Calmodulin Binding Site Research Articles

N-terminal and C-terminal domains of calmodulin mediate FADD and TRADD interaction.

FADD (Fas–associated death domain) and TRADD (Tumor Necrosis Factor Receptor 1-associated death domain) proteins are important regulators of cell fate in mammalian cells. They are both involved in death receptors mediated signaling pathways and have been linked to the Toll-like receptor family and innate immunity. Here we identify and characterize by database search analysis, mutagenesis and calmodulin (CaM) pull-down assays a calcium-dependent CaM binding site in the α-helices 1–2 of TRADD death domain. We also show that oxidation of CaM methionines drastically reduces CaM affinity for FADD and TRADD suggesting that oxidation might regulate CaM-FADD and CaM-TRADD interactions. Finally, using Met-to-Leu CaM mutants and binding assays we show that both the N- and C-terminal domains of CaM are important for binding.

Human Adenosine A2A Receptor Binds Calmodulin with High Affinity in a Calcium-Dependent Manner

Understanding how ligands bind to G-protein-coupled receptors and how binding changes receptor structure to affect signaling is critical for developing a complete picture of the signal transduction process. The adenosine A2A receptor (A2AR) is a particularly interesting example, as it has an exceptionally long intracellular carboxyl terminus, which is predicted to be mainly disordered. Experimental data on the structure of the A2AR C-terminus is lacking, because published structures of A2AR do not include the C-terminus. Calmodulin has been reported to bind to the A2AR C-terminus, with a possible binding site on helix 8, next to the membrane. The biological meaning of the interaction as well as its calcium dependence, thermodynamic parameters, and organization of the proteins in the complex are unclear. Here, we characterized the structure of the A2AR C-terminus and the A2AR C-terminus-calmodulin complex using different biophysical methods, including native gel and analytical gel filtration, isothermal titration calorimetry, NMR spectroscopy, and small-angle X-ray scattering. We found that the C-terminus is disordered and flexible, and it binds with high affinity (Kd = 98 nM) to calmodulin without major conformational changes in the domain. Calmodulin binds to helix 8 of the A2AR in a calcium-dependent manner that can displace binding of A2AR to lipid vesicles. We also predicted and classified putative calmodulin-binding sites in a larger group of G-protein-coupled receptors.

Uncoupling PIP2-calmodulin regulation of Kv7.2 channels by an assembly destabilizing epileptogenic mutation.

We show that the combination of an intracellular bi-partite calmodulin (CaM)-binding site and a distant assembly region affect how an ion channel is regulated by a membrane lipid. Our data reveal that regulation by phosphatidylinositol(4,5)bisphosphate (PIP2) and stabilization of assembled Kv7.2 subunits by intracellular coiled-coil regions far from the membrane are coupled molecular processes. Live-cell fluorescence energy transfer measurements and direct binding studies indicate that remote coiled-coil formation creates conditions for different CaM interaction modes, each conferring different PIP2 dependency to Kv7.2 channels. Disruption of coiled-coil formation by epilepsy-causing mutation decreases apparent CaM-binding affinity and interrupts CaM influence on PIP2 sensitivity.

EPR with Rigidly Bound Spin Labels used to Probe the Interaction of Calmodulin with the Ryanodine Receptor

We have used site-directed spin labeling and EPR spectroscopy to probe the structural dynamics of calmodulin bound to a ryanodine receptor target peptide. Calmodulin binds to a conserved 30-amino acid sequence on the sarcoplasmic reticulum calcium release channel (ryanodine receptor, RyR) and directly modulate its activity in muscle contraction. In order to characterize the structural details of this crucial interaction, a bifunctional spin label was used to rigidly label di-cysteine mutant calmodulin at positions 34 and 38. Solid-phase peptide synthesis was used to create a peptide corresponding to the calmodulin binding site on the ryanodine receptor channel (RyRp, residues 3614-3643 in RyR1) with a TOAC spin label rigidly coupled to the backbone. The intermolecular distance distribution between the two spin probes was determined by EPR, using both continuous wave dipolar broadening and dipolar electron-electron resonance (DEER). The center of the distance distribution was in good agreement with the crystal structure of the complex (18 A), but the width of the distribution was ∼ 1 nm, comparable to that observed previously within calmodulin itself. Thus calmodulin retains significant inter-lobe domain motion, even after RyRp binding. These results are consistent with a model in which calmodulin binds RyRp, bringing its two terminal lobes close together but preventing them from physical contact with each other, allowing flexibility in each of the lobe.

