Insights into voltage-gated calcium channel regulation from the structure of the Ca_V1.2 IQ domain–Ca²⁺/calmodulin complex

Abstract

Changes in activity-dependent calcium flux through voltage-gated calcium channels (Ca_Vs) drive two self-regulatory calcium-dependent feedback processes that require interaction between Ca²⁺/calmodulin (Ca²⁺/CaM) and a Ca_V channel consensus isoleucine-glutamine (IQ) motif: calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF). Here, we report the high-resolution structure of the Ca²⁺/CaM–Ca_V1.2 IQ domain complex. The IQ domain engages hydrophobic pockets in the N-terminal and C-terminal Ca²⁺/CaM lobes through sets of conserved ‘aromatic anchors.’ Ca²⁺/N lobe adopts two conformations that suggest inherent conformational plasticity at the Ca²⁺/N lobe–IQ domain interface. Titration calorimetry experiments reveal competition between the lobes for IQ domain sites. Electrophysiological examination of Ca²⁺/N lobe aromatic anchors uncovers their role in Ca_V1.2 CDF. Together, our data suggest that Ca_V subtype differences in CDI and CDF are tuned by changes in IQ domain anchoring positions and establish a framework for understanding CaM lobe–specific regulation of Ca_Vs.

Voltage-gated calcium channels are the ion channels that define excitable cells¹. These channels control cellular calcium entry in response to changes in membrane potential and are pivotal in the generation of cardiac action potentials, excitation-contraction coupling, hormone and neurotransmitter release and activity-dependent transcription initiation^1,2. Ca_Vs are multisubunit complexes composed of three essential channel subunits², Ca_Vα₁, Ca_Vβ and Ca_Vα₂δ, plus the ubiquitous intracellular calcium sensor calmodulin (CaM)³. An additional subunit, Ca_Vγ, is associated with skeletal muscle channels, but its general importance in other tissues is unsettled⁴.

The Ca_Vα₁ subunits are single polypeptide chains of ∼1,800–2,200 residues in which the ion-conducting pore is formed from four homologous repeats that each bear six transmembrane segments². There are three Ca_V subfamilies, which have diverse physiological and pharmacological properties that depend largely on the Ca_Vα₁-subunit: Ca_V 1.x (L-type), Ca_V2.x (2.1, P/Q-type; 2.2, N-type; 2.3, R-type) and Ca_V3.x (T-type)¹. Large interdomain intracellular loops bridge the four transmembrane repeats of the Ca_Vα₁ subunit and serve as docking sites for auxiliary subunits and regulatory molecules that control channel activity and connect Ca_V channels to larger macromolecular complexes and cellular signaling pathways^5,6.

Calcium influx is a potent activator of intracellular signaling pathways but is toxic in excess^1,7. Because Ca_Vs are major sources of calcium influx, Ca_V activity is strongly controlled by both self-regulatory and extrinsic mechanisms that tune channel action in response to electrical excitation, neurotransmitter stimulation and hormonal cues^1,2,8. This intense regulation uses multiple mechanisms and classes of intracellular signaling molecules. The bi-lobed calcium sensor CaM has a preeminent role in the intrinsic mechanisms of Ca_V calcium-dependent regulation^9–15. Activity-dependent changes in intracellular calcium levels from flux through Ca_V channel pores drive two self-regulatory calcium-dependent feedback mechanisms that limit or enhance calcium entry through the channel: CDI and CDF⁸. Both require the interaction of Ca²⁺/CaM with a consensus IQ motif in the Ca_Vα₁ C-terminal cytoplasmic tail^9,11–16.

Multiple lines of evidence suggest that CaM is constitutively tethered to the channel to detect calcium in the vicinity of the channel pore^12,17,18 and to permit calcium ions to exert control over their own entry through Ca_Vs. The nature of the tethering site remains unclear. It may comprise a region immediately adjacent to the IQ domain on the N-terminal side, termed the Pre-IQ region^17,19,20, or a composite of the Pre-IQ and IQ domain^18,20.

The IQ domain is believed to be the site of action of Ca²⁺/CaM. Numerous studies show that Ca²⁺/CaM binds IQ peptides from different Ca_V channels in vitro^{9,11–14,17,20–22} and that IQ domain mutations in full-length channels can eliminate CDI^9,13–15,23 and CDF^9,14.

Although CDI and CDF are well documented, the details are complex, differ among channel subtypes and have not been readily predictable given the primary-sequence similarities among the Ca_V CaM-binding domains. Elegant experiments have demonstrated that CDI and CDF are controlled independently by the abilities of the N-terminal and C-terminal CaM lobes (the N lobe and C lobe) to bind calcium^{9,10,12,16,23}. CDI is present in both L-type (Ca_V1.2) and non–L-type (Ca_V2.1, Ca_V2.2 and Ca_V2.3) channels but arises from the action of opposite CaM lobes in these two cases^9,23. In the L-type channel Ca_V1.2, CDI is governed by the binding of calcium ions to the C lobe¹², whereas in non–L-type channels (Ca_V2s), CDI is triggered by the binding of calcium ions to the N lobe^9,11,23. In the non–L-type channel Ca_V2.1, CDF arises from interactions between calcium ions and the C lobe. Despite the fact that the C lobe–mediated processes elicit different channel behaviors in different channel subtypes, CDI in L-type and CDF in non–L-type channels, both C lobe processes share insensitivity to the fast calcium chelator BAPTA²³. This shared resistance suggests that in both cases the C lobe captures calcium that is local to the channel pore²³. In contrast, Ca_V2 N lobe–mediated CDF is sensitive to the slow calcium chelator EGTA, suggesting that it detects global changes in calcium concentration^9,23. These lobe-specific calcium sensitivities have been suggested to provide mechanisms for Ca_Vs to sense, decode and distinguish local calcium changes due to calcium permeation through the channel from global calcium changes due to aggregate cellular signals^9,23.

