The Calmodulin-Binding,Short Linear Motif,NSCaTE Is Conserved in L-Type Channel Ancestors of Vertebrate Cav1.2 and Cav1.3 Channels

NSCaTE is a short linear motif of (xWxxx(I or L)xxxx), composed of residues with a high helix-forming propensity within a mostly disordered N-terminus that is conserved in L-type calcium channels from protostome invertebrates to humans. NSCaTE is an optional, lower affinity and calcium-sensitive binding site for calmodulin (CaM) which competes for CaM binding with a more ancient, C-terminal IQ domain on L-type channels. CaM bound to N- and C- terminal tails serve as dual detectors to changing intracellular Ca²⁺ concentrations, promoting calcium-dependent inactivation of L-type calcium channels. NSCaTE is absent in some arthropod species, and is also lacking in vertebrate L-type isoforms, Ca_v1.1 and Ca_v1.4 channels. The pervasiveness of a methionine just downstream from NSCaTE suggests that L-type channels could generate alternative N-termini lacking NSCaTE through the choice of translational start sites. Long N-terminus with an NSCaTE motif in L-type calcium channel homolog LCa_v1 from pond snail Lymnaea stagnalis has a faster calcium-dependent inactivation than a shortened N-termini lacking NSCaTE. NSCaTE effects are present in low concentrations of internal buffer (0.5 mM EGTA), but disappears in high buffer conditions (10 mM EGTA). Snail and mammalian NSCaTE have an alpha-helical propensity upon binding Ca²⁺-CaM and can saturate both CaM N-terminal and C-terminal domains in the absence of a competing IQ motif. NSCaTE evolved in ancestors of the first animals with internal organs for promoting a more rapid, calcium-sensitive inactivation of L-type channels.     

The Calmodulin Regulator Protein,PEP-19, Sensitizes ATP-induced Ca2+ Release

PEP-19 is a small, intrinsically disordered protein that binds to the C-domain of calmodulin (CaM) via an IQ motif and tunes its Ca²⁺ binding properties via an acidic sequence. We show here that the acidic sequence of PEP-19 has intrinsic Ca²⁺ binding activity, which may modulate Ca²⁺ binding to CaM by stabilizing an initial Ca²⁺-CaM complex or by electrostatically steering Ca²⁺ to and from CaM. Because PEP-19 is expressed in cells that exhibit highly active Ca²⁺ dynamics, we tested the hypothesis that it influences ligand-dependent Ca²⁺ release. We show that PEP-19 increases the sensitivity of HeLa cells to ATP-induced Ca²⁺ release to greatly increase the percentage of cells responding to sub-saturating doses of ATP and increases the frequency of Ca²⁺ oscillations. Mutations in the acidic sequence of PEP-19 that inhibit or prevent it from modulating Ca²⁺ binding to CaM greatly inhibit its effect on ATP-induced Ca²⁺ release. Thus, this cellular effect of PEP-19 does not depend simply on binding to CaM via the IQ motif but requires its acidic metal binding domain. Tuning the activities of Ca²⁺ mobilization pathways places PEP-19 at the top of CaM signaling cascades, with great potential to exert broad effects on downstream CaM targets, thus expanding the biological significance of this small regulator of CaM signaling.     

Arrhythmogenic Calmodulin Mutations Affect the Activation and Termination of Cardiac Ryanodine Receptor-mediated Ca2+ Release

