Chemical Shift Assignments of a Calmodulin Intermediate with Two Ca²⁺ Bound in Complex with the IQ-motif of Voltage-gated Ca²⁺ Channels (Ca_V1.2)

Abstract

Calcium-dependent inactivation (CDI) of neuronal voltage-gated Ca²⁺ channels (Ca_V1.2) is important for synaptic plasticity, which is associated with learning and memory. The Ca²⁺-dependent binding of calmodulin (CaM) to Ca_V1.2 is essential for CDI. Here we report NMR assignments for a CaM mutant (D21A/D23A/D25A/E32Q/D57A/D59A/N61A/E68Q, called CaM^EF12) that contains two Ca²⁺ bound at the third and fourth EF-hands (EF3 and EF4) and is bound to the IQ-motif (residues 1644–1665) from Ca_V1.2 (BMRB accession no. 27692).

Biological Context

The neuronal L-type voltage-gated Ca²⁺ channel (Ca_V1.2) controls the excitability of the postsynaptic membrane in hippocampal neurons, which plays an important role in learning and memory (Hell et al, 1993; Moosmang et al, 2005; Vogl et al, 2015). The cytosolic C-terminal region of Ca_V1.2 (residues 1644–1665, called IQ-motif) is important for promoting Ca²⁺-dependent inactivation (CDI) of Ca_V1.2 (Erickson et al, 2001; Zuhlke et al, 1999). Ca²⁺-saturated CaM (with 4 Ca²⁺ bound) binds tightly to the IQ-motif (Findeisen et al, 2013), and the Ca_V1.2 mutation I1654A that disables Ca²⁺/CaM binding also prevents CDI (Ben Johny et al, 2013). The Ca²⁺-free form of calmodulin (apoCaM) has been suggested to bind to Ca_V1.2 under basal conditions, which may explain how Ca_V1.2 is pre-associated with CaM (Adams et al, 2014). Atomic resolution structures are currently known for the IQ-motif bound to both apoCaM (Gabelli et al, 2014) and Ca²⁺-saturated CaM (Van Petegem et al, 2005), but no structural information is known for the IQ-motif bound to the half-saturated intermediate state of CaM (with Ca²⁺ bound at EF3 and EF4 but not bound at EF1 and EF2). This CaM intermediate state is functionally present under basal conditions (Wang et al, 2018) and may play a role in CDI (Simms & Zamponi, 2014). To study the structure of the half-saturated CaM intermediate state, we first constructed a mutant protein (D21A/D23A/D25A/E32Q/D57A/D59A/N61A/E68Q, called CaM^EF12) that disables Ca²⁺ binding to EF1 and EF2 but retains Ca²⁺-binding solely at EF3 and EF4. This CaM intermediate state is also important for regulation of ryanodine receptors (Sondergaard et al, 2015) and nitric oxide synthase (Piazza et al, 2016). We report detailed NMR resonance assignments for the CaM^EF12 mutant with two Ca²⁺ bound (called Ca²⁺₂/CaM^EF12) that is bound to the IQ-motif of Ca_V1.2 (the complex is called Ca²⁺₂/CaM^EF12-IQ) as a first step toward solving the structure of this complex.

Methods and Experiments

Preparation of Ca²⁺₂/CaM^EF12 bound to the Ca_V1.2 IQ-motif.

A cDNA of Homo sapiens calmodulin that contained the mutations (D21A/D23A/D25A/E32Q/D57A/D59A/N61A/E68Q) was subcloned into pET-11b vector (Novagen) that produced recombinant CaM^EF12 without any affinity tag or extra residues. Uniformly ¹⁵N-labeled and ¹³C,¹⁵N-labeled CaM^EF12 were expressed in bacterial cells grown on M9 minimal media supplemented with ¹⁵N-labeled NH₄Cl (0.5 g per liter of cell culture) and ¹³C-labeled glucose (3 g per liter). The recombinant CaM^EF12 protein was purified using Ca²⁺-dependent hydrophobic interaction chromatography (Gopalakrishna & Anderson, 1982). A peptide fragment of the Ca_v1.2 IQ-motif (residues 1642–1665) was purchased from GenScript, dissolved in DMSO-d₆ and quantified using UV-Vis absorption. A 1.5-fold excess of Ca_v1.2 IQ-motif was added to Ca²⁺-bound CaM^EF12 and the complex (Ca²⁺₂/CaM^EF12-IQ) was subsequently concentrated to 500 μM in the presence of 2 mM CaCl₂ using a 3K Amicon concentrator.

