Calmodulin-binding Region Research Articles

Identification of Additional Calmodulin Binding Regions in Connexin43

Gap junction, allowing the intercellular transmission of molecules through its specialized cell membrane channels, plays a major role in intercellular calcium signaling between adjacent cells. Connexin43 (Cx43), the most ubiquitous connexin, belongs to α family of gap junction proteins expressed in heart where are essential for normal heart development. Calmodulin (CaM) has been implicated in mediating the Ca2+-dependent regulation of gap junctions. We have reported CaM binding site in the second half of intracellular loop of Cx43. In this study, two additional CaM binding regions in cytosol loop and C-termini of Cx43 have been identified by biophysical studies. Our results indicate that in the presence of Ca2+, synthesized Cx peptide fragments encompassing predicted CaM binding regions are able to bind with high affinity to CaM using NMR spectroscopy. Our results elucidate the molecular level of regulation of Cx43 by multiple CaM targeting regions and provide insight into molecular basis of gap junction gating mechanism.

Reduced CaM/FLIP binding by a single point mutation in c-FLIPL modulates Fas-mediated apoptosis and decreases tumorigenesis

We have previously demonstrated that calmodulin (CaM) binds directly to c-FLIPL in a Ca2+-dependent manner. Deletion of the CaM-binding region (amino acid 197–213) results in reduced CaM binding, and increased Fas-mediated apoptosis and decreased tumorigenesis of cholangiocarcinoma cells. The present studies were designed to identify the precise amino acids between 197 and 213 that are responsible for CaM/FLIP binding, and their roles in mediating the anti-apoptotic function of c-FLIPL. Sequence analysis of the CaM-binding region at 197–213 predicted three unique positively charged residues at 204, 207 and 209, which might be responsible for the CaM/FLIP binding. A point mutation at H204 of c-FLIPL was found to markedly reduce CaM binding, whereas point mutation at R207 or K209 did not affect c-FLIPL binding to CaM. Decreased CaM/FLIP binding was confirmed in cholangiocarcinoma cells overexpressing the H204 c-FLIPL mutant. Reduced CaM binding by the H204 mutant resulted in increased sensitivity to Fas-mediated apoptosis and inhibited tumor growth in mice compared with wild-type c-FLIPL. Death-inducing signaling complex (DISC) analysis showed that the reduced CaM binding to H204 mutant resulted in less c-FLIPL recruited into the DISC. Concurrently, increased caspase 8 was recruited to the DISC, which resulted in increased cleavage and activation of caspase 8, activation of downstream caspase 3 and increased apoptosis. Therefore, these results demonstrate that the H204 residue is responsible for c-FLIPL binding to CaM, which mediates the anti-apoptotic function of c-FLIPL, most likely through affecting recruitment of caspase 8 into the DISC and thus caspase 8 activation. These studies further characterized CaM/FLIP interaction and its function in regulating Fas-mediated apoptosis and tumorigenesis, which may provide new therapeutic targets for cancer therapy.

Structural Basis for Activation of Calcineurin by Calmodulin

The highly conserved phosphatase calcineurin (CaN) plays vital roles in numerous processes including T-cell activation, development and function of the central nervous system, and cardiac growth. It is activated by the calcium sensor calmodulin (CaM). CaM binds to a regulatory domain (RD) within CaN, causing a conformational change that displaces an autoinhibitory domain (AID) from the active site, resulting in activation of the phosphatase. This is the same general mechanism by which CaM activates CaM-dependent protein kinases. Previously published data have hinted that the RD of CaN is intrinsically disordered. In this work, we demonstrate that the RD is unstructured and that it folds upon binding CaM, ousting the AID from the catalytic site. The RD is 95 residues long, with the AID attached to its C-terminal end and the 24-residue CaM binding region toward the N-terminal end. This is unlike the CaM-dependent protein kinases that have CaM binding sites and AIDs immediately adjacent in sequence. Our data demonstrate that not only does the CaM binding region folds but also an ∼25- to 30-residue region between it and the AID folds, resulting in over half of the RD adopting α-helical structure. This appears to be the first observation of CaM inducing folding of this scale outside of its binding site on a target protein.