To delineate the ways in which CDI and CDF arise, we set out to determine the molecular basis for Ca²⁺/CaM interactions. Here, we report the high-resolution crystal structure of the human Ca²⁺/CaM–Ca_V1.2 IQ domain complex. By using the structural data to inform functional experiments, we uncover an unexpected competition between the Ca²⁺/CaM lobes for a common site on the IQ domain and demonstrate a previously unknown role for the Ca²⁺/N lobe anchors in Ca_V1.2 CDF. This structure provides the first insight into the molecular machinery that underlies Ca²⁺/CaM regulation of Ca_Vs.

Results

Structure of the Ca²⁺/CaM–Ca_V1.2 IQ domain complex

We solved the crystal structure of the Ca²⁺/CaM–Ca_V1.2 IQ domain complex at 2.00-Å resolution using a three-wavelength MAD experiment on a selenomethionine (SeMet)-substituted protein crystal. The asymmetric unit contains three independent 1:1 Ca²⁺/CaM–IQ domain complexes (complexes A, B and C) that have two principal conformations, represented by complexes A and C (Fig. 1a,b). Complex B is similar to complex A but has poorly defined density for the C-terminal half of the IQ domain (Supplementary Fig. 1 online). Thus, our analysis focuses on the well-defined structures of complexes A and C. Both are compact structures in which Ca²⁺/CaM embraces the largely α-helical IQ domain in a parallel orientation (Fig. 1a,b) through extensive interactions that involve twenty-two (complex A) or twenty-one (complex C) contiguous residues in the IQ domain, and both bury ∼3,100 Ų total surface area of which roughly 1,650 Ų is hydrophobic (Fig. 1c). The observed parallel orientation in which Ca²⁺/N lobe binds the N-terminal portion of the target helix and Ca²⁺/C lobe binds the C-terminal portion is unusual and known in only one other Ca²⁺/CaM peptide structure, the Ca²⁺/CaM-dependent kinase peptide complex^24,25. A high density of positively charged side chains project from the Ca_V1.2 IQ helix near the C terminus and make electrostatic interactions with negatively charged residues that ring the Ca²⁺/CaM exit tunnel (Fig. 2). These interactions are consistent with the proposal that the distribution of positive charges on Ca²⁺/CaM-binding peptides is an important determinant of binding orientation²⁴.

Figure 1.

Structure of the Ca²⁺/CaM–Ca_V1.2 IQ domain complex. (a) Ribbon diagram of the complex. Green, CaM Ca²⁺/N lobe; blue, Ca²⁺/C lobe; red, IQ domain, with residues Ile1624 and Q1625 from complex A in stick representation; darker shades, complex A; lighter shades, complex C. The complexes were superposed using the Ca²⁺/C lobes and IQ domains. Labels indicate termini of components in complex A. Dashed lines indicate regions absent from the structures. (b) 90° rotation of a. The shift in Ca²⁺/N lobe Ca²⁺ position in EF-hand 2 is indicated. (c) Sequence alignment of IQ regions from each Ca_V1 and Ca_V2 isoform. Zigzag, α-helical regions; straight line, nonhelical residues; dashed lines, residues present in the crystallized construct but absent from the electron density. Residues contacting CaM (≤4 Å) for conformation A and conformation C are highlighted as follows: green, contacts to Ca²⁺/N lobe; cyan, Ca²⁺/C lobe; purple, Ca²⁺/N lobe and Ca²⁺/C lobe. Asterisks indicate principal aromatic anchor positions. Sequences are human Ca_V1.2 1609–1647, human Ca_V1.1 1514–1552, rat Ca_V1.3 1641–1679, human Ca_V1.4 1563–1602, human Ca_V2.1 1947–1985, human Ca_V2.2 1845–1883 and rat Ca_V2.3 1811–1850.

Figure 2.

Lobe-specific Ca²⁺/CaM–Ca_V1.2 IQ domain interactions. (a) Ca²⁺/CaM C lobe from complex A bound to the IQ domain. Buried surface area = 1,819 Ų (965 Ų hydrophobic). (b) Ca²⁺/CaM N lobe from complex A bound to the IQ domain. Buried surface area = 1,450 Ų (743 Ų hydrophobic). (c) Ca²⁺/CaM N lobe from complex C bound to the IQ domain. Buried surface area = 1,491 Ų (500 Ų hydrophobic). IQ domain is shown in stick representation with aromatic anchor residues in white. CaM lobes are shown in surface representation with residues that contribute hydrophobic (yellow), negatively charged (red), positively charged (blue) and polar (green) side chain contacts (≤4 Å) to the IQ domain indicated. Select residues are labeled to orient the reader. IQ domain residue labels are boxed.

The Ca²⁺/C lobes of complexes A and C are nearly identical (r.m.s. deviation for Cα atoms is 0.475) and are anchored similarly to the IQ domain helix (Figs. 1 and 2). The Ca²⁺/N lobes are also similar (r.m.s. deviation for Cα atoms is 0.97) and have only small changes in the positions of side chains involved in direct contacts with the IQ peptide (Supplementary Fig. 2 online). Superposition of Ca²⁺/C lobes and IQ helices shows that the Ca²⁺/N lobe adopts two distinct binding modes (Fig. 1a,b). In complex A, the Ca²⁺/N lobe is positioned further along the N-terminal end of the IQ domain, whereas in complex C, the Ca²⁺/N lobe is tilted and shifted toward the C-terminal end of the IQ domain, leading to a more compact conformation. The conformational differences cause relative displacements in the positions of the two Ca²⁺ ions in the Ca²⁺/N lobe EF-hands of ∼8.6 Å and ∼9.5 Å and increase the bend present in both IQ domain helices at Ile1624 by ∼10° in complex C.