The intracellular Ca²⁺ sensor calmodulin (CaM) regulates the cardiac Ca²⁺ release channel/ryanodine receptor 2 (RyR2), and mutations in CaM cause arrhythmias such as catecholaminergic polymorphic ventricular tachycardia (CPVT) and long QT syndrome. Here, we investigated the effect of CaM mutations causing CPVT (N53I), long QT syndrome (D95V and D129G), or both (CaM N97S) on RyR2-mediated Ca²⁺ release. All mutations increased Ca²⁺ release and rendered RyR2 more susceptible to store overload-induced Ca²⁺ release (SOICR) by lowering the threshold of store Ca²⁺ content at which SOICR occurred and the threshold at which SOICR terminated. To obtain mechanistic insights, we investigated the Ca²⁺ binding of the N- and C-terminal domains (N- and C-domain) of CaM in the presence of a peptide corresponding to the CaM-binding domain of RyR2. The N53I mutation decreased the affinity of Ca²⁺ binding to the N-domain of CaM, relative to CaM WT, but did not affect the C-domain. Conversely, mutations N97S, D95V, and D129G had little or no effect on Ca²⁺ binding to the N-domain but markedly decreased the affinity of the C-domain for Ca²⁺. These results suggest that mutations D95V, N97S, and D129G alter the interaction between CaM and the CaMBD and thus RyR2 regulation. Because the N53I mutation minimally affected Ca²⁺ binding to the C-domain, it must cause aberrant regulation via a different mechanism. These results support aberrant RyR2 regulation as the disease mechanism for CPVT associated with CaM mutations and shows that CaM mutations not associated with CPVT can also affect RyR2. A model for the CaM-RyR2 interaction, where the Ca²⁺-saturated C-domain is constitutively bound to RyR2 and the N-domain senses increases in Ca²⁺ concentration, is proposed.     

The neuronal voltage-dependent sodium channel type II IQ motif lowers the calcium affinity of the C-domain of calmodulin

Calmodulin (CaM) is the primary calcium sensor in eukaryotes. Calcium binds cooperatively to pairs of EF-hand motifs in each domain (N and C). This allows CaM to regulate cellular processes via calcium-dependent interactions with a variety of proteins, including ion channels. One neuronal target is NaV1.2, voltage-dependent sodium channel type II, to which CaM binds via an IQ motif within the NaV1.2 C-terminal tail (residues 1901-1938) [Mori, M., et al. (2000) Biochemistry 39, 1316-1323]. Here we report on the use of circular dichroism, fluorescein emission, and fluorescence anisotropy to study the interaction between CaM and NaV1.2 at varying calcium concentrations. At 1 mM MgCl2, both full-length CaM (CaM1-148) and a C-domain fragment (CaM76-148) exhibit tight (nanomolar) calcium-independent binding to the NaV1.2 IQ motif, whereas an N-domain fragment of CaM (CaM1-80) binds weakly, regardless of calcium concentration. Equilibrium calcium titrations of CaM at several concentrations of the NaV1.2 IQ peptide showed that the peptide reduced the calcium affinity of the CaM C-domain sites (III and IV) without affecting the N-domain sites (I and II) significantly. This leads us to propose that the CaM C-domain mediates constitutive binding to the NaV1.2 peptide, but that interaction then distorts calcium-binding sites III and IV, thereby reducing their affinity for calcium. This contrasts with the CaM-binding domains of voltage-dependent Ca2+ channels, kinases, and phosphatases, which increase the calcium binding affinity of the C-domain of CaM.     

Properties of Calmodulin Binding to NaV1.2 IQ Motif and Its Autism-Associated Mutation R1902C

Voltage-gated sodium channels (VGSCs) are fundamental to the initiation and propagation of action potentials in excitable cells. Ca²⁺/calmodulin (CaM) binds to VGSC type II (Na_V1.2) isoleucine and glutamine (IQ) motif. An autism-associated mutation in Na_V1.2 IQ motif, Arg1902Cys (R1902C), has been reported to affect the combination between CaM and the IQ motif compared to that of the wild type IQ motif. However, the detailed properties for the Ca²⁺-regulated binding of CaM to Na_V1.2 IQ (1901Lys-1927Lys, IQ_wt) and mutant IQ motif (IQ_R1902C) remains unclear. Here, the binding ability of CaM and CaM's constituent proteins including N- and C lobe to the IQ motif of Na_V1.2 and its mutant was investigated by protein pull-down experiments. We discovered that the combination between CaM and the IQ motif was U-shaped with the highest at [Ca²⁺] ≈ free and the lowest at 100 nM [Ca²⁺]. In the IQ_R1902C mutant, Ca²⁺-dependence of CaM binding was nearly lost. Consequently, the binding of CaM to IQ_R1902C at 100 and 500 nM [Ca²⁺] was increased compared to that of IQ_wt. Both N- and C lobe of CaM could bind with Na_V1.2 IQ motif and IQ_R1902C mutant, with the major effect of C lobe. Furthermore, CaMKII had no impact on the binding between CaM and Na_V1.2 IQ motif. This research offers novel insight to the regulation of Na_V1.2 IQ_wt and IQ_R1902C motif, an autism-associated mutation, by CaM.

