Volume 15, Issue 12, Pages 1603-1617 (December 2007) The NMDA Receptor NR1 C1 Region Bound to Calmodulin: Structural Insights into Functional Differences between Homologous Domains  Zeynep Akyol Ataman, Lokesh Gakhar, Brenda R. Sorensen, Johannes W. Hell, Madeline A. Shea  Structure  Volume 15, Issue 12, Pages 1603-1617 (December 2007) DOI: 10.1016/j.str.2007.10.012 Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 1 CaM Binding to NMDAR NR1 (A) Schematic diagram of NR1 indicating relative positions of intracellular regions C0, C1, and C2/C2′, and sequences of C0 and C1. The CaMBD sequence of C0 (residues 838–865) shows the single tryptophan (presumed anchor) residue boxed. The sequence of C1 (residues 875–898) is shown with the ER retention signal (RRR) underlined, the PKC sites boxed and shaded, and residues F880 and T886 boxed. (B) Binding of CaM to NR1C1p monitored by fluorescence anisotropy of Fl-NR1C1p (intrinsic value of 0.04) to a final concentration of 51.5 μM apo CaM (open; KD = 158 μM) or 0.76 μM (Ca2+)4-CaM (filled; KD = 1.99 nM). The asterisk indicates that the anisotropy of Fl-NR1C1p titrated with apo CaM was normalized to the value (0.13) observed after saturation with calcium. (C) Simulation of apo CaM (dashed black) and (Ca2+)4-CaM (solid black) binding to NR1C1p with equilibrium constants from (B). For comparison, binding of CaM to NR1C0p (gray) simulated with a KD of 87 nM (Ehlers et al., 1996b) for (Ca2+)4-CaM (solid) and a KD of 2.25 μM (Akyol et al., 2004) for apo CaM (dashed). Structure 2007 15, 1603-1617DOI: (10.1016/j.str.2007.10.012) Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 2 Crystal Structure of the CaM-NR1C1p Complex (A) NR1C1p sequence and structure superimposed on its electron density map contoured at 1.0σ. (B and C) Alternate views of CaM-NR1C1p (2HQW) showing the CaM N-domain backbone (blue), the C domain (red), Ca2+ ions and binding sites (yellow), and NR1C1p (gray). The figure was made with MacPymol. (D and E) Alignment of 17 canonical CaM-target complexes by their Cα atoms of the (D) N-domain (residues 5–72; 68 atoms) and (E) C-domain (residues 84–146; 63 atoms) FLMM residues as described in Experimental Procedures. Structure 2007 15, 1603-1617DOI: (10.1016/j.str.2007.10.012) Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 3 Solvent Accessibility of S890 (A) Surface (CaM) and stick (NR1C1p) diagram of 2HQW colored as a gradient from blue (buried) to red (exposed) according to % SASA values: S890, 41%; R893, 89%; R894, 72%; R895, 92%; and S896, 87%. (B) Ball-and-stick diagram of S890 and CaM N-domain residues (M36, M51, and Q41). The figure was made with MacPymol. Structure 2007 15, 1603-1617DOI: (10.1016/j.str.2007.10.012) Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 4 Distribution of CaM N- and C-Domain Contacts in the CaM-NR1C1p Complex (A) N-domain residues ≤ 4.5 Å of NR1C1p shown as sticks; 17 contacts were made with NR1 residues 875–885 (gray), and 19 contacts were made with residues 885–896 (black). (B) Sequence map of CaM residues ≤ 4.5 Å of NR1C1p. Residues in NR1C1p that make the highest number of contacts exclusively with the C domain (F880) and the N domain (T886) are boxed; the ER retention signal is underlined. (C) C-domain residues ≤ 4.5 Å of NR1C1p shown as sticks; 27 contacts were made with residues 875–885, and 7 contacts were made with residues 885–896. Ca2+ ions and binding sites (yellow in [A] and [C]) are designated I, II, III, and IV. The figure was made with MacPymol. Structure 2007 15, 1603-1617DOI: (10.1016/j.str.2007.10.012) Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 5 Comparison of FLMM Tetrads in N and C Domains of CaM (A) Sequence alignment of the CaM N domain (1–75) and C domain (76–148). Blue boxes highlight F19, L32, M51, and M71; red boxes indicate F92, L105, M124, and M144. Yellow boxes indicate calcium-binding sites. Residues contacting K875 are purple and underlined. (B) The CaM N domain (blue; residues 8–73) and C domain (red; residues 81–146) aligned according to Cα atoms (green) of their FLMM tetrad residues. Ca2+ ions and binding sites are yellow. (C) Comparison of FLMM residue side chains (sticks) after alignment of Cα atoms (green