The NMDA receptor NR1 C1 region bound to calmodulin: structural insights into functional differences between homologous domains

Abstract

Calmodulin (CaM) regulates tetrameric N-methyl-D-aspartate receptors (NMDARs) by binding tightly to the C0 and C1 regions of its NR1 subunit. A crystal structure (2HQW; 1.96 A) of calcium-saturated CaM bound to NR1C1 (peptide spanning 875-898) showed that NR1 S890, whose phosphorylation regulates membrane localization, was solvent protected, whereas the endoplasmic reticulum retention motif was solvent exposed. NR1 F880 filled the CaM C-domain pocket, whereas T886 was closest to the N-domain pocket. This 1-7 pattern was most similar to that in the CaM-MARCKS complex. Comparison of CaM-ligand wrap-around conformations identified a core tetrad of CaM C-domain residues (FLMM(C)) that contacted all ligands consistently. An identical tetrad of N-domain residues (FLMM(N)) made variable sets of contacts with ligands. This CaM-NR1C1 structure provides a foundation for designing mutants to test the role of CaM in NR1 trafficking as well as insights into how the homologous CaM domains have different roles in molecular recognition.

Figure 1

CaM Binding to NMDAR NR1 (A) Schematic diagram of NR1 indicating relative positions of intracellular regions C0, C1, C2/C2′, and sequences of C0 and C1. The CaMBD sequence of C0 (residues 838–865) shows the single tryptophan (presumed anchor) residue boxed. 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 final concentration of 51.5 μM apo CaM (open; K_d 158 μM) or 0.76 μM (Ca²⁺)₄CaM (filled; K_d 1.99 nM). 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⁺)₄CaM (solid black) binding to NR1C1p using equilibrium constants from B. For comparison, binding of CaM to NR1C0p (gray) simulated with K_d of 87 nM (Ehlers et al., 1996b) for (Ca²⁺)₄CaM (solid) and K_d of 2.25 μM (Akyol et al., 2004) for apo CaM (dashed).

Figure 2

Crystal structure of the CaM-NR1C1p complex. (A) NR1C1p sequence and structure superimposed on its electron density map contoured at 1.0 σ. (B, C) Alternate views of CaM-NR1C1p (2HQW) showing CaM N-domain backbone (blue), C-domain (red), Ca²⁺ ions and binding sites (yellow), and NR1C1p (gray). Figure made with MacPymol™. 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.

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). Figures made with MacPymol™.

Figure 4

Distribution of CaM N- (blue) and C-domain (red) contacts in the CaM-NR1C1p complex. (A) N-domain residues ≤ 4.5 Å of NR1C1p shown as sticks; 17 contacts made with NR1 residues 875–885 (gray), 19 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 N-domain (T886) are boxed; ER retention signal is underlined. (C) C-domain residues ≤ 4.5 Å of NR1C1p shown as sticks; 27 contacts with residues 875–885, 7 with residues 885–896. Ca²⁺ ions and binding sites (yellow in (A) and (C)) are designated I, II, III, and IV. Figures made using MacPymol™.

Figure 5

Comparison of FLMM tetrads in N- and C-domains of CaM. (A) Sequence alignment of 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) CaM N-domain (blue; residues 8–73) and C-domain (red; residues 81–146) aligned according to C_α atoms (green) of their FLMM tetrad residues. Ca²⁺ ions and binding sites are yellow. (C) Comparison of FLMM residue sidechains (sticks) after alignment of Cα atoms (green spheres). (D) Electron density of FLMM_C and F880 shown at a contour level of 1.0 σ. Figures made with MacPymol™.

Figure 6

Statistical Analysis of CaM-Target Interfaces. Histograms showing residues in the (A) C-domain (residues 84 to 146) or (C) N-domain (residues 5 to 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) FLMM_C (red) or (D) FLMM_N (blue) residues; their sidechains 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 sidechains in FLMM_N and FLMM_C in 16 CaM-target structures compared to the corresponding residue in 2HQW (see Table S3). Bins represent increments of 0.2 Å. Comparison of sidechain 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 1NIW (orange) and 1MXE (green). Figures made with MacPymol™.

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 FLMM_C residues; peptide residue that contacted the majority of FLMM_N residues is boxed in blue. Numbers above sequences denote the spacing between these two residues. Ten peptides bind to CaM in an anti-parallel orientation; sequences are shown using the standard convention (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–C) Alignment of 13 CaM-peptide complexes by the Cα atoms of their FLMM residues. These residues in 2HQW are shown as red spheres; sidechains of the primary contact residue of the target is 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. FLMM_C (red) and FLMM_N (blue) residues of CaM, and F880 (black), T886 (black) and F891 (gray) of NR1C1p, in 2HQW 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 CaM N-domain is black and the C-domain is green; transparency of domains was 0.5. Figures made with MacPymol™.

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 reside contacting the highest number of N-domain residues.

Figure 9

Comparison of CaM Sequences from 102 Eukaryotes. (A) Number of species different from human CaM vs. residue position; residues 1 to 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 FLMM_N and FLMM_C is shown in black bars; in gray are substitutions for M51 (L), M71 (L), L105 (V, W), and M144 (V, I, L).