¹H, ¹³C, and ¹⁵N Chemical Shift Assignments of Neuronal Calcium Sensor Protein, Hippocalcin

Abstract

Hippocalcin, a member of the neuronal calcium sensor (NCS) subclass of the calmodulin superfamily, serves as an important calcium sensor for the slow afterhyperpolarizing (sAHP) current in the hippocampus, which underlies some forms of learning and memory. Hippocalcin is also a calcium sensor for hippocampal long-term depression (LTD) and genetically linked to neurodegenerative diseases. We report NMR chemical shift assignments of Ca²⁺-free hippocalcin (BMRB no. 18627).

Biological Context

Hippocalcin (Kobayashi et al., 1993) belongs to the NCS branch of the calmodulin superfamily (Ames et al., 1996; Burgoyne, 2007) (Fig. 1) and confers Ca²⁺-induced activation of sAHP channels in hippocampal neurons that are important for learning and memory (Tzingounis et al., 2007; Villalobos and Andrade, 2010). Hippocalcin also functions as a calcium sensor for hippocampal LTD by binding to the beta2-adaptin subunit of the AP2 adaptor complex that couples NMDA receptor-dependent activation to regulated endocytosis of AMPA receptors during LTD (Jo et al., 2010).

Figure 1.

Alignment of the primary sequence of human hippocalcin, bovine recoverin, and S. pombe NCS-1. Secondary structural elements indicated schematically were derived from analysis of NMR data (³J_HNHα, chemical shift index (CSI) (Wishart et al., 1992) and sequential NOE patterns). The four EF-hands (EF1, EF2, EF3 and EF4) are highlighted green, salmon, cyan, and yellow, respectively. Residues that contact the myristoyl group are colored blue (recoverin) and magenta (NCS-1).

Three-dimensional structures are known for many NCS proteins, including recoverin (Ames et al., 1997; Flaherty, 1993), frequenin (Bourne et al., 2001), Frq1 (Strahl et al., 2007), neurocalcin (Vijay-Kumar and Kumar, 1999), NCS-1 (Lim et al., 2011), and guanylate cyclase activating proteins (GCAPs) (Ames et al., 1999; Stephen et al., 2007). The Ca²⁺-bound NCS proteins all share a common fold with four EF-hands arranged in a tandem array and an exposed N-terminus. Binding of Ca²⁺ to recoverin leads to extrusion of its myristoyl group, termed the calcium-myristoyl switch, that enables recoverin to bind to retinal membrane targets only at high Ca²⁺ levels (Dizhoor et al., 1993; Zozulya and Stryer, 1992). A similar Ca²⁺-myristoyl switch has been reported for hippocalcin in the hippocampus (O’Callaghan et al., 2003).

The N-terminal myristoyl group attached to NCS proteins is sequestered inside unique protein environments in the Ca²⁺-free forms of recoverin (Tanaka et al., 1995), NCS-1 (Lim et al., 2011), and GCAP1 (Stephen et al., 2007). The structure of Ca²⁺-free recoverin contains a covalently attached myristoyl group buried inside a protein hydrophobic cavity formed by the first and second EF-hands in the N-terminal domain (residues highlighted blue in Fig. 1). The structure of Ca²⁺-free myristoylated NCS-1 contains a covalently attached myristoyl group buried in a cavity located near the C-terminus (see residues highlighted magenta in Fig. 1) and looks very different from that of recoverin. Finally, the structure of myristoylated GCAP1 (Stephen et al., 2007) contains a sequestered myristoyl group flanked by both N-terminal and C-terminal helices that looks different from the myristoyl environment in either recoverin and NCS-1. Thus, the myristoylated NCS proteins each fold differently around the covalently attached myristoyl group, which helps explain how these highly homologous proteins adopt unique structures to carry out distinct biological functions. Atomic-resolution structures of other myristoylated NCS proteins are needed to better define the range and different types of NCS protein-myristate interactions. We report here NMR resonance assignments of myristoylated hippocalcin in the Ca²⁺-free state as a first step toward elucidating its protein structure and environment around the N-terminal myristoyl group.