Structure of Human Na+/H+ Exchanger NHE1 Regulatory Region in Complex with Calmodulin and Ca2+

The ubiquitous mammalian Na(+)/H(+) exchanger NHE1 has critical functions in regulating intracellular pH, salt concentration, and cellular volume. The regulatory C-terminal domain of NHE1 is linked to the ion-translocating N-terminal membrane domain and acts as a scaffold for signaling complexes. A major interaction partner is calmodulin (CaM), which binds to two neighboring regions of NHE1 in a strongly Ca(2+)-dependent manner. Upon CaM binding, NHE1 is activated by a shift in sensitivity toward alkaline intracellular pH. Here we report the 2.23 Å crystal structure of the NHE1 CaM binding region (NHE1(CaMBR)) in complex with CaM and Ca(2+). The C- and N-lobes of CaM bind the first and second helix of NHE1(CaMBR), respectively. Both the NHE1 helices and the Ca(2+)-bound CaM are elongated, as confirmed by small angle x-ray scattering analysis. Our x-ray structure sheds new light on the molecular mechanisms of the phosphorylation-dependent regulation of NHE1 and enables us to propose a model of how Ca(2+) regulates NHE1 activity.

KCNE4 Juxtamembrane Region Is Required for Interaction with Calmodulin and for Functional Suppression of KCNQ1

Voltage-gated potassium (K(V)) channels, such as KCNQ1 (K(V)7.1), are modulated by accessory subunits and regulated by intracellular second messengers. Accessory subunits belonging to the KCNE family exert diverse functional effects on KCNQ1, have been implicated in the pathogenesis of various genetic disorders of heart rhythm, and contribute to transducing intracellular signaling events into changes in K(V) channel activity. We investigated the interactions between calmodulin (CaM), the ubiquitous Ca(2+)-transducing protein that binds and confers Ca(2+) sensitivity to the biophysical properties of KCNQ1, and KCNE4. These studies were motivated by the observed similarities between the suppression of KCNQ1 function by pharmacological disruption of KCNQ1-CaM interactions and the effects of KCNE4 co-expression on the channel. We determined that KCNE4, but not KCNE1, can biochemically interact with CaM and that this interaction is Ca(2+)-dependent and requires a tetraleucine motif in the juxtamembrane region of the KCNE4 C terminus. Furthermore, disruption of the KCNE4-CaM interaction either by mutagenesis of the tetraleucine motif or by acute Ca(2+) chelation impairs the ability of KCNE4 to inhibit KCNQ1. Our findings have potential relevance to KCNQ1 regulation both by KCNE accessory subunits and by an important intracellular signaling molecule.

Structural Studies of Calmodulin Activation of Estrogen Receptor Alpha

The goal is to determine the mechanism of calcium-dependent activation of estrogen receptor alpha (ERa) by calmodulin (CaM) and to ascertain how oxidative stress and oxidative modifications mediate the interactions between CaM, ERa and antiestrogens. Systemic endocrine/antiestrogen therapy is among the most common treatments for estrogen-dependent breast cancers. ERa is the primary target for antiestrogen therapies, and antiestrogen drugs such as tamoxifen and its metabolites (4-hydroxytamoxifen, endoxifen) bind tightly to ERa and inhibit its ability to activate transcription. Recently, it was demonstrated that CaM is an obligate ERa activator. Interestingly, antiestrogens that bind tightly to ERa also bind tightly to CaM. It has been suggested that therapeutic benefit of antiestrogens for estrogen-dependent breast cancers may derive partially from CaM antagonism. Towards our goal of understanding how CaM activates ERa, we have localized the CaM binding region of ERa and initiated structural studies to determine the structure of the complex of CaM with the ERa CaM binding region. Using NMR and SAXS we find that CaM bound to ERa is somewhat extended structurally compared to high affinity CaM complexes. Circular dichroism and fluorescence studies indicate high affinity between CaM and the CaM binding domain of ERa and that upon binding to CaM the CaM binding region of ERa adopts only partial helical character. Binding of CaM to hydrophobic antiestrogens (tamoxifen, 4-hydroxytamoxifen, endoxifen, raloxifene) is eliminated when methionine residues in CaM are oxidized. However, oxidation does not eliminate binding to ERa. Control experiments with CaM mutants (leucine for methionine) indicate methionine residues are not essential for antiestrogen binding. The results are important for understanding CaM activation of ERa and the link between oxidative stress and antiestrogen resistance development. (Supported by the DOD, the Georgia Cancer Coalition, Bruker AXS and the University of Georgia).