spheres). (D) Electron density of FLMMC and F880 shown at a contour level of 1.0σ. The figure was made with MacPymol. Structure 2007 15, 1603-1617DOI: (10.1016/j.str.2007.10.012) Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 6 Statistical Analysis of CaM-Target Interfaces (A–D) Histograms showing residues in the (A) C domain (residues 84–146) or (C) N domain (residues 5–72) of CaM ≤ 4.5 Å from a bound peptide or drug in more than 11 of 17 compact CaM-target structures. Bars for residues in the FLMM tetrads are black; others are gray. Only residues defined in all 17 structures were analyzed. Alignment of 13 CaM-peptide complexes by the Cα atoms of their (B) FLMMC (red) or (D) FLMMN (blue) residues; their side chains are shown as sticks. The domain surface of 2HQW is colored pink (C domain) or light blue (N domain). (E) A histogram of rmsds for residue side chains in FLMMN and FLMMC in 16 CaM-target structures compared to the corresponding residue in 2HQW (see Table S3). (F–H) Bins represent increments of 0.2 Å. Comparison of side chain orientations of representative FLMM residues with high rmsds from 2HQW (black): (F) F19 in 2BCX (blue), 1SY9 (orange), and 1MXE (green); (G) L32 in 2BCX (blue), 2BE6 (red), 1CKK (orange), and 1CDL (green); and (H) F92 in 1NIW (orange) and 1MXE (green). The figure was made with MacPymol. Structure 2007 15, 1603-1617DOI: (10.1016/j.str.2007.10.012) Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 7 Distribution and Orientation of Target Residues Contacting CaM in Compact CaM-Peptide Complexes (A) Sequences of 13 peptides aligned by the residue (red box) that contacted the majority of FLMMC residues; the peptide residue that contacted the majority of FLMMN residues is boxed in blue. The numbers above the sequences denote the spacing between these 2 residues. Ten peptides bind to CaM in an antiparallel orientation; sequences are shown by using the standard convention (the N-terminal residue is leftmost). Three peptides noted by an asterisk (∗) and listed last bind to CaM in a parallel orientation; their sequences are shown in reverse. (B and C) Alignment of 13 CaM-peptide complexes by the Cα atoms of their FLMM residues. These residues in 2HQW are shown as red spheres; the side chains of the primary contact residue of the target are shown as black sticks. The domain surface of 2HQW is colored pink (C domain) or light blue (N domain). (D) FLMM pocket occupancy in 2HQW. FLMMC (red) and FLMMN (blue) residues of CaM, as well as F880 (black), T886 (black), and F891 (gray) of NR1C1p in 2HQW are shown as sticks. (E) Drug occupancy of the CaM domains. Alignment of the N domain (blue, residues 8–73) and C domain (red, residues 81–146) of CaM in a 1:2 DPD:CaM (1QIV.pdb) by the Cα atoms of their FLMM residues. DPD bound to the CaM N domain is black, and DPD bound to the C domain is green; the transparency of domains was 0.5. The figure was made with MacPymol. Structure 2007 15, 1603-1617DOI: (10.1016/j.str.2007.10.012) Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 8 Interface Analysis of 13 CaM-Peptide Complexes Residues in the N domain (gray) and C domain (black) of CaM within 4.5 Å of a peptide residue determined with CSU. Red indicates the peptide residue contacting the highest number of C-domain residues; blue indicates the peptide residue contacting the highest number of N-domain residues. Structure 2007 15, 1603-1617DOI: (10.1016/j.str.2007.10.012) Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 9 Comparison of CaM Sequences from 102 Eukaryotes (A) The number of species different from human CaM versus residue position; residues 1–75 (blue), 76–79 (gray), 80–148 (red). (B) FLMM tetrad residues compared by BLAST (Altschul et al., 1997) on ExPASy server (Gasteiger et al., 2003) (http://ca.expasy.org) searching the SWISS-PROT knowledgebase (see Table S1 for accession numbers, molecular identifications, and sequence substitutions for each species). Conservation of residues of FLMMN and FLMMC is shown in black bars; in gray are substitutions for M51 (L), M71 (L), L105 (V, W), and M144 (V, I, L). Structure 2007 15, 1603-1617DOI: (10.1016/j.str.2007.10.012) Copyright © 2007 Elsevier Ltd Terms and Conditions