Aromatic anchors mediate IQ domain–Ca²⁺/CaM contacts

The Ca_V1.2 IQ helix engages Ca²⁺/CaM through a set of ‘aromatic anchor’ residues. The C-terminal portion of the IQ helix displays three aromatic anchors (Tyr1627, Phe1628 and Phe1631) that bind hydrophobic Ca²⁺/C lobe pockets. Two anchors (Tyr1627 and Phe1628) are deeply buried (Fig. 2a). The N-terminal part of the IQ helix presents three aromatic residues (Phe1618, Tyr1619 and Phe1622) that make hydrophobic interactions with the Ca²⁺/N lobe; these residues reside on the opposite helical face from the C-terminal anchors. Phe1618 makes the most extensive contacts and binds a deep hydrophobic pocket (Fig. 2b,c). Despite the alternative positions of the Ca²⁺/N lobe, Phe1618 remains buried in the same Ca²⁺/N lobe hydrophobic pocket in both complexes by adopting different side chain rotamers (Fig. 2b,c and Supplementary Fig. 2).

Ca²⁺/CaM interactions with IQ domain consensus residues

IQ domains are defined by the consensus sequence (I/L/V) QXXXRXXXX(R/K) (where X is any residue)^26,27. The Ca²⁺/CaM–Ca_V1.2 structure reveals the ways in which the hallmark residues of the IQ domain interact with the Ca²⁺/CaM lobes. The Ile1624 side chain is completely buried (solvent-exposed area = 0.7 Ų) and contacts the hydrophobic surface of the C lobe (Fig. 2a and Supplementary Figs. 2 and 3 online). The Gln1625 side chain has many contacts to the Ca²⁺/N lobe. This residue also makes side chain and main chain water-mediated hydrogen bonds to the Ca²⁺/C lobe (Supplementary Figs. 2 and 3). The central arginine, Arg1629, and terminal basic residue, Arg1634, form salt bridges with Glu11 and Glu14 of the Ca²⁺/N lobe (Supplementary Figs. 2 and 3) and Glu127 of the Ca²⁺/C lobe (Supplementary Fig. 3), respectively. Thus, all IQ hallmark residues seem important for IQ domain–Ca²⁺/CaM interactions. These residues, together with the aromatic anchors and other positions throughout the IQ domain, establish the extensive network of contacts in the complex (Supplementary Fig. 3).

Isothermal titration calorimetry

We used isothermal titration calorimetry (ITC) to gain insight into the thermodynamics of lobe-specific Ca²⁺/CaM–IQ domain interactions (Fig. 3). Ca²⁺/C lobe binds the IQ domain with 1:1 stoichiometry and a high affinity (K_d = 2.63 × 10⁻⁹ M) that is driven by favorable enthalpic and entropic contributions (Fig. 3a and Table 1). Similar favorable components drive Ca²⁺/N lobe binding; however, the isotherms revealed that there are two different binding sites, a medium-affinity (K_d = 5.76 × 10⁻⁸ M) and low-affinity (K_d = 1.92 × 10⁻⁵ M) site (Fig. 3b and Table 1). Both Ca²⁺/N lobe–IQ domain interactions are substantially weaker than the Ca²⁺/C lobe–IQ domain interaction. Titration of Ca²⁺/N lobe into a solution containing Ca²⁺/C lobe–IQ domain complexes yielded no further binding energy (Fig. 3c). These data directly demonstrate that the high-affinity Ca²⁺/C lobe–IQ domain interaction occludes both measurable Ca²⁺/N lobe–binding sites.

Figure 3.

ITC characterization of Ca²⁺/CaM–Ca_V1.2 IQ domain interactions. (a) 70 μM IQ domain into 7 μM Ca²⁺/C lobe. (b) 500 μM Ca²⁺/N lobe into 50 μM IQ domain. (c) 200 μM Ca²⁺/N lobe into a solution of 20 μM IQ domain and 37 μM C lobe. (d) 60 μM IQ domain F1628A mutant into 6 μM Ca²⁺/C lobe. (e) 500 μM Ca²⁺/N lobe into a solution of 5 μM IQ domain F1628A mutant. Isotherms are fit to a single binding site model for a and d and a double binding site model for b and e. Panels show addition of 10 μl aliquots of titrant into the target solution (top) and binding isotherms (bottom).

Table 1. Thermodynamic parameters for Ca_V1.2 IQ domain–Ca²⁺/CaM lobe interactions.

	WT–Ca2+/C lobe	WT–Ca2+/N lobe	F1628A–Ca2+/C lobe	F1628A–Ca2+/N lobe
N1	0.98 ± 0.18	0.85 ± 0.05	0.92 ± 0.04	0.96 ± 0.13
Kd1 (nM)	2.63 ± 0.07	57.6 ± 35.5	2.59 ± 0.24	1,003 ± 567
ΔH1 (kcal mol−1)	−6.77 ± 0.21	−1.91 ± 0.14	−3.69 ± 0.17	0.45 ± 0.02
ΔS1 (cal mol−1 K−1)	15.75 ± 0.78	26.70 ± 0.85	26.45 ± 0.78	29.20 ± 1.13
ΔG1 (kcal mol−1)	−11.31 ± 0.01	−9.60 ± 0.38	−11.31 ± 0.06	−7.96 ± 0.35
N2		1.04 ± 0.0		1.0 ± 0.0
Kd2 (nM)		19,190 ± 3,600		9,812 ± 8,220
ΔH2 (kcal mol−1)		−1.42 ± 0.13		−0.67 ± 0.22
ΔS2 (cal mol−1 K−1)		16.60 ± 0.84		21.00 ± 2.69
ΔG2 (kcal mol−1)		−6.20 ± 0.11		−6.71 ± 0.55

Thermodynamic parameters of Ca²⁺/C lobe and Ca²⁺/N lobe binding to wild-type (WT) and F1628A Ca_V1.2 IQ domain at pH 7.4 in the presence of 1 mM CaCl₂. Each value corresponds to the mean of two separate experiments with different batches of both components (± s.d.).