     

Calmodulin interactions with IQ peptides from voltage-dependent calcium channels

Calmodulin (CaM) functions as a Ca²⁺ sensor for inactivation and, in some cases, facilitation of a variety of voltage-dependent Ca²⁺ channels. A crucial determinant for CaM binding to these channels is the IQ motif in the COOH-terminal tail of the channel-forming subunit. The binding of CaM to IQ peptides from Lc-, P/Q-, and R-type, but not N-type, voltage-dependent Ca²⁺ channels increases the Ca²⁺ affinity of both lobes of CaM, producing similar N- and C-lobe Ca²⁺ affinities. Ca²⁺ associates with and dissociates from the N-lobe much more rapidly than the C-lobe when CaM is bound to the IQ peptides. Compared with the other IQ peptides, CaM-bound Lc-IQ has the highest Ca²⁺ affinity and the most rapid rates of Ca²⁺ association at both lobes, which is likely to make Ca²⁺ binding to CaM, bound to this channel, less sensitive than other channels to intracellular Ca²⁺ buffers. These kinetic differences in Ca²⁺ binding to the lobes of CaM when bound to the different IQ motifs may explain both the ability of CaM to perform multiple functions in these channels and the differences in CaM regulation of the different voltage-dependent Ca²⁺ channels. Ca²⁺-dependent inactivation; Ca²⁺-dependent facilitation; apocalmodulin     

Calcium binding and conformational properties of calmodulin complexed with peptides derived from myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP)

The myristoylated alanine-rich C kinase substrate (MARCKS) and the MARCKS-related protein (MRP) are members of a distinct family of protein ki-nase C (PKC) substrates that bind calmodulin (CaM) in a manner regulated by Ca²⁺ and phosphorylation by PKC. The CaM binding region overlaps with the PKC phosphorylation sites, suggesting a potential coupling between Ca²⁺-CaM signalling and PKC-mediated phosphorylation cascades. We have studied Ca²⁺ binding of CaM complexed with CaM binding peptides from MARCKS and MRP using flow dialysis, NMR and circular dichroism (CD) spectroscopy. The wild-type MARCKS and MRP peptides induced significant increases in the Ca²⁺ affinity of CaM (pCa 6.1 and 5.8, respectively, compared to 5.2, for CaM in the absence of bound peptides), whereas a modified MARCKS peptide, in which the four serine residues susceptible to phosphorylation in the wild-type sequence have been replaced with aspartate residues to mimic phosphorylation, had smaller effect (pCa 5.6). These results are consistent with the notions that phosphorylation of MARCKS reduces its binding affinity for CaM and that the CaM binding affinity of the peptides is coupled to the Ca²⁺ affinity of CaM. All three MARCKS/MRP peptides perturbed the backbone NMR resonances of residues in both the N- and C-terminal domains of CaM and, in addition, the wild-type MARCKS and the MRP peptides induced strong positive cooperativity in Ca²⁺ binding by CaM, suggesting that the peptides interact with the amino- and carboxy-terminal domains of CaM simultaneously. NMR analysis of the Ca²⁺-CaM-MRP peptide complex, as well as CD measurements of Ca²⁺-CaM in the presence and absence of MARCKS/MRP peptides suggest that the peptide bound to CaM is non-helical, in contrast to the α-helical conformation found in the CaM binding regions of myosin light-chain kinase and CaM-dependent protein kinase II. The adaptation of the CaM molecule for binding the peptide requires disruption of its central helical linker between residues Lys-75 and Glu-82. Received: 26 September 1996 / 22 October 1996     

Molecular dynamics studies on conformational thermodynamics of Orai1–calmodulin complex