Regulation of Intercellular Regulation of Signaling via Gap Junction

Gap junction, allowing the intercellular transmission of molecules through its specialized cell membrane channels, plays a major role in intercellular calcium signaling between adjacent cells. Connexin 43, 44 and 50 (Cx43, Cx44 and Cx50) are members of the α family of gap junction proteins expressed in heart and the lens of the eye where are essential for normal heart and lens development. We have identified calmodulin (CaM) binding sites in Cx43, Cx44 and Cx50. Biophysical results indicate that in the presence of calcium, synthesized Cx peptide fragments encompassing predicted CaM binding regions are able to bind with high affinity to CaM using NMR spectroscopy and fluorescence method with dansylated CaM. In addition, electrophysiological studies demonstrate that elevated intracellular Ca2+ concentration inhibits Cx50 gap junctions with the 95% decline in junctional conductance (gj). Inclusion of either CaM inhibitor or the designed CaM binding Cx peptide is able to prevent this calcium-dependent inhibition of Cx50 gap junctions. Further, we have predicted several calcium binding pockets using our developed MUG algorithm. We have further revealed the site-specific calcium binding capability of the predicted calcium binding site in gap junctions by grafting approach. Our results elucidate the molecular mechanism of regulation of gap junction by both calcium and calcium dependent CaM actions and provide insight into molecular basis of human diseases.

Inhibition of endothelial- and neuronal-type, but not inducible-type, nitric oxide synthase by the oxidized cholesterol metabolite secosterol aldehyde: Implications for vascular and neurodegenerative diseases

The cholesterol ozonolysis products secosterol-A and its aldolization product secosterol-B were recently detected in human atherosclerotic tissues and brain specimens, and have been postulated to play pivotal roles in the pathogenesis of atherosclerosis and neurodegenerative diseases. We examined several oxidized cholesterol metabolites including secosterol-A, secosterol-B, 25-hydroxycholesterol, 5β,6β-epoxycholesterol and 7-ketocholesterol for their effects on the activities of three nitric oxide synthases. In contrast to other oxidized metabolites, secosterol-A was found to be a potent inhibitor against the neuronal- and endothelial-type, but not the inducible-type nitric oxide synthase, with IC50 values of 22 ± 1 and 50 ± 5 µM, respectively. The calmodulin-binding regions of the neuronal- and endothelial-nitric oxide synthases contain lysine residues which are not present in the inducible-type nitric oxide synthase. Secosterol-A modifies proteins through the formation of a Schiff base with the lysine epsilon-amino group. It is possible that secosterol-A modifies lysine residues of constitutive nitric oxide synthases, leading to the inhibition of enzymatic activities. As nitric oxide is a critical signaling molecule in vascular function and in long-term potentiation, its reduced production through inhibition of constitutive nitric oxide synthases by secosterol-A may contribute to the development of atherosclerosis and memory impairment in particular neurodegenerative diseases.