To investigate further whether the Ca²⁺/N lobe– and Ca²⁺/C lobe–binding sites depend on interactions observed in the crystal structure, we examined the consequence of a mutation in the IQ domain's aromatic anchor for Ca²⁺/C lobe, F1628A. The F1628A mutant binds Ca²⁺/C lobe with an affinity that is identical to the wild-type domain (K_d = 2.59 × 10⁻⁹ M), but with a reduced enthalpy (Fig. 3d and Table 1). This type of enthalpy-entropy compensation is a common feature in protein-protein interactions²⁸ and reflects a loss of key interactions in the bound state that is compensated by increased disorder. In contrast, the F1628A mutation causes a substantial perturbation of Ca²⁺/N lobe medium-affinity binding (K_d = 1.003 × 10⁻⁶ M for F1628A compared to K_d = 5.76 × 10⁻⁸ M for the wild-type domain) and causes an unfavorable binding enthalpy. These data directly demonstrate that the medium-affinity Ca²⁺/N lobe–binding site requires interactions with residues that comprise the crystallographically observed Ca²⁺/C lobe site.

N lobe anchors have a role in CDF

Although Ca²⁺/C lobe has an established role in Ca_V1.2 CDI^12,16,17, no role has yet been defined for Ca²⁺/N lobe. Therefore, we tested whether the crystallographically observed Ca²⁺/N lobe interface has a role in channel function by using two-electrode voltage clamp to interrogate mutant channels that were heterologously expressed in Xenopus laevis oocytes. A triple mutant lacking the three aromatic anchors for Ca²⁺/N lobe (F1618A Y1619A F1622A), ‘TripleA,’ showed no appreciable difference in Ca_V1.2 inactivation when either Ca²⁺ or Ba²⁺ was the charge carrier (Fig. 4a,b). Thus, Ca²⁺/N lobe–aromatic anchor interactions do not seem important for Ca_V1.2 CDI. This result agrees with the observation that overexpression of the CaM mutant CaM₁₂, in which the N lobe EF-hands have diminished calcium binding, has little effect on CDI^12,16. TripleA channels consistently gave lower current amplitudes compared to wild-type channels. Previous reports have suggested a role for the IQ domain in Ca_V trafficking²⁹; our observation raises the possibility that aromatic anchors for Ca²⁺/N lobe may be important in this process.

Figure 4.

Lobe-specific interactions affect CDI and CDF. (a) Voltage-activated Ba²⁺ and Ca²⁺ currents from wild-type (WT) and TripleA channels during a 600-ms depolarizing step from −90 mV to +20 mV. Traces are normalized to the peak current to facilitate comparison. Tail currents are not shown. (b) r₃₀₀ values (current fraction 300 ms after depolarization) in Ba²⁺ (black) and Ca²⁺ (grey). Error bars show s.d. (c) I1624A and I1624A TripleA Ca²⁺ currents in a 3-Hz 40-pulse train (50-ms steps to +20-mV from −90-mV holding potential). The first pulse currents are scaled to the same level for comparison. (d) Relative current increase between last and first pulses for I1624A and I1624A TripleA. Error bars show s.d.

Because the aromatic anchors for Ca²⁺/N lobe are not crucial for Ca_V1.2 CDI, we asked whether they might have a role in CDF. CDF is a prominent property of Ca_V2.1 channels^9,10,30. Although CDF is not readily detectable in wild-type Ca_V1.2 channels, robust CDF is unmasked by the I1624A mutation^14,15. The I1624A mutation in the TripleA background (I1624A TripleA) results in channels lacking both CDI (Fig. 4b) and CDF (Fig. 4c,d). Lowering expression of a I1624A-only mutant such that current magnitudes were equivalent to those of the I1624A TripleA mutant still produced clear CDF (data not shown). This result excludes the possibility that CDF loss in I1624A TripleA is a consequence of the lower current magnitudes. Together, these experiments suggest that the aromatic anchors for the Ca²⁺/N lobe have a previously unappreciated role in Ca_V1.2 CDF.

Discussion

Calcium ions are potent chemical effectors of many cellular processes^1,7. The opening of Ca_Vs in response to changes in membrane potential is a major source of calcium influx¹ and thus couples two forms of biological signals, electrical and chemical. The Ca_V activity that drives this powerful signaling combination is subject to a variety of control mechanisms. A diverse set of proteins that includes auxiliary channel subunits, G-proteins, synaptic vesicle components, kinases, phosphatases and calcium sensors interact with and modify the behavior of the pore-forming subunit to limit or enhance calcium influx^1,2.

Two types of feedback regulation in which calcium ions affect their own entry through Ca_Vs and control local calcium levels have been intensively studied for more than two decades⁸. CDI, which limits calcium flux through Ca_Vs, and CDF, which enhances calcium flux through Ca_Vs, both result from interactions between calcium ions, a channel-resident CaM and the Ca_Vα₁ subunit's C-terminal cytoplasmic IQ domain^9,11–16,31. Further dissection of the molecular origins of CDI and CDF has shown that each process arises from the binding of specific calcium-bound CaM lobes to the IQ domain^10,12–14; however, the precise details have remained unknown.