Molecular understanding of bio-macromolecular binding is a challenging task due to large sizes of the molecules and presence of variety of interactions. Here, we study the molecular mechanism of calmodulin (CaM) binding to Orai1 that regulates Ca²⁺-dependent inactivation process in eukaryotic cells. Although experimental observations indicate that Orai1 binds to the C-terminal of Ca²⁺-loaded CaM, it is not decisive if N-domain of CaM interacts with Orai1. We address the issue of interaction of different domains of CaM with Orai1 using conformational thermodynamic changes, computed from histograms of dihedral angles over simulated trajectories of CaM, CaM-binding domain of Orai1 and complexes of CaM with Orai1. The changes for all residues of both C and N terminal domains of CaM upon Orai1 binding are compared. Our analysis shows that Orai1binds to both C-terminal and N-terminal domains of CaM, indicating 1:2 stoichiometry. The Orai1 binding to N-terminal domain of CaM is less stable than that to the C-terminal domain. The binding residues are primarily hydrophobic. These observations are in qualitative agreement to the experiments. The conformational thermodynamic changes thus provide a useful computational tool to provide atomic details of interactions in bio-macromolecular binding.     

Thermodynamic linkage between calmodulin domains binding calcium and contiguous sites in the C-terminal tail of Ca(V)1.2

Calmodulin (CaM) binding to the intracellular C-terminal tail (CTT) of the cardiac L-type Ca²⁺ channel (Ca_V1.2) regulates Ca²⁺ entry by recognizing sites that contribute to negative feedback mechanisms for channel closing. CaM associates with Ca_V1.2 under low resting [Ca²⁺], but is poised to change conformation and position when intracellular [Ca²⁺] rises. CaM binding Ca²⁺, and the domains of CaM binding the CTT are linked thermodynamic functions. To better understand regulation, we determined the energetics of CaM domains binding to peptides representing pre-IQ sites A₁₅₈₈, and C₁₆₁₄ and the IQ motif studied as overlapping peptides IQ₁₆₄₄ and IQ′₁₆₅₀ as well as their effect on calcium binding. (Ca²⁺)₄-CaM bound to all four peptides very favorably (K_d ≤ 2 nM). Linkage analysis showed that IQ_1644-1670 bound with a K_d ~ 1 pM. In the pre-IQ region, (Ca²⁺)₂-N-domain bound preferentially to A₁₅₈₈, while (Ca²⁺)₂-C-domain preferred C₁₆₁₄. When bound to C₁₆₁₄, calcium binding in the N-domain affected the tertiary conformation of the C-domain. Based on the thermodynamics, we propose a structural mechanism for calcium-dependent conformational change in which the linker between CTT sites A and C buckles to form an A-C hairpin that is bridged by calcium-saturated CaM.     

Solution NMR Structure of the Ca2+-bound N-terminal Domain of CaBP7: A REGULATOR OF GOLGI TRAFFICKING*

Calcium-binding protein 7 (CaBP7) is a member of the calmodulin (CaM) superfamily that harbors two high affinity EF-hand motifs and a C-terminal transmembrane domain. CaBP7 has been previously shown to interact with and modulate phosphatidylinositol 4-kinase III-β (PI4KIIIβ) activity in in vitro assays and affects vesicle transport in neurons when overexpressed. Here we show that the N-terminal domain (NTD) of CaBP7 is sufficient to mediate the interaction of CaBP7 with PI4KIIIβ. CaBP7 NTD encompasses the two high affinity Ca²⁺ binding sites, and structural characterization through multiangle light scattering, circular dichroism, and NMR reveals unique properties for this domain. CaBP7 NTD binds specifically to Ca²⁺ but not Mg²⁺ and undergoes significant conformational changes in both secondary and tertiary structure upon Ca²⁺ binding. The Ca²⁺-bound form of CaBP7 NTD is monomeric and exhibits an open conformation similar to that of CaM. Ca²⁺-bound CaBP7 NTD has a solvent-exposed hydrophobic surface that is more expansive than observed in CaM or CaBP1. Within this hydrophobic pocket, there is a significant reduction in the number of methionine residues that are conserved in CaM and CaBP1 and shown to be important for target recognition. In CaBP7 NTD, these residues are replaced with isoleucine and leucine residues with branched side chains that are intrinsically more rigid than the flexible methionine side chain. We propose that these differences in surface hydrophobicity, charge, and methionine content may be important in determining highly specific interactions of CaBP7 with target proteins, such as PI4KIIIβ.     