Methods and Experiments

Expression and Purification of hippocalcin

Recombinant myristoylated hippocalcin was uniformly ¹⁵N- or ¹⁵N/¹³C-labeled by co-expressing hippocalcin and yeast N-myristoyl CoA-transferase in E. coli strain, BL21(DE3) grown on M9 medium supplemented with ¹⁵N-NH₄Cl and/or ¹³C₆-glucose (Ames et al., 1994). Recombinant protein expression was induced by exogenously adding myristic acid (10 mg/L) and 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) to cells grown overnight at 25 °C. Typically, a 1-L culture yields about 10 mg of myristoylated protein. Detailed procedures for purifying myristoylated NCS proteins are described elsewhere (Zozulya et al., 1995). Triply labeled (perdeuterated) ²H/¹⁵N/¹³C-labeled hippocalcin was expressed in the similar way described above with the exception that ¹H₂O and ¹³C₆-glucose were substituted with ²H₂O and ¹³C₆-²H₇-glucose.

NMR spectroscopy

Samples of recombinant Ca²⁺-free myristoylated hippocalcin (0.5 mM) were prepared in 90%/10% H₂O/D₂O or 100% D₂O with 5 mM Tris-d₁₁ (pH 7.4), 4 mM DTT-d₁₁ and 0.3 mM EDTA-d₁₂. NMR experiments were conducted using Bruker Advance 600 or 800 MHz spectrometer equipped with a triple resonance cryogenic probe. All experiments were performed at 310 K. Backbone and side-chain chemical shift assignments were obtained using ¹⁵N-HSQC, HNCO, HNCACB, CBCACONH, HBHACONH and ¹⁵N-HSQC-TOCSY (mixing time of 60 ms) spectra (Ikura et al., 1990). Methyl group side-chain resonances were assigned using ¹³C-CT-HSQC and ¹³C-HCCH-TOCSY. For aromatic side-chain chemical shift assignments, HBCBCGCDHD, HBCBCGCDCEHE, ¹³C-CT-HSQC-TOCSY spectra along with ¹³C-HSQC-NOESY, recorded with a mixing time of 120 ms, were used. NMR data were processed using NMRPipe software package and analyzed using SPARKY.

Assignments and Data Deposition

The ¹⁵N-¹H HSQC spectrum of Ca²⁺-free myristoylated hippocalcin (Fig. 2A) illustrates representative backbone resonance assignments. A ¹³C-¹H constant-time HSQC spectrum (Fig. 2B) shows resonance assignments for side-chain methyl groups. NMR assignments were based on 3D heteronuclear NMR experiments performed on ²H/¹³C/¹⁵N-labeled hippocalcin (residues 2-193). The protein sample in this study consists of 193 native residues with an N-terminal myristoyl group covalently attached at Gly 2. Most of the non-proline residues exhibited strong backbone amide resonances with uniform intensities, indicative of a well-defined three-dimensional protein structure. More than 95% of the backbone resonances (¹HN, ¹⁵N, ¹³Cα, ¹³Cβ, and ¹³CO) and 82% of side-chain resonances were assigned. The chemical shift assignments (¹H, ¹⁵N, ¹³C) of Ca²⁺-free hippocalcin have been deposited in the BioMagResBank (http://www.bmrb.wisc.edu) under accession number 18627.

Figure 2.

Two-dimensional NMR spectra (¹⁵N-HSQC, A and ¹³C-¹H constant-time HSQC, B) of Ca²⁺-free myristoylated hippocalcin at 800 MHz proton frequency. Chemical shift assignments are shown for backbone amide groups (A) and side-chain methyl groups (B). The inset shows an expanded view of the spectrally crowded region. A complete list of assignments can be found at the BMRB repository (accession no. 18627).

The chemical shift index (CSI) of each amino acid residue reveals a protein secondary structure in hippocalcin (Fig. 1) similar to that observed previously in recoverin (Tanaka et al., 1995) and NCS-1 (Ames et al., 1999). Hippocalcin contains 10 α-helices and two antiparallel β-sheets (α1: 10-17; β1: 40-43; α2: 25-35; α3: 44-56; β2: 78-81; α4: 62-72; α5: 82-91; β3: 114-117; α6: 101-110; α7: 118-131; β4: 162-165; α8: 146-157; α9: 166-175; α10: 178-187). The NMR assignments reported here for Ca²⁺-free hippocalcin are overall different from those reported previously for recoverin (Tanaka et al., 1995), NCS-1 (Lim et al., 2011) and GCAP1 (Ames et al., 1999). The most noteworthy chemical shift differences are observed for residues known to interact with the N-terminal myristoyl group (highlighted blue for recoverin and magenta for NCS-1 in Fig. 1), consistent with the proposal that N-terminal myristoylation creates a unique binding site environment in each highly homologous NCS protein (Ames and Lim, 2012; Lim et al., 2011).