IQ domains are protein segments that contain the 11-residue consensus sequence (I/L/V)QXXXRXXXX(R/K) and comprise a family of Ca²⁺/CaM– and apo CaM–binding motifs found in diverse proteins including molecular motors, voltage-gated calcium channels, voltage-gated sodium channels and phosphatases^26,27. The Ca²⁺/CaM–IQ domain structure presented here offers the first high-resolution picture of the molecular details of a Ca²⁺/CaM–IQ domain interaction. A previous work has proposed that Ile1624 is exposed in the Ca²⁺/CaM–Ca_V1.2 complex to provide hydrophobic contacts to other parts of the channel-gating machinery³². Contrary to this prediction, the structure shows that Ile1624 is completely buried (solvent-exposed area = 0.7 Ų) and contacts the hydrophobic surface of the C lobe (Fig. 2a and Supplementary Figs. 2 and 3). Other hallmark residues of the IQ domain engage in extensive hydrophobic and polar interactions with the CaM lobes that establish the importance of these residues for the Ca²⁺/CaM–IQ domain interaction (Fig. 1 and Supplementary Figs. 2 and 3).

The aromatic anchors that form the IQ domain's principal contacts to the Ca²⁺/CaM lobes were not anticipated from prior studies. Comparison of the Ca_V IQ domain defined by the crystal structure with other IQ motifs reveals that many anchor positions are unique to Ca_Vs. Such an array of aromatic anchors, particularly the N-terminal anchors, is lacking in the C-terminal cytoplasmic IQ domains of the closely related family of voltage-gated sodium channels (Na_Vs)^33–35 (Supplementary Fig. 4 online). This observation suggests that CaM binding to Ca_V and Na_V IQ domains and subsequent modulatory effects may be fundamentally different despite overall similarities in Na_V and Ca_V archictecture¹.

Examination of the Ca²⁺/CaM–Ca_V1.2 IQ domain complexes shows interesting differences in the ways Ca²⁺/N lobe and Ca²⁺/C lobe interact with the IQ domain: the number of binding conformations differs, with two for Ca²⁺/N lobe versus one for Ca²⁺/C lobe; the number of deep anchor positions differs, with one for Ca²⁺/N lobe (Phe1618) versus two for Ca²⁺/C lobe (Tyr1627 and Phe1628); and the buried surface areas are not equivalent, as Ca²⁺/C lobe buries more total and more hydrophobic surface than Ca²⁺/N lobe (Fig. 2). These differences suggest that the Ca²⁺/C lobe binds more tightly to the IQ domain. As it is not simple to infer directly the energetic importance of molecular interactions from structural data alone³⁶, we pursued experiments to determine whether these observed differences in modes of interaction have functional consequences.

We used ITC to probe the binding between the individual CaM lobes and the IQ domain. ITC experiments directly determine the thermodynamic parameters that underlie binding reactions and provide a degree of resolution for studying macromolecular interactions that is unmatched by other methods³⁷. The experiments showed that the Ca²⁺/C lobe binds the IQ domain with high affinity (K_d = 2.6 nM). To our surprise, we found multiple binding sites for the Ca²⁺/N lobe on the IQ domain (Fig. 5a). The lack of appreciable Ca²⁺/N lobe binding to the Ca²⁺/C lobe–IQ domain complex (Fig. 3c), together with the large reduction in Ca²⁺/N lobe binding affinity by the aromatic-anchor mutation F1628A (Fig. 3e), indicates that the medium-affinity Ca²⁺/N lobe–binding site overlaps directly with the Ca²⁺/C lobe–binding site (Fig. 5). The competition experiment (Fig. 3c) also showed that the binding of Ca²⁺/C lobe to the IQ domain lowers the affinity of the second Ca²⁺/N lobe–binding site to an undetectable level (K_d > 200 μM, given the concentrations used in the experiment³⁷). The dominance of Ca²⁺/C lobe in IQ domain binding energetics agrees with the eminent role of the Ca²⁺/C lobe in CDI of L-type channels^12,16,17. Given that the medium-affinity Ca²⁺/N lobe site overlaps with the high-affinity Ca²⁺/C lobe site and that Ca²⁺/C lobe binding to its high-affinity site weakens the avidity of Ca²⁺/N lobe for its low-affinity site, the thermodynamic data suggest that the crystallographically observed Ca²⁺/N lobe interactions correspond to the Ca²⁺/N lobe low-affinity site. These interactions arise from the high effective concentration³⁸ of the Ca²⁺/N lobe relative to the peptide caused by Ca²⁺/C lobe binding and the CaM interlobe linker (Fig. 5d).

Figure 5.

Schematic of Ca²⁺/CaM lobe multiple binding modes on the Ca_V1.2 IQ domain. (a) Ca²⁺/N lobe (green) has medium-affinity and low-affinity binding sites on the IQ domain. (b) Ca²⁺/C lobe (blue) has a high-affinity binding site on the IQ domain. (c) Binding of Ca²⁺/C lobe to the IQ domain blocks Ca²⁺/N lobe access to the Ca²⁺/N lobe medium-affinity site (black X) and reduces Ca²⁺/N lobe binding to the Ca²⁺/N lobe low-affinity site (grey X). (d) Representation of how Ca²⁺/C lobe binding to the IQ domain tethers Ca²⁺/N lobe near the Ca²⁺/N lobe low-affinity site. Yellow line indicates the CaM interlobe linker. In all panels, the approximate position of aromatic anchor Phe1628, which is shared by the Ca²⁺/N lobe medium-affinity site and the Ca²⁺/C lobe high-affinity site, is indicated by the white hexagon.