Two EF-hand motifs in ryanodine receptor calcium release channels contribute to isoform-specific regulation by calmodulin

The mammalian ryanodine receptor Ca²⁺ release channel (RyR) has a single conserved high affinity calmodulin (CaM) binding domain. However, the skeletal muscle RyR1 is activated and cardiac muscle RyR2 is inhibited by CaM at submicromolar Ca²⁺. This suggests isoform-specific domains are involved in RyR regulation by CaM. To gain insight into the differential regulation of cardiac and skeletal muscle RyRs by CaM, RyR1/RyR2 chimeras and mutants were expressed in HEK293 cells, and their single channel activities were measured using a lipid bilayer method. All RyR1/RyR2 chimeras and mutants were inhibited by CaM at 2 μM Ca²⁺, consistent with CaM inhibition of RyR1 and RyR2 at micromolar Ca²⁺ concentrations. An RyR1/RyR2 chimera with RyR1 N-terminal amino acid residues (aa) 1–3725 and RyR2 C-terminal aa 3692–4968 were inhibited by CaM at <1 μM Ca²⁺ similar to RyR2. In contrast, RyR1/RyR2 chimera with RyR1 aa 1–4301 and RyR2 4254–4968 was activated at <1 μM Ca²⁺ similar to RyR1. Replacement of RyR1 aa 3726–4298 with corresponding residues from RyR2 conferred CaM inhibition at <1 μM Ca²⁺, which suggests RyR1 aa 3726–4298 are required for activation by CaM. Characterization of additional RyR1/RyR2 chimeras and mutants in two predicted Ca²⁺ binding motifs in RyR1 aa 4081–4092 (EF1) and aa 4116–4127 (EF2) suggests that both EF-hand motifs and additional sequences in the large N-terminal regions are required for isoform-specific RyR1 and RyR2 regulation by CaM at submicromolar Ca²⁺ concentrations.     

Acidic/IQ motif regulator of calmodulin

The small IQ motif proteins PEP-19 (62 amino acids) and RC3 (78 amino acids) greatly accelerate the rates of Ca(2+) binding to sites III and IV in the C-domain of calmodulin (CaM). We show here that PEP-19 decreases the degree of cooperativity of Ca(2+) binding to sites III and IV, and we present a model showing that this could increase Ca(2+) binding rate constants. Comparative sequence analysis showed that residues 28 to 58 from PEP-19 are conserved in other proteins. This region includes the IQ motif (amino acids 39-62), and an adjacent acidic cluster of amino acids (amino acids 28-40). A synthetic peptide spanning residues 28-62 faithfully mimics intact PEP-19 with respect to increasing the rates of Ca(2+) association and dissociation, as well as binding preferentially to the C-domain of CaM. In contrast, a peptide encoding only the core IQ motif does not modulate Ca(2+) binding, and binds to multiple sites on CaM. A peptide that includes only the acidic region does not bind to CaM. These results show that PEP-19 has a novel acidic/IQ CaM regulatory motif in which the IQ sequence provides a targeting function that allows binding of PEP-19 to CaM, whereas the acidic residues modify the nature of this interaction, and are essential for modulating Ca(2+) binding to the C-domain of CaM.     