Transmembrane and Trans-subunit Regulation of Ectodomain Shedding of Platelet Glycoprotein Ibα

Ectodomain shedding of transmembrane proteins may be regulated by their cytoplasmic domains. To date, the effecting cytoplasmic domain and the shed extracellular domain have been in the same polypeptide. In this study, shedding of GPIbα, the ligand-binding subunit of the platelet GPIb-IX complex and a marker for platelet senescence and storage lesion, was assessed in Chinese hamster ovary cells with/without functional GPIbα sheddase ADAM17. Mutagenesis of the GPIb-IX complex, which contains GPIbα, GPIbβ, and GPIX subunits, revealed that the intracellular membrane-proximal calmodulin-binding region of GPIbβ is critical for ADAM17-dependent shedding of GPIbα induced by the calmodulin inhibitor, W7. Perturbing the interaction between GPIbα and GPIbβ subunits further lessened the restraint of GPIbβ on GPIbα shedding. However, contrary to the widely accepted model of calmodulin regulation of ectodomain shedding, the R152E/L153E mutation in the GPIbβ cytoplasmic domain disrupted calmodulin binding to GPIbβ but had little effect on GPIbα shedding. Analysis of induction of GPIbα shedding by membrane-permeable GPIbβ-derived peptides implicated the association of GPIbβ with an unidentified intracellular protein in mediating regulation of GPIbα shedding. Overall, these results provide evidence for a novel trans-subunit mechanism for regulating ectodomain shedding.

Identification and characterization of PRG-1 as a neuronal calmodulin-binding protein

Intracellular Ca2+-dependent cellular responses are often mediated by the ubiquitous protein CaM (calmodulin), which, upon binding Ca2+, can interact with and alter the function of numerous proteins. In the present study, using a newly developed functional proteomic screen of rat brain extracts, we identified PRG-1 (plasticity-related gene-1) as a novel CaM target. A CaM-overlay and an immunoprecipitation assay revealed that PRG-1 is capable of binding the Ca2+/CaM complex in vitro and in transfected cells. Surface plasmon resonance and zero-length cross-linking showed that the C-terminal putative cytoplasmic domain (residues 466-766) of PRG-1 binds equimolar amounts of CaM in a Ca2+-dependent manner, with a relatively high affinity (a Kd value for Ca2+/CaM of 8 nM). Various PRG-1 mutants indicated that the Ca2+/CaM-binding region of PRG-1 is located between residues Ser554 and Gln588, and that Trp559 and Ile578 potentially anchor PRG-1 to CaM. This is supported by pronounced changes in the fluorescence emission spectrum of Trp559 in the PRG-1 peptide (residues 554-588) upon binding to Ca2+/CaM, showing the stoichiometrical binding of the PRG-1 peptide with Ca2+/CaM. Immunoblot analyses revealed that the PRG-1 protein is abundant in brain, but is weakly expressed in the testes. Immunohistochemical analysis revealed that PRG-1 is highly expressed in forebrain structures and in the cerebellar cortex. Furthermore, PRG-1 localizes at the postsynaptic compartment of excitatory synapses and dendritic shafts of hippocampal neurons, but is not present in presynaptic nerve terminals. The combined observations suggest that PRG-1 may be involved in postsynaptic functions regulated by intracellular Ca2+-signalling.

S100A1: a physiological modulator of RYR1, Ca2+ release, and contractility in skeletal muscle. Focus on “S100A1 promotes action potential-initiated calcium release flux and force production in skeletal muscle”

SKELETAL MUSCLES adjust their contractile output over an exceptionally wide dynamic range, as needed to produce movements ranging from sitting and standing, to athletics and dance. At the single fiber level, the force generated by skeletal muscles is determined, ultimately, by the formation of cycling cross bridges between actin and myosin. This interaction requires Ca 2 and is governed by the simple relationship: force is proportional to intracellular ionized Ca 2 . Cross-bridge formation is inhibited in resting muscle when Ca 2 is sequestered within the sarcoplasmic reticulum (SR); it is activated when stored Ca 2 is released into the cytosol, allowing actin-myosin interactions to proceed. Contraction stops as Ca 2 is actively resequestered by the Ca 2 -ATPase of the SR (SERCA1). Thus Ca 2 is the central messenger in muscle activation, and the amplitude and time course of the intracellular Ca 2 change is the major determinant of contractile output. The key mechanisms that activate Ca 2 release from the SR are now well defined. This sequence, termed excitation-contraction coupling, begins when an excitatory input from the motor nerve triggers a propagating action potential on the muscle membrane. The action potential rapidly depolarizes the entire sarcolemma, including transverse tubules, which invaginate from the surface membrane and form specialized junctions (triads) with the SR along much of their length. The triad junction serves as a platform for assembling sarcolemmal calcium