As Ca²⁺/C lobe binds tighter than Ca²⁺/N lobe to the site that includes Phe1628, Ca²⁺/N lobe is not expected to occupy this part of the IQ domain when both CaM lobes are loaded with calcium. However, Ca²⁺/N lobe may occupy this position in channel functional states in which the N lobe is loaded with calcium but the C lobe is in the apo state. This property may be particularly relevant for Ca_V2s, where functional experiments have demonstrated that CaM mutants that have diminished C lobe calcium binding but preserve N lobe calcium binding maintain CDI^9,11,23.

Functional studies indicate lobe-specific tasks for CaM in Ca_V regulation^{9,12,16,17,23}. The interactions that bring about lobe-specific regulation by CaM have remained obscure despite the investigation of an abundance of mutations to the IQ domain^{13–15,17,18,32}. In the context of the structure, it is clear that most of the reported Ca_V1.2 mutants, many of which involve multiple residue changes, alter contacts to both lobes simultaneously and therefore cannot be used to decipher the roles of binding to each lobe individually. The exceptions are mutants having changes at two single positions that contact the Ca²⁺/C lobe, Ile1624 and Phe1628 (refs. 14,15). Ile1624 mutations that perturb the residue's hydrophobicity and size disrupt CDI¹⁵. F1628A changes channel inactivation properties¹⁵. Together, these results indicate that the crystallographically defined IQ domain interface that contacts the Ca²⁺/C lobe is important for channel inactivation.

Both CaM lobes have specific functional roles in modulation of Ca_V2 channels^9,11,23. There is a clear role for the Ca²⁺/C lobe in Ca_V1 CDI. Ca_V1s and Ca_V2s are highly homologous in the region that interacts with CaM to mediate CDF and CDI. Despite the similarity, no role has yet been reported for the N lobe in Ca_V1 modulation. We tested whether the Ca²⁺/N lobe–anchoring residues observed in the Ca²⁺/CaM–IQ domain structure had a functional role. Simultaneous mutation of all three aromatic anchors for the N lobe to alanine did not affect Ca_V1.2 CDI (Fig. 4a,b). In contrast, these same mutations in the background of the I1624A mutation, which unmasks Ca_V1.2 CDF, completely disrupted Ca_V1.2 CDF, demonstrating a clear role for this Ca²⁺/N lobe interface in CDF (Fig. 4c,d). These experiments define a previously unknown role for the N lobe interface and suggest that the Ca²⁺/N lobe conformations seen in the structure are important for CDF. Apo CaM can also bind the IQ domain^18,20. It remains possible that the functional effects we observed have more complex origins that arise from interplay between Ca²⁺/CaM– and apo CaM–IQ domain binding.

The dual binding mode of Ca²⁺/N lobe observed in the structure suggests a level of conformational plasticity in Ca²⁺/N lobe–channel interactions. Regions on the N-terminal side of the IQ domain bind Ca²⁺/CaM in vitro^{17,19–21,39}, but with lower affinity than the IQ domain does^17,20. Anchoring of the Ca²⁺/C lobe to the IQ domain may permit Ca²⁺/CaM to remain bound while other regions of the channel compete for Ca²⁺/N lobe or the Ca²⁺/N lobe aromatic anchors during various states of channel operation^17,39,40. Alternatively, the observed conformational plasticity may provide a means for the complex to accommodate conformational changes that facilitate interactions with other channel domains while maintaining the Ca²⁺/N lobe–IQ interaction.

Ca_V2s show lobe-specific Ca²⁺/CaM modulation that seems inverted relative to Ca_V1.2 (refs. 9,11,23). Ca_V2 CDI relies on the Ca²⁺/N lobe^9,11,23, whereas the prominent CDF of Ca_V2.1 originates with the Ca²⁺/C lobe^9,11. The ITC data show that the Ca²⁺/N lobe–binding sites in the Ca_V1.2 IQ domain overlap with the Ca²⁺/C lobe–binding site. It is notable that three aromatic anchors identified here, Phe1618, Phe1622 and Phe1631, including Ca²⁺/N lobe aromatic anchor Phe1618, are conserved among Ca_V1s but not between Ca_V1s and Ca_V2s (Fig. 1c). Amino acid substitutions at these positions, together with changes at Gln1625 and Lys1633 (Fig. 1c), may tilt the Ca²⁺/N lobe– and Ca²⁺/C lobe–IQ domain affinity differences in favor of the Ca²⁺/N lobe. Such changes, in concert with interactions to other parts of the channel, may underlie the fundamental differences in lobe-specific modulation between Ca_V1.2 and Ca_V2s in a way that exploits the prodigious adaptability of CaM to recognize varied targets⁴¹.

The Ca²⁺/CaM–Ca_V1.2 IQ domain structure presented here is the first high-resolution view of the components that transduce Ca²⁺-dependent Ca_V regulation, and it provides a necessary molecular framework for detailed dissection of the transitions that drive the rich regulation of Ca_Vs by CaM.

Methods

Purification

The IQ domain of human Ca_v1.2 (Ca_Vα₁c77) (residues 1611–1644), human CaM N lobe (residues 1–78) and human CaM C lobe (residues 79–148) were cloned into a modified pET28 vector (Novagen) denoted HMT⁴² that contains, in sequence, a His₆-tag, maltose-binding protein and a cleavage site for the tobacco etch virus (TEV) protease. Full-length CaM was cloned into pEGST⁴³ without using any affinity tag. The F1628A mutation in the IQ domain was obtained using the QuikChange protocol (Stratagene).