Calmodulin Binding to a Novel Site in the AB Module of Kv7.2 Subunits Regulates Surface Expression

Calmodulin (CaM) binding to the AB module underlie multiple mechanisms governing the function of Kv7.2 subunits, which are the main component of the non-inactivating K+ M-current, a key controller of neuronal excitability. Simultaneous binding to helix A and B is crucial for trafficking to the plasma membrane. We have identified a novel CaM-binding site located downstream of helix A, reminiscent of the TW motif of CaM-gated SK2 K+ channels, with W360 as pivotal for surface expression. In the context of the known CaM-binding modes and previous studies, our data suggest a model for traffic control that involves alternative CaM docking to either helix B, the novel TW motif, or the IQ-site of helix A.

Ca(2+) binding protein S100A1 competes with calmodulin and PIP2 for binding site on the C-terminus of the TPRV1 receptor.

Transient receptor potential vanilloid 1 ion channel (TRPV1) belongs to the TRP family of ion channels. These channels play a role in many important biological processes such as thermosensation and pain transduction. The TRPV1 channel was reported to be also involved in nociception. Ca(2+) ions are described to participate in the regulation of TRP channels through the interaction with Ca(2+)-binding proteins, such as calmodulin or S100A1. Calmodulin is involved in the Ca(2+)-dependent regulation of TRPV1 via its binding to the TRPV1 C-terminal region. However, the role of the Ca(2+)-binding protein S100A1 in the process of TRP channel regulation remains elusive. Here we characterized a region on the TRPV1 C-terminus responsible for the interaction with S100A1 using biochemical and biophysical tools. We found that this region overlaps with previously identified calmodulin and PIP2 binding sites and that S100A1 competes with calmodulin and PIP2 for this binding site. We identified several positively charged residues within this region, which have crucial impact on S100A1 binding, and we show that the reported S100A1-TRPV1 interaction is calcium-dependent. Taken together, our data suggest a mechanism for the mutual regulation of PIP2 and the Ca(2+)-binding proteins S100A1 and calmodulin to TRPV1.

Negatively Charged Amino Acids Near and in Transient Receptor Potential (TRP) Domain of TRPM4 Channel Are One Determinant of Its Ca2+ Sensitivity

Transient receptor potential (TRP) channel melastatin subfamily member 4 (TRPM4) is a broadly expressed nonselective monovalent cation channel. TRPM4 is activated by membrane depolarization and intracellular Ca(2+), which is essential for the activation. The Ca(2+) sensitivity is known to be regulated by calmodulin and membrane phosphoinositides, such as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Although these regulators must play important roles in controlling TRPM4 activity, mutation analyses of the calmodulin-binding sites have suggested that Ca(2+) binds to TRPM4 directly. However, the intrinsic binding sites in TRPM4 remain to be elucidated. Here, by using patch clamp and molecular biological techniques, we show that there are at least two functionally different divalent cation-binding sites, and the negatively charged amino acids near and in the TRP domain in the C-terminal tail of TRPM4 (Asp-1049 and Glu-1062 of rat TRPM4) are required for maintaining the normal Ca(2+) sensitivity of one of the binding sites. Applications of Co(2+), Mn(2+), or Ni(2+) to the cytosolic side potentiated TRPM4 currents, increased the Ca(2+) sensitivity, but were unable to evoke TRPM4 currents without Ca(2+). Mutations of the acidic amino acids near and in the TRP domain, which are conserved in TRPM2, TRPM5, and TRPM8, deteriorated the Ca(2+) sensitivity in the presence of Co(2+) or PI(4,5)P2 but hardly affected the sensitivity to Co(2+) and PI(4,5)P2. These results suggest a novel role of the TRP domain in TRPM4 as a site responsible for maintaining the normal Ca(2+) sensitivity. These findings provide more insights into the molecular mechanisms of the regulation of TRPM4 by Ca(2+).