Integrated Protein Array Screening and High Throughput Validation of 70 Novel Neural Calmodulin-binding Proteins

Calmodulin is an essential regulator of intracellular processes in response to extracellular stimuli mediated by a rise in Ca(2+) ion concentration. To profile protein-protein interactions of calmodulin in human brain, we probed a high content human protein array with fluorophore-labeled calmodulin in the presence of Ca(2+). This protein array contains 37,200 redundant proteins, incorporating over 10,000 unique human neural proteins from a human brain cDNA library. We designed a screen to find high affinity (K(D) < or = 1 microm) binding partners of calmodulin and identified 76 human proteins from all intracellular compartments of which 72 are novel. We measured the binding kinetics of 74 targets with calmodulin using a high throughput surface plasmon resonance assay. Most of the novel calmodulin-target complexes identified have low dissociation rates (k(off) < or = 10(-3) s(-1)) and high affinity (K(D) </= 1 mum), consistent with the design of the screen. Many of the identified proteins are known to assemble in neural tissue, forming assemblies such as the spectrin scaffold and the postsynaptic density. We developed a microarray of the identified target proteins with which we can characterize the biochemistry of calmodulin for all targets in parallel. Four novel targets were verified in neural cells by co-immunoprecipitation, and four were selected for exploration of the calmodulin-binding regions. Using synthetic peptides and isothermal titration calorimetry, calmodulin binding motifs were identified in the potassium voltage-gated channel Kv6.1 (residues 474-493), calmodulin kinase-like vesicle-associated protein (residues 302-316), EF-hand domain family member A2 (residues 202-216), and phosphatidylinositol-4-phosphate 5-kinase, type I, gamma (residues 400-415).

Probing Myosin-VI Processivity using Artificial Lever Arms

The lever arm of myosin VI has an unusual composition in which two different calmodulin-binding domains, a globular three-helix bundle, and an extended single alpha-helix domain may all contribute structural roles. We wish to understand which properties of this lever arm are important for mediating intra-head communication, preventing dissociation from the actin filament, and determining the distribution of stride sizes in a processively stepping dimer. We have replaced parts of the myosin VI lever arm with alpha-actinin repeats in a series of chimeric constructs. In dimers with artifical levers arms, we have found that processivity is surprisingly robust to dramatic changes in the properties of the lever arm, even when the stride size is altered (Liao et al., JMB 2009). In new chimeric constructs, we show that limited processivity is possible even in the absence of both calmodulin-binding regions. We examine the importance of intra-head coordination for processive motion in myosin VI by comparing the predictions of simple kinetic models to measurements of run length distributions for chimeric myosins and for control contructs.

Localization of Potential Calmodulin Binding Sequences onto the Three Dimensional Structure of the Cardiac Ryanodine Receptor Reveals A Binding Pocket for Calmodulin

Calmodulin (CaM), a 16 kDa ubiquitous calcium-sensing protein, is known to bind tightly to the cardiac calcium release channel/ryanodine receptor (RyR2) at low and high Ca2+ concentrations, and modulate the function of the channel. CaM binding studies using RyR fragments or synthetic peptides have revealed that multiple regions in the RyR's primary sequence may be involved in CaM binding. However, the locations of these potential CaM binding regions in the three dimensional structure of RyRs have yet to be determined.