All proteins were expressed in Escherichia coli BL21(DE3)pLysS grown in 2×YT media at 37 °C. Cells were lysed in 250 mM KCl, 10 mM K-HEPES (pH 7.4) and 1 mM CaCl₂ (buffer A) supplemented with 1 mM PMSF. The complex of CaM and IQ domain was obtained by coexpression and was purified on a Poros20MC column (Perseptive Biosystems), washed with buffer A and eluted with buffer A plus 500 mM imidazole (buffer B). After cleavage with His-tagged TEV protease⁴⁴ at room temperature for ∼ 12 h and dialysis against 100 mM KCl, 10 mM Tris-HCl (pH 8.8) and 1 mM CaCl₂ (buffer C), the complex was purified on a Hiload HQ column (Amersham) in buffer C with a linear gradient to 30% buffer D (1 M KCl, 10 mM Tris-HCl (pH 8.8), 1 mM CaCl₂). Trace amounts of residual maltose-binding protein were removed by collecting the flow-through from an additional passage of the purified material over a Poros20MC column in buffer A. Finally, the sample was dialyzed against 20 mM KCl, 10 mM K-HEPES (pH 7.4) and 1 mM CaCl₂.

SeMet-substituted complex was expressed in BL21(DE3)pLysS cells in M9 minimal medium containing 20% (w/v) glucose, with the methionine biosynthesis pathway inhibited⁴⁵. Purification was as described above with all buffers supplemented with 5 mM methionine and 10 mM β-mercaptoethanol.

The first steps of purifying HMT–CaM N lobe and HMT–CaM C lobe fusions were similar to the CaM–IQ domain purification. After TEV cleavage, the material was dialyzed against buffer E (10 mM Tris-HCl (pH 8.8), 10 mM KCl, 1 mM CaCl₂) and purified on a Hiload HQ column with a gradient from 0 to 50% buffer F (1 M KCl, 10 mM Tris-HCl (pH 8.8), 1 mM CaCl₂). The proteins were purified further on a Poros20MC column in buffer A, and the concentrated flow-through was applied to a TSK2000 column (Tosoh Biosep) run in buffer A.

To obtain free IQ domain we purified the complex with CaM as described above and separated the two components on a preparative C18 HPLC column (Vydac) with a linear gradient from 35 to 50% acetonitrile in 0.1% (v/v) trifluoroacetic acid over 15 column volumes. The purity was verified by MALDI-TOF mass spectrometry.

Crystallization and structure determination

Complexes of native and SeMet-substituted CaM–IQ domain were crystallized by sitting or hanging drop vapor diffusion⁴⁶ at 20 °C by mixing equal volumes of protein (∼ 10 mg ml⁻¹) and well solution containing 0.1 M Bis-Tris (pH 6.5) and 20–30% (w/v) PEG 3350. After transfer to Paratone oil (Hampton Research) and flash-freezing, diffraction data were collected at Beamline 8.3.1 (Advanced Light Source, Lawrence Berkeley National Laboratories) and processed using HKL2000 (ref. 47). A three-wavelength MAD experiment was performed on crystals of SeMet-substituted protein (Table 2). Twenty-five initial selenium positions were located using ShelxD⁴⁸. Subsequent refinement, substructure completion and phasing were performed using SHARP⁴⁹ (Supplementary Fig. 1). After density modification the figure of merit was 0.852. An initial model was built using RESOLVE⁵⁰ and ARP/wARP⁵¹, manually extended with XtalView⁵² and refined against 2.00-Å native data using REFMAC5 (ref. 53). TLS parameters were used throughout the refinement. Side chains and full residues with missing electron densities were not modeled. The final model consists of three Ca²⁺/CaM–IQ domain complexes in the asymmetric unit with 95.2% of the residues in the core region of the Ramachandran plot and none in disallowed regions. A Ni²⁺ ion is observed in all three complexes and most likely was introduced into the sample from the Ni²⁺ affinity column (Poros20MC). Its presence in the crystal was confirmed by a fluorescence wavelength scan. The Ni²⁺ binds at the C lobe outer surface distal from the IQ domain and is not expected to interfere with Ca²⁺/CaM–IQ domain binding. Refinement data and statistics are shown in Table 2.

Table 2. Data collection, phasing and refinement statistics.

	Native		SeMet
Data collection
Space group	P21		P21
Cell dimensions
a, b, c (Å)	84.73, 37.24, 86.86		82.55, 37.01, 87.55
α, β, γ (°)	90, 97.77, 90		90, 98.10, 90
		Peak	Inflection	Remote
Wavelength	1.116	0.97957	0.97972	1.01986
Resolution (Å)	30.0–2.0 (2.07–2.0)	30–2.5 (2.59–2.50)	30–2.8 (2.90–2.80)	30–2.4 (2.49–2.40)
Rsym	6.6 (33.1)	9.5 (42.8)	7.4 (40.1)	6.6 (34.3)
I / σI	19.8 (2.2)	19.0 (2.5)	14.2 (2.1)	15.0 (1.7)
Completeness (%)	99.2 (98.3)	97.1 (99.4)	99.9 (100)	87.1 (89.6)
Redundancy	3.5	7.8	3.8	2.6
Refinement
Resolution (Å)	30.0–2.0
No. reflections	36,436
Rwork / Rfree	20.23 / 25.47
No. atoms
Protein	3,845
Ligand/ion	15
Water	260
B-factors
Protein	31.50
Ligand/ion	38.16
Water	47.05
R.m.s. deviations
Bond lengths (Å)	0.016
Bond angles (°)	1.363

One native and one SeMet crystal were used for structure solution. Highest-resolution shell is shown in parentheses.