NMR spectroscopy.

Samples of Ca²⁺₂/CaM^EF12-IQ complex for NMR analysis were prepared by exchanging the protein complex into a buffer containing 20 mM Tris-d₁₁ (pH 7.5) with 1 mM CaCl₂, and 92% H₂O/8% D₂O. All NMR experiments were performed at 303K on a Bruker Avance 800 MHz spectrometer equipped with a four channel interface and triple resonance cryogenic (TCI) probe. The ¹⁵N-¹H HSQC spectrum (Fig. 1) was recorded with 256 × 2048 complex points for ¹⁵N(F1) and ¹H(F2), respectively. Assignment of backbone resonances was obtained by analyzing the following spectra: HNCA, HNCACB, CBCA(CO)NH, HNCO (Ikura et al, 1990). Side chain resonances were assigned by analyzing HCCH-TOCSY (Ikura et al, 1991). The NMR data were processed using NMRPipe (Delaglio et al, 1995) and analyzed using Sparky NMRFAM (Lee et al, 2015).

Fig. 1:

Two-dimensional ¹⁵N-¹H HSQC NMR spectrum of ¹⁵N-labeled Ca²⁺₂/CaM^EF12 bound to unlabeled Ca_v1.2 IQ peptide at pH 7.5 recorded at 800-MHz ¹H frequency (A). Expanded view of the crowded spectral region in the center of the spectrum (B). Constant-time ¹³C-¹H HSQC spectrum of 13C-labeled Ca²⁺₂/CaM^EF12 bound to unlabeled Ca_v1.2 IQ peptide (C). Representative assignments are indicated; complete assignments are available as BMRB accession no. 27692.

Assignments and Data Deposition

A two-dimensional ¹⁵N-¹H HSQC NMR spectrum of Ca²⁺₂/CaM^EF12 bound to unlabeled Ca_v1.2 IQ peptide illustrates representative NMR assignments (Fig. 1). NMR assignments were based on 3D heteronuclear NMR spectra recorded from ¹³C/¹⁵N-labeled Ca²⁺₂/CaM^EF12 bound to unlabeled IQ peptide (residues 1642–1665). The NMR spectra of Ca²⁺₂/CaM^EF12-IQ exhibited well-dispersed peaks with uniform intensities indicative of a stably folded protein complex. A few amide resonances exhibit noteworthy downfield shifts, including T29, I101, and N138 that are each located in β-strands (see arrows in Fig. 2) and are expected to form antiparallel β-sheets with strong backbone amide hydrogen bonds. Two downfield peaks assigned to G99 (EF3) and G135 (EF4) are likely caused by a strong hydrogen bond between the backbone NH of G99 and G135 with the side chain carboxyl group of D94 and D130, respectively caused by Ca²⁺ binding to EF3 and EF4. The absence of corresponding downfield shifts for G26 (EF1) and G62 (EF2) is consistent with a lack of Ca²⁺ binding at EF1 and EF2. More than 92% of the main chain ¹³C resonances (¹³Cα, ¹³Cβ, and ¹³CO), 90% of backbone amide resonances (¹HN, ¹⁵N), and 74% of methyl side chain resonances were assigned. The unassigned residues (14–15, 30–32, 43, 59–61, 75, 78, 94) had overlapped backbone amide resonances and/or weak NMR intensities that obscured their assignment. In particular, the EF-hand loop residues in EF1 (residues 30–32) and EF2 (residues 58–61) could not be assigned due to weak NMR intensities, perhaps caused by conformational disorder in these Ca²⁺-free binding loops. A complete listing of the chemical shift assignments (¹H, ¹⁵N, ¹³C) of Ca²⁺₂/CaM^EF12 bound to unlabeled Ca_v1.2 IQ peptide have been deposited in the BioMagResBank (http://www.bmrb.wisc.edu) under accession number 27692.