Neurogranin controls the spatiotemporal pattern of postsynaptic Ca2+/CaM signaling

Neurogranin (Ng) is a postsynaptic IQ-motif containing protein that accelerates Ca²⁺ dissociation from calmodulin (CaM), a key regulator of long-term potentiation and long-term depression in CA1 pyramidal neurons. The exact physiological role of Ng, however, remains controversial. Two genetic knockout studies of Ng showed opposite outcomes in terms of the induction of synaptic plasticity. To understand its function, we test the hypothesis that Ng could regulate the spatial range of action of Ca²⁺/CaM based on its ability to accelerate the dissociation of Ca²⁺ from CaM. Using a mathematical model constructed on the known biochemistry of Ng, we calculate the cycle time that CaM molecules alternate between the fully Ca²⁺ saturated state and the Ca²⁺ unbound state. We then use these results and include diffusion of CaM to illustrate the impact that Ng has on modulating the spatial profile of Ca²⁺-saturated CaM within a model spine compartment. Finally, the first-passage time of CaM to transition from the Ca²⁺-free state to the Ca²⁺-saturated state was calculated with or without Ng present. These analyses suggest that Ng regulates the encounter rate between Ca²⁺ saturated CaM and its downstream targets during postsynaptic Ca²⁺ transients.     

Structure of the Small Dictyostelium discoideum Myosin Light Chain MlcB Provides Insights into MyoB IQ Motif Recognition

Dictyostelium discoideum MyoB is a class I myosin involved in the formation and retraction of membrane projections, cortical tension generation, membrane recycling, and phagosome maturation. The MyoB-specific, single-lobe EF-hand light chain MlcB binds the sole IQ motif of MyoB with submicromolar affinity in the absence and presence of Ca²⁺. However, the structural features of this novel myosin light chain and its interaction with its cognate IQ motif remain uncharacterized. Here, we describe the NMR-derived solution structure of apoMlcB, which displays a globular four-helix bundle. Helix 1 adopts a unique orientation when compared with the apo states of the EF-hand calcium-binding proteins calmodulin, S100B, and calbindin D_9k. NMR-based chemical shift perturbation mapping identified a hydrophobic MyoB IQ binding surface that involves amino acid residues in helices I and IV and the functional N-terminal Ca²⁺ binding loop, a site that appears to be maintained when MlcB adopts the holo state. Complementary mutagenesis and binding studies indicated that residues Ile-701, Phe-705, and Trp-708 of the MyoB IQ motif are critical for recognition of MlcB, which together allowed the generation of a structural model of the apoMlcB-MyoB IQ complex. We conclude that the mode of IQ motif recognition by the novel single-lobe MlcB differs considerably from that of stereotypical bilobal light chains such as calmodulin.     

Pull-down of Calmodulin-binding Proteins

Calcium (Ca²⁺) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca²⁺ signaling is mediated through the calcium-binding protein known as calmodulin (CaM)^1,2. CaM is involved at multiple levels in almost all cellular processes, including apoptosis, metabolism, smooth muscle contraction, synaptic plasticity, nerve growth, inflammation and the immune response. A number of proteins help regulate these pathways through their interaction with CaM. Many of these interactions depend on the conformation of CaM, which is distinctly different when bound to Ca²⁺ (Ca²⁺-CaM) as opposed to its Ca²⁺-free state (ApoCaM)³.While most target proteins bind Ca²⁺-CaM, certain proteins only bind to ApoCaM. Some bind CaM through their IQ-domain, including neuromodulin⁴, neurogranin (Ng)⁵, and certain myosins⁶. These proteins have been shown to play important roles in presynaptic function⁷, postsynaptic function⁸, and muscle contraction⁹, respectively. Their ability to bind and release CaM in the absence or presence of Ca²⁺ is pivotal in their function. In contrast, many proteins only bind Ca²⁺-CaM and require this binding for their activation. Examples include myosin light chain kinase¹⁰, Ca²⁺/CaM-dependent kinases (CaMKs)¹¹ and phosphatases (e.g. calcineurin)¹², and spectrin kinase¹³, which have a variety of direct and downstream effects¹⁴.The effects of these proteins on cellular function are often dependent on their ability to bind to CaM in a Ca²⁺-dependent manner. For example, we tested the relevance of Ng-CaM binding in synaptic function and how different mutations affect this binding. We generated a GFP-tagged Ng construct with specific mutations in the IQ-domain that would change the ability of Ng to bind CaM in a Ca²⁺-dependent manner. The study of these different mutations gave us great insight into important processes involved in synaptic function^8,15. However, in such studies, it is essential to demonstrate that the mutated proteins have the expected altered binding to CaM.Here, we present a method for testing the ability of proteins to bind to CaM in the presence or absence of Ca²⁺, using CaMKII and Ng as examples. This method is a form of affinity chromatography referred to as a CaM pull-down assay. It uses CaM-Sepharose beads to test proteins that bind to CaM and the influence of Ca²⁺ on this binding. It is considerably more time efficient and requires less protein relative to column chromatography and other assays. Altogether, this provides a valuable tool to explore Ca²⁺/CaM signaling and proteins that interact with CaM.     

