), and Ca 2+ sequestration into stores (Berridge 2002;Clapham 2007). Ligandgated opening of NMDAR channels at the postsynaptic membrane causes intracellular Ca 2+ levels to increase into the micromolar range (Wadel et al. 2007), causing a wide range of Ca 2+ -dependent processes (Luscher and Malenka 2012;Kunz et al. 2013;Puri 2020). Prolonged elevation of intracellular Ca 2+ levels is cytotoxic (Stanika et al. 2012), and NMDAR channels are negatively regulated by a process known as Ca 2+ -dependent inactivation (CDI) (Iacobucci and Popescu 2017, 2019, 2020. The Ca 2+ -dependent inactivation of NMDA receptors requires CaM binding to the cytosolic C0 domain in GluN1 (Zhang and Majerus 1998;Iacobucci and Popescu 2017, 2019, 2020. Ca 2+ -free CaM (apoCaM) is believed to be pre-associated with the C0 domain that may cause channel activation at low Ca 2+ levels (Wang et al. 2008;Iacobucci and Popescu 2017, 2019, 2020. Neurotransmitter binding to NMDAR causes channel opening, which triggers a rise in intracellular Ca 2+ that promotes a conformational change in the NMDAR/CaM complex, leading to CDI (Krupp et al. 1999;Wang et al. 2008;Iacobucci and Popescu 2020).
Atomic-level structures of NMDARs have been solved by x-ray crystallography (Karakas and Furukawa 2014;Lee et al. 2014) and cryo-EM (Jalali-Yazdi et al. 2018;Regan et al. 2018;Chou et al. 2020) that show detailed inter-subunit interactions between the extracellular ligand-binding domain and transmembrane channel domain. However, the C-terminal cytosolic domain (that mediates CDI) is not visible in any of the published structures and its structure has remained elusive. The cytosolic region of GluN1 (residues 830-938) is comprised of helical domains called C0 (residues 841-865) and C1 (residues 875-898) that interact with CaM (Ehlers et al. 1996;Krupp et al. 1999;Ataman et al. 2007). A crystal structure of CaM bound to the C1 domain has been reported (Ataman et al. 2007), but a structure of CaM bound to C0 is currently not known. We report here NMR resonance assignments of Ca 2+ -saturated CaM bound to the C0 domain of GluN1 (hereafter called CaM/GluN1 1 3 C0). These assignments are an important step toward elucidating the complete structure of CaM bound to GluN1, which may be important for understanding the mechanism of CDI.

Expression and purification of CaM
Human CaM was overexpressed in E. coli strain BL21(DE3) using pET11b (Novagen) and the expressed protein was purified as described previously (Bej and Ames 2022a). The CaM protein samples typically have greater than 99% purity as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A peptide fragment of the GluN1 C0 domain of the NMDA receptor (residues 841-865) was purchased from GenScript, dissolved in DMSO-d 6 , and quantified using UV-Vis absorption spectroscopy (ε 280 = 5500 M −1 cm −1 ). A 2.5-fold excess of the peptide was added to Ca 2+ -bound CaM, incubated at room temperature for 30 min, and concentrated to 0.4 mM in a final volume of 0.3 ml.