Cam Interaction and Binding Mode Study with Peptide from Intracellular Loop of Cx50

Connexin 50 (Cx50) is a member of the α family of gap junction proteins expressed in the lens of the eye where it has been shown to be essential for normal lens development. We have identified calmodulin (CaM) binding sites in the intracellular loop near to the 3rd transmembrane region of Connexin43 (Cx43) and Connexin44 (Cx44) which belong to the same α connexin family as Cx50. Sequence alignment of the candidate CaM binding regions of Cx43 and Cx44, with Cx50 identified a region encompassing residues 141-166 of Cx50 with a high predicted affinity for CaM. A peptide Cx50141-166 was synthesized to study the interaction of CaM with this domain of Cx50. Biophysical results indicate that in the presence of Ca2+, Cx50141-166 binds with high affinity (0.14 μM) to CaM, as monitored by NMR spectroscopy and measured by the fluorescence intensity change of IAEDANS that was covalently attached to the C-terminal Cys of CaM. Electrophysiological data support the hypothesis that elevated intracellular Ca2+ concentration inhibits Cx50 gap junctions because omission of Ca2+ from the 1 μM ionomycin bath saline prevented the 95% decline in junctional conductance (gj) observed in the presence of 1.8 mM CaCl2. This is likely CaM-mediated, because inclusion of a CaM inhibitor also prevented this Ca2+-dependent inhibition of Cx50 gap junctions. The involvement of the Cx50 CaM binding domain in this Ca2+/CaM-dependent regulation was further demonstrated by inclusion ofthe Cx50141-166 peptide in both whole cell patch pipettes, which effectively prevented the usual decrease in Cx50 gj. These results demonstrate that the binding of Ca2+ -CaM to the intracellular loop of Cx50 is critical for mediating the Ca2+-dependent inhibition of Cx50 gap junctions in the lens of the eye.

SDSL-EPR Study of a C-terminal Segment of Myelin Basic Protein in a Myelin Mimetic Environment

Myelin basic protein (MBP) is a peripheral membrane protein and a major component of human central nervous system myelin. It is a multifunctional, highly positively charged, intrinsically disordered protein that undergoes extensive post-translational modifications. Its multifunctionality includes among others, binding of cytoskeletal proteins to a membrane surface and polymerization and bundling of cytoskeletal proteins. These interactions are modulated by interaction of MBP with Ca2+-calmodulin. We report here the use of site-directed spin labeling and continuous wave power saturation electron paramagnetic resonance spectroscopy (SDSL-EPR) to examine the conformation of segment A141-L154 of a recombinant 18.5 kDa murine isoform of MBP in a reconstituted membrane environment. This segment overlaps the primary calmodulin binding region (T147-D158) of MBP and is predicted, on the basis of helical wheel constructs, to form an amphipathic alpha helix. Solution NMR investigations also showed this region to exist as a transient alpha helix in 30% TFE. Our measurements using SDSL-EPR reveal that this region forms an extended helix with a period of ∼3.8 residues per turn and with a tilt angle of ∼4.3 degrees with respect to the plane of the lipid bilayer. The N-terminal end of the helix appears to be buried more deeply in the membrane bilayer than the C-terminal end. The C-terminal region of the segment bears two Lys residues whose side chains interact with the lipid head groups causing this end to be more exposed. The accessibility of the C-terminal region of A141-L154, which forms a part of the primary calmodulin binding segment, could facilitate interaction of this region with cytosolic proteins viz., calmodulin and modulate cytoskeletal binding to the oligodendrocyte plasma membrane.(Supported by CIHR, NSERC and MS Society of Canada)

Regulation of Interdomain Interactions by Calmodulin in Inducible Nitric-oxide Synthase

Nitric-oxide synthases (NOSs) catalyze the conversion of l-arginine to nitric oxide and citrulline. There are three NOS isozymes, each with a different physiological role: neuronal NOS, endothelial NOS, and inducible NOS (iNOS). NOSs consist of an N-terminal oxygenase domain and a C-terminal reductase domain, linked by a calmodulin (CaM)-binding region. CaM is required for NO production, but unlike other NOS isozymes, iNOS binds CaM independently of the exogenous Ca(2+) concentration. We have co-expressed CaM and the FMN domain of human iNOS, which includes the CaM-binding region. The Ca(2+)-bound protein complex (CaCaMxFMN) forms an air-stable semiquinone when reduced with NADPH and reduces cytochrome c when reconstituted with the iNOS FAD/NADPH domain. We have solved the crystal structure of the CaCaMxFMN complex in four different conformations, each with a different relative orientation, between the FMN domain and the bound CaM. The CaM-binding region together with bound CaM forms a hinge, pivots on the conserved Arg(536), and regulates electron transfer from FAD to FMN and from FMN to heme by adjusting the relative orientation and distance among the three cofactors. In addition, the relative orientations of the N- and C-terminal lobes of CaM are also different among the four conformations, suggesting that the flexibility between the two halves of CaM also contributes to the fine tuning of the orientation/distance between the redox centers. The data demonstrate a possible mode for precise control of electron transfer by altering the distance and orientation of redox centers in a protein displaying domain movement.