Structural Insights into Activation of the Retinal L-type Ca2+ Channel (Cav1.4) by Ca2+-binding Protein 4 (CaBP4)

CaBP4 modulates Ca²⁺-dependent activity of L-type voltage-gated Ca²⁺ channels (Cav1.4) in retinal photoreceptor cells. Mg²⁺ binds to the first and third EF-hands (EF1 and EF3), and Ca²⁺ binds to EF1, EF3, and EF4 of CaBP4. Here we present NMR structures of CaBP4 in both Mg²⁺-bound and Ca²⁺-bound states and model the CaBP4 structural interaction with Cav1.4. CaBP4 contains an unstructured N-terminal region (residues 1–99) and four EF-hands in two separate lobes. The N-lobe consists of EF1 and EF2 in a closed conformation with either Mg²⁺ or Ca²⁺ bound at EF1. The C-lobe binds Ca²⁺ at EF3 and EF4 and exhibits a Ca²⁺-induced closed-to-open transition like that of calmodulin. Exposed residues in Ca²⁺-bound CaBP4 (Phe¹³⁷, Glu¹⁶⁸, Leu²⁰⁷, Phe²¹⁴, Met²⁵¹, Phe²⁶⁴, and Leu²⁶⁸) make contacts with the IQ motif in Cav1.4, and the Cav1.4 mutant Y1595E strongly impairs binding to CaBP4. We conclude that CaBP4 forms a collapsed structure around the IQ motif in Cav1.4 that we suggest may promote channel activation by disrupting an interaction between IQ and the inhibitor of Ca²⁺-dependent inactivation domain.     

Characterization of novel calmodulin binding domains within IQ motifs of IQGAP1

IQ motif-containing GTPase-activating protein 1 (IQGAP1), which is a well-known calmodulin (CaM) binding protein, is involved in a wide range of cellular processes including cell proliferation, tumorigenesis, adhesion, and migration. Interaction of IQGAP1 with CaM is important for its cellular functions. Although each IQ domain of IQGAP1 for CaM binding has been characterized in a Ca²⁺-dependent or -independent manner, it was not clear which IQ motifs are physiologically relevant for CaM binding in the cells. In this study, we performed immunoprecipitation using 3xFLAGhCaM in mammalian cell lines to characterize the domains of IQGAP1 that are key for CaM binding under physiological conditions. Interestingly, using this method, we identified two novel domains, IQ(2.7–3) and IQ(3.5–4.4), within IQGAP1 that were involved in Ca²⁺-independent or -dependent CaM binding, respectively. Mutant analysis clearly showed that the hydrophobic regions within IQ(2.7–3) were mainly involved in apoCaM binding, while the basic amino acids and hydrophobic region of IQ(3.5–4.4) were required for Ca²⁺/CaM binding. Finally, we showed that IQ(2.7–3) was the main apoCaM binding domain and both IQ(2.7–3) and IQ(3.5–4.4) were required for Ca²⁺/CaM binding within IQ(1-2-3-4). Thus, we identified and characterized novel direct CaM binding motifs essential for IQGAP1. This finding indicates that IQGAP1 plays a dynamic role via direct interactions with CaM in a Ca²⁺-dependent or -independent manner.     