Calcium Binding to Calmodulin by Molecular Dynamics with Effective Polarization.

Calcium represents a key biological signaling ion with the EF-hand loops being its most prevalent binding motif in proteins. We show using molecular dynamics simulations with umbrella sampling that including electronic polarization effects via ionic charge rescaling dramatically improves agreements with experiment in terms of the strength of calcium binding and structures of the calmodulin binding sites. The present study thus opens way to accurate calculations of interactions of calcium and other computationally difficult high-charge-density ions in biological contexts.

Evaluation of the calmodulin-SOX9 interaction by "magnetic fishing" coupled to mass spectrometry.

Disruption of calmodulin (CaM)-based protein interactions has been touted as a potential means for modulating several disease pathways. Among these is SOX9, which is a DNA binding protein that is involved in chrondrocyte differentiation and regulation of the hormones that control sexual development. In this work, we employed a "magnetic fishing"/mass spectrometry assay in conjunction with intrinsic fluorescence to examine the interaction of CaM with the CaM-binding domain of SOX9 (SOX-CAL), and to assess the modulation of this interaction by known anti-CaM compounds. Our data show that there is a high affinity interaction between CaM and SOX-CAL (27±9 nM), and that SOX-CAL bound to the same location as the well-known CaM antagonist melittin; unexpectedly, we also found that addition of CaM-binding small molecules initially produced increased SOX-CAL binding, indicative of binding to both the well-known high-affinity CaM binding site and a second, lower-affinity binding site.

Identification and expression analyses of calmodulin-binding transcription activator genes in soybean

Environmental stresses are major hurdles for the production of soybean (Glycine max), a major food crop that is able to acquire N and P nutrients through symbiotic association with rhizobia and arbuscular mycorhizal fungi. The calmodulin-binding transcription activators (CAMTA) belong to a transcription factor family that plays critical roles in plant responses to both abiotic and biotic stresses such as drought, cold and attacks from pathogens and insects. Our current knowledge about CAMTA genes in soybean is scarce, but is of critical significance to the improvement of stress tolerance and production of soybean, a worldwide major protein-rich food crop. In this study, we first searched for all the CAMTA homologous genes in the whole genome of soybean. We then surveyed the distribution of stress-related cis-regulatory elements in the −1.5 kb promoter regions of all GmCAMTA genes. Next, we analyzed the expression patterns of all these GmCAMTAs in root and leaf tissues and studied how they respond to various stress treatments by quantitative RT-PCR. Fifteen genes in the soybean genome were identified to encode CAMTA proteins, and their gene structures and protein sequences were highly similar to that in Arabidopsis. Like their counterparts in other plants and animals, all GmCAMTAs contain a CG-1 DNA-binding domain in the N terminal region, followed by a TIG domain, ankyrin repeats, IQ motifs and a calmodulin binding site. All GmCAMTA genes were found to be expressed in root and leaf tissues. The expression of all GmCAMTAs was induced by salinity. Most of the GmCAMTA genes were induced by several stresses and hormone signals including dehydration, cold, H2O2, abscisic acid, salicylic acid, and methyl jasmonate. Only four GmCAMTAs (GmCAMTA5, 6, 7 and 10) were found to be repressed by H2O2 treatment. Consistent with their responsiveness to all these stimuli, many stress-related cis-elements were found in the promoter regions of all the GmCAMTA genes. These results indicate that GmCAMTA genes are responsive to various stress and hormone signals, and likewise, GmCAMTAs could act as critical components in regulating soybean tolerances to various environmental stresses.