Extent of assignments and data deposition
Backbone resonance assignments of CaM/GluN1 C0 are illustrated in the 15 N-1 H HSQC spectrum of 15 N-labeled CaM bound to unlabeled C0 peptide (Fig. 1A, B). Side chain aliphatic resonance assignments are shown by labeled peaks in the constant-time 13 C-1 H HSQC spectrum (Fig. 1C).
NMR assignments were derived from the analysis of 3D heteronuclear NMR experiments performed on 13 C/ 15 N-labeled CaM bound to unlabeled C0 peptide. The highly dispersed spectral peaks and uniform peak intensities suggest that CaM/GluN1 C0 complex is stable in solution and properly folded. The downfield amide peaks assigned to G26, G62, G99 and G135 indicate that Ca 2+ is bound to each of the four EF-hands (Fig. 1A). Upfield-shifted side chain methyl peaks Fig. 1 NMR spectra of Ca 2+ -saturated CaM bound to unlabeled GluN1 C0 peptide. A 15 N-1 H HSQC spectrum recorded at 800 MHz 1 H frequency. The black-dashed box highlights spectrally crowded region. B Expanded view of the resonance assignment from the spectrally crowded region highlighted with black-dashed box. C Constanttime 13 C-1 H HSQC spectrum of Ca 2+ -saturated CaM bound to the peptide assigned to I28, L33, V36, L40, M72, I101, V109 and V143 (Fig. 1C) suggest these residues may interact with aromatic side chain atoms located in the hydrophobic core. At least 97% of the non-proline backbone resonances ( 1 HN, 15 N, 13 Cα, 13 Cβ, and 13 CO) and 97% of side-chain resonances were assigned. Only K78 in the second EF-hand of CaM remains unassigned, because its HSQC peak could not be detected. The chemical shift assignments ( 1 H, 15 N, 13 C) for CaM/GluN1 C0 have been deposited in the BioMagResBank (http:// www. bmrb. wisc. edu) under accession number 51715.
The amino acid sequence of GluN1 C0 when aligned with the IQ-motif of L-type Ca 2+ channels (CaV1.1 and CaV1.2) reveal critical conserved hydrophobic residues that likely contact CaM (Fig. 4A). The GluN1 C0 residues L850, A854 and W858 align with conserved hydrophobic residues of the IQ-motif that contact the CaM C-lobe (highlighted red in Fig. 4A) as seen in the known structures of CaM bound to the IQ-motif of CaV1.2 (Fig. 4B) (Fallon et al. 2005) and CaV1.1 (Halling et al. 2009). Therefore, we predict that these residues in GluN1 C0 likely contact the CaM C-lobe. In addition, the GluN1 C0 residues M848, A851 and F852 align with conserved hydrophobic residues (highlighted blue in Fig. 4A) that contact the CaM N-lobe in the crystal structure of CaM bound to the CaV1.2 IQ peptide (Fig. 4B), suggesting that these residues from GluN1 C0 may contact the CaM N-lobe. Future NMR studies are needed to determine the NMR structure of CaM/GluN1 C0 and test whether it is similar to the collapsed structure of CaM bound to CaV1.2 IQ (Fig. 4B). The NMR assignments of CaM/GluN1 C0 presented here are an important first step toward determining the full three-dimensional structure of CaM bound to GluN1 C0.

Fig. 2
Secondary structure and RCI order parameters (S 2 ) of Ca 2+ -saturated CaM bound to GluN1 C0 peptide predicted from the assigned backbone chemical shifts. A Probability of secondary structural elements (cyan for helix and magenta for strand) and B RCI S 2 of Ca 2+ -saturated CaM bound to the GluN1 C0 peptide were predicted using TALOS + server (Shen et al. 2009). The wire diagram depicting the secondary structural elements (cyan cylinder for helix and magenta triangle for strand) was obtained from the CaM structure (PDB ID: 2VAY (Halling et al. 2009)) Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes CSP = √ (ΔH) 2 + (0.14 × ΔC) 2 . ΔH and ΔC are the observed difference in the 1 H and 13 C methyl chemical shifts, respectively between peptide-bound and unbound Ca 2+ -saturated CaM. CSP values are mapped on to the CaM structure (PDB ID: 2VAY (Halling et al. 2009)). Residues with significant CSP are showed in spheres and labeled accordingly. Residues, without CSP values including proline, amino acids without methyl group, or unassigned resonances, are colored as gray Fig. 4 Conserved residues in GluN1 C0 that may contact CaM. A Sequence alignment of GluN1 C0 with the IQ motif of CaV1.1 and CaV1.2. Residues contacting the N-lobe and C-lobe of CaM are highlighted in blue and red, respectively. B Crystal structure of CaM bound to CaV1.2 IQ peptide (PDB: 2F3Y (Fallon et al. 2005)). Key residues involved in interaction with CaM are labeled were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.