Calmodulin Mediates the Ca-Dependent Regulation of Cx44 Gap Junctions

We have shown previously that the Ca²⁺-dependent inhibition of lens epithelial cell-to-cell communication is mediated in part by the direct association of calmodulin (CaM) with connexin43 (Cx43), the major connexin in these cells. We now show that elevation of [Ca²⁺]_i in HeLa cells transfected with the lens fiber cell gap junction protein sheep Cx44 also results in the inhibition of cell-to-cell dye transfer. A peptide comprising the putative CaM binding domain (aa 129-150) of the intracellular loop region of this connexin exhibited a high affinity, stoichiometric interaction with Ca²⁺-CaM. NMR studies indicate that the binding of Cx44 peptide to CaM reflects a classical embracing mode of interaction. The interaction is an exothermic event that is both enthalpically and entropically driven in which electrostatic interactions play an important role. The binding of the Cx44 peptide to CaM increases the CaM intradomain cooperativity and enhances the Ca²⁺-binding affinities of the C-domain of CaM more than twofold by slowing the rate of Ca²⁺ release from the complex. Our data suggest a common mechanism by which the Ca²⁺-dependent inhibition of the α-class of gap junction proteins is mediated by the direct association of an intracellular loop region of these proteins with Ca²⁺-CaM.     

Intra- and Interdomain Effects Due to Mutation of Calcium-binding Sites in Calmodulin

The IQ-motif protein PEP-19, binds to the C-domain of calmodulin (CaM) with significantly different k_on and k_off rates in the presence and absence of Ca²⁺, which could play a role in defining the levels of free CaM during Ca²⁺ transients. The initial goal of the current study was to determine whether Ca²⁺ binding to sites III or IV in the C-domain of CaM was responsible for affecting the kinetics of binding PEP-19. EF-hand Ca²⁺-binding sites were selectively inactivated by the common strategy of changing Asp to Ala at the X-coordination position. Although Ca²⁺ binding to both sites III and IV appeared necessary for native-like interactions with PEP-19, the data also indicated that the mutations caused undesirable structural alterations as evidenced by significant changes in amide chemical shifts for apoCaM. Mutations in the C-domain also affected chemical shifts in the unmodified N-domain, and altered the Ca²⁺ binding properties of the N-domain. Conversion of Asp⁹³ to Ala caused the greatest structural perturbations, possibly due to the loss of stabilizing hydrogen bonds between the side chain of Asp⁹³ and backbone amides in apo loop III. Thus, although these mutations inhibit binding of Ca²⁺, the mutated CaM may not be able to support potentially important native-like activity of the apoprotein. This should be taken into account when designing CaM mutants for expression in cell culture.     

Calcium-dependent Association of Calmodulin with the Rubella Virus Nonstructural Protease Domain

The rubella virus (RUBV) nonstructural (NS) protease domain, a Ca²⁺- and Zn²⁺-binding papain-like cysteine protease domain within the nonstructural replicase polyprotein precursor, is responsible for the self-cleavage of the precursor into two mature products, P150 and P90, that compose the replication complex that mediates viral RNA replication; the NS protease resides at the C terminus of P150. Here we report the Ca²⁺-dependent, stoichiometric association of calmodulin (CaM) with the RUBV NS protease. Co-immunoprecipitation and pulldown assays coupled with site-directed mutagenesis demonstrated that both the P150 protein and a 110-residue minidomain within NS protease interacted directly with Ca²⁺/CaM. The specific interaction was mapped to a putative CaM-binding domain. A 32-mer peptide (residues 1152–1183, denoted as RUBpep) containing the putative CaM-binding domain was used to investigate the association of RUBV NS protease with CaM or its N- and C-terminal subdomains. We found that RUBpep bound to Ca²⁺/CaM with a dissociation constant of 100–300 nm. The C-terminal subdomain of CaM preferentially bound to RUBpep with an affinity 12.5-fold stronger than the N-terminal subdomain. Fluorescence, circular dichroism and NMR spectroscopic studies revealed a “wrapping around” mode of interaction between RUBpep and Ca²⁺/CaM with substantially more helical structure in RUBpep and a global structural change in CaM upon complex formation. Using a site-directed mutagenesis approach, we further demonstrated that association of CaM with the CaM-binding domain in the RUBV NS protease was necessary for NS protease activity and infectivity.