Ginseng gintonin activates the human cardiac delayed rectifier K+ channel: involvement of Ca2+/calmodulin binding sites.

Gintonin, a novel, ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand, elicits [Ca2+]i transients in neuronal and non-neuronal cells via pertussis toxin-sensitive and pertussis toxin-insensitive G proteins. The slowly activating delayed rectifier K+ (IKs) channel is a cardiac K+ channel composed of KCNQ1 and KCNE1 subunits. The C terminus of the KCNQ1 channel protein has two calmodulin-binding sites that are involved in regulating IKs channels. In this study, we investigated the molecular mechanisms of gintonin-mediated activation of human IKs channel activity by expressing human IKs channels in Xenopus oocytes. We found that gintonin enhances IKs channel currents in concentration- and voltage-dependent manners. The EC50 for the IKs channel was 0.05 ± 0.01 μg/ml. Gintonin-mediated activation of the IKs channels was blocked by an LPA1/3 receptor antagonist, an active phospholipase C inhibitor, an IP3 receptor antagonist, and the calcium chelator BAPTA. Gintonin-mediated activation of both the IKs channel was also blocked by the calmodulin (CaM) blocker calmidazolium. Mutations in the KCNQ1 [Ca2+]i/CaM-binding IQ motif sites (S373P, W392R, or R539W)blocked the action of gintonin on IKs channel. However, gintonin had no effect on hERG K+ channel activity. These results show that gintonin-mediated enhancement of IKs channel currents is achieved through binding of the [Ca2+]i/CaM complex to the C terminus of KCNQ1 subunit.

Structural Mapping of Divergent Regions in the Type 1 Ryanodine Receptor Using Fluorescence Resonance Energy Transfer

Ryanodine receptors (RyRs) release Ca(2+) to initiate striated muscle contraction. Three highly divergent regions (DRs) in the RyR protein sequence (DR1, DR2, and DR3) may confer isoform-specific functional properties to the RyRs. We used cell-based fluorescence resonance energy transfer (FRET) measurements to localize these DRs to the cryoelectron microscopic (cryo-EM) map of the skeletal muscle RyR isoform (RyR1). FRET donors were targeted to RyR1 using five different FKBP12.6 variants labeled with Alexa Fluor 488. FRET was then measured to the FRET acceptors, Cy3NTA or Cy5NTA, targeted to decahistidine tags introduced within the DRs. DR2 and DR3 were localized to separate positions within the "clamp" region of the RyR1 cryo-EM map, which is presumed to interface with Cav1.1. DR1 was localized to the "handle" region, near the regulatory calmodulin-binding site on the RyR. These localizations provide insights into the roles of DRs in RyR allosteric regulation during excitation contraction coupling.

Drosophilapericentrin requires interaction with calmodulin for its function at centrosomes and neuronal basal bodies but not at sperm basal bodies

Pericentrin is a critical centrosomal protein required for organizing pericentriolar material (PCM) in mitosis. Mutations in pericentrin cause the human genetic disorder Majewski/microcephalic osteodysplastic primordial dwarfism type II, making a detailed understanding of its regulation extremely important. Germaine to pericentrin's function in organizing PCM is its ability to localize to the centrosome through the conserved C-terminal PACT domain. Here we use Drosophila pericentrin-like-protein (PLP) to understand how the PACT domain is regulated. We show that the interaction of PLP with calmodulin (CaM) at two highly conserved CaM-binding sites in the PACT domain controls the proper targeting of PLP to the centrosome. Disrupting the PLP-CaM interaction with single point mutations renders PLP inefficient in localizing to centrioles in cultured S2 cells and Drosophila neuroblasts. Although levels of PCM are unaffected, it is highly disorganized. We also demonstrate that basal body formation in the male testes and the production of functional sperm does not rely on the PLP-CaM interaction, whereas production of functional mechanosensory neurons does.

Calmodulation meta-analysis: predicting calmodulin binding via canonical motif clustering.

The