Fig. 2:

Secondary structure of Ca²⁺₂/CaM^EF12 bound to Ca_v1.2 IQ. Normalized order parameter (upper panel) and ANN-secondary structure probability (lower panel) plotted as a function of residue number. Secondary structure elements (rectangle for helix and arrow for β-strand) were calculated on the basis of chemical shift index and sequential NOE patterns.

The secondary structure of Ca²⁺₂/CaM^EF12 in the complex was calculated based on the chemical shift index (Wishart et al, 1992) of each assigned amino acid residue and ANN-secondary structure prediction using TALOS (Shen et al, 2009) (Fig. 2). As expected, Ca²⁺₂/CaM^EF12 contains four EF-hands comprised of eight α-helices. The helices are named α1 (residues 8–20), α2 (residues 30–39), α3 (residues 46–56), α4 (residues 66–76), α5 (residues 83–93), α6 (residues 103–113), α7 (residues 119–129) and α8 (residues 139–146) depicted by blue rectangles in Fig. 2. In addition, a short and conserved β-strand was observed in EF1 (residues 27–29), EF2 (residues 63–65), EF3 (residues 100–102) and EF4 (residues 136–138) shown by orange arrows in Fig. 2. The overall secondary structure of Ca²⁺₂/CaM^EF12 is similar to that observed in the crystal structure of Ca²⁺-saturated CaM bound to IQ peptide (Van Petegem et al, 2005). A plot of the amide chemical shift perturbation caused by the binding of the IQ peptide reveals that CaM^EF12 residues in the N-terminal domain (residues 1–80 in EF1 and EF2) exhibit relatively small chemical shift differences (CSDs) upon binding to the IQ peptide (Fig. 3). In stark contrast, the CaM residues in the C-terminal domain (residues 81–149) exhibit much larger CSDs induced by IQ binding (Fig. 3). The larger CSD values for the C-terminal domain residues suggest that the IQ-motif is bound to the C-terminal domain (EF3 and EF4). The relatively smaller CSD values for the N-terminal domain residues suggest that the IQ peptide may not make contact with the CaM N-terminal domain (EF1 and EF2), in contrast to what is seen in the crystal structure of Ca²⁺-saturated CaM bound to IQ. The NMR assignments of Ca²⁺₂/CaM^EF12 bound to the CaV1.2 IQ-motif presented here are an important first step toward determining the full three-dimensional structure of Ca²⁺₂/CaM^EF12 bound to Ca_V1.2.

Fig. 3:

Amide chemical shift perturbation for Ca²⁺₂/CaM^EF12 in the presence versus absence of bound IQ peptide. The chemical shift difference (CSD) was calculated as (CSD = {(HN_A – HN_B)² + (¹⁵N_A – ¹⁵N_B)²}^1/2 HN_A and HN_B are amide proton chemical shift of Ca²⁺₂/CaM^EF12 in the presence and absence of IQ peptide respectively, and ¹⁵N_A and ¹⁵N_B are amide ¹⁵N chemical shift of Ca²⁺₂/CaM^EF12 in the presence and absence of IQ peptide respectively. Chemical shift assignments for Ca²⁺₂/CaM¹² (without IQ) were derived from wildtype apo-CaM (BMRB 27190) for N-terminal domain residues (1–79) and from wild type Ca²⁺-saturated CaM (BMRB 547) for the C-terminal domain residues (80–149). The CSD values of the mutated residues in EF1 and EF2 (21, 23, 25, 57, 59, and 61) are not meaningful (due to the mutation) and are not shown.