Calmodulin Is a Functional Regulator of Cav1.4 L-type Ca2+ Channels

Cav1.4 channels are unique among the high voltage-activated Ca2+ channel family because they completely lack Ca2+-dependent inactivation and display very slow voltage-dependent inactivation. Both properties are of crucial importance in ribbon synapses of retinal photoreceptors and bipolar cells, where sustained Ca2+ influx through Cav1.4 channels is required to couple slow graded changes of the membrane potential with tonic glutamate release. Loss of Cav1.4 function causes severe impairment of retinal circuitry function and has been linked to night blindness in humans and mice. Recently, an inhibitory domain (ICDI: inhibitor of Ca2+-dependent inactivation) in the C-terminal tail of Cav1.4 has been discovered that eliminates Ca2+-dependent inactivation by binding to upstream regulatory motifs within the proximal C terminus. The mechanism underlying the action of ICDI is unclear. It was proposed that ICDI competitively displaces the Ca2+ sensor calmodulin. Alternatively, the ICDI domain and calmodulin may bind to different portions of the C terminus and act independently of each other. In the present study, we used fluorescence resonance energy transfer experiments with genetically engineered cyan fluorescent protein variants to address this issue. Our data indicate that calmodulin is preassociated with the C terminus of Cav1.4 but may be tethered in a different steric orientation as compared with other Ca2+ channels. We also find that calmodulin is important for Cav1.4 function because it increases current density and slows down voltage-dependent inactivation. Our data show that the ICDI domain selectively abolishes Ca2+-dependent inactivation, whereas it does not interfere with other calmodulin effects.

Ral GTPase interacts with the N-terminal in addition to the C-terminal region of PLC-δ1

Previously, we have shown that RalA, a calmodulin (CaM)-binding protein, binds to the C2 region in the C-terminal of PLC-δ1, and increases its enzymatic activity. Since PLC-δ1 contains a CaM-like region in its N-terminus, we have investigated if RalA can also bind to the N-terminus of PLC-δ1. Therefore, we created a GST-PLC-δ1 construct consisting of the first 294 amino acids of PLC-δ1 (GST-PLC-δ1 1–294). In vitro binding experiments confirmed that PLC-δ1 1–294 was capable of binding directly to RalA. W-7 coupled to polyacrylamide beads bound pure PLC-δ1, demonstrating that PLC-δ1 contains a CaM-like region. Competition assays with W-7, peptides representing RalA and the newly identified RalB CaM-binding regions, or the IQ peptide from PLC-δ1 were able to inhibit RalA binding to PLC-δ1 1–294. This study demonstrates that there are two binding sites for RalA in PLC-δ1 and provides further insight into the role of Ral GTPase in the regulation of PLC-δ1 function.

Structural interactions of calmodulin with peptide from calmodulin binding regions of various enzymes

Calmodulin (CaM) is an important calcium (Ca2+) sensor in the cell and is able to activate over three hundred proteins to initiate cellular activities. CaM is a highly dynamic protein with a flexible central linker region joining the N‐terminal and C‐terminal domains. In each domain are two Ca2+ binding sites that cause structural changes to CaM when Ca2+ binds. These changes include the CaM central linker elongating and methionine rich hydrophobic patches being exposed, allowing for CaM to bind to the large population of target proteins. The structural interaction of CaM to short peptides from the CaM binding region of various target enzymes were investigated, including the nitric oxide synthase (NOS) enzyme.