Isothermal titration calorimetry

Samples were concentrated and dialyzed twice against 5 mM KCl, 10 mM HEPES (pH 7.4) and 1mM CaCl₂. Higher salt concentrations reduced the solubility of the IQ domain. Samples were degassed for 5 min and titrations were performed on a VP-ITC calorimeter (MicroCal) at 15 °C. Data were processed with MicroCal Origin 7.0. For fitting of the F1628A N lobe data, the stoichiometry for the second binding site was set equal to 1. Because of the high titrant concentration required to measure weak interactions, and owing to limited solubility of the IQ domain, N lobe was titrated into peptide and not vice versa.

Protein concentrations were determined by absorbance⁵⁴, except for N lobe CaM (which has no aromatic residues), where the BCA method was used, using known concentrations of full-length CaM and CaM C lobe as references. Each ITC experiment was repeated with different batches of purified protein, yielding similar thermodynamic parameters and stoichiometry values. Control injections, consisting of titrating one component into buffer, were used to adjust the baseline of each experiment.

Electrophysiology

Constructs for electrophysiology consisted of human Ca_V1.2 (splice variant α₁c77) in pcDNA3.1(+)/hygro (Invitrogen), Ca_Vβ_2a in pGEM (Promega) and rabbit Ca_Vα₂δ in pcDNA3 (Invitrogen). Mutants of the α₁c77 subunit were made using the QuikChange protocol (Stratagene). RNA transcripts were prepared using a T7 mMessage mMachine kit (Ambion). 50 nl of a complementary RNA mixture containing 30–100 nM Ca_V1.2 α₁c77, 33 nM Ca_Vβ_2a and 33 nM Ca_Vα₂δ was microinjected into Xenopus oocytes, which were then kept at 18 °C in ND96 medium supplemented with penicillin and streptomycin. Recordings were performed 3–7 d after injection. Before recording, oocytes were injected with 25–50 nl 100 mM BAPTA to minimize contaminating Ca²⁺-activated Cl⁻ current. During recordings, the oocytes were superfused using a Valvelink 16 (Automate Scientific) controller with either a Ba²⁺-containing solution (40 mM Ba(OH)₂, 50 mM NaOH, 1 mM KOH, 0.4% (w/v) niflumic acid, 10 mM HEPES) or a Ca²⁺-containing solution (Ba(OH)₂ replaced with Ca(NO₃)₂). Both solutions were adjusted to pH 7.4 using HNO₃. Two-electrode voltage-clamp experiments were performed using a GeneClamp 500B amplifier (Axon Instruments) controlled by a computer with a 1,200 MHz processor (Celeron, Gateway) using CLAMPEX 8.2.0.224 (Axon Instruments) and digitized at 1 kHz with a Digidata1332A (Axon Instruments). Electrodes were filled with 3 M KCl and had resistances of 0.1–1.5 MΩ. Leak currents were subtracted using a P/4 protocol. Ionic currents were analyzed with Clampfit 8.2 (Axon Instruments).

Supplementary Material

Supplementary Fig. 1 a, Experimentally phased and density modified electron density map, contoured at 1.2 σ, shown for the IQ domain in complex A. b, Residual difference electron density map contoured at 2 σ for the IQ domain in complex B. N-lobe and C-lobe are displayed in green and blue, respectively. IQ domain residues are shown as red sticks.

Supplementary Fig. 2 a-g, Details of interactions of Ca_V1.2 IQ domain residues with Ca²⁺/CaM. IQ domain residues are displayed in red, Ca²⁺/N-lobe residues in green, and Ca²⁺/C-lobe residues in blue. Dark colours correspond to complex A and light colours correspond to complex C. For the Ca²⁺/N-lobe interaction figures, the Ca²⁺/N-lobes of complex A and complex C have been superposed, outlining the differences in positions of IQ domain residues. For the Ca²⁺/C-lobe interactions, only chain A is shown, as the interactions in complex A and C are very similar. h, Superposition of complex A Ca²⁺/C-lobe (blue) with the Ca²⁺/C-lobe of CaM complexed to a CaMKII peptide (PDB entry 1CDM) (yellow), showing the different conformation of Met109 to produce a pocket for the deep anchor F1628. The CamKII peptide is not shown for clarity.

Supplementary Fig. 3 Overview of mainchain and sidechain interactions a, complex A and b, complex C. CaM residues ≤ 4 Å to the IQ domain are shown. IQ domain residues (red) are in three rows: contacts to Ca²⁺/N-lobe only (upper), none or both (middle) or Ca²⁺/C-lobe only (lower). Because contacts that are just below 4Å in one complex but just above 4 Å in the other would appear only in one figure and hinder appreciation of the main differences, contacts ≤ 4.2 Å are included, provided they are ≤ 4.0 Å in the other complex. Water-mediated hydrogen bonds are indicated (o). Residues N-terminal or C-terminal to the interaction site with are not shown.

Supplementary Fig. 4 Sequence alignment of the IQ domains of Ca_V1, Ca_V2, and Na_V1 channels. Ca_V residues are coloured according to their corresponding contacts in complex A: green, Ca²⁺/N-lobe; cyan, Ca²⁺/C-lobe; purple, Ca²⁺/N-lobe and Ca²⁺/C-lobe. Na_V residues are coloured according to their degree of conservation: grey, identical; orange, conserved. Asterisks indicate principal aromatic anchor positions of the Ca_V1.2 IQ domain. The consensus IQ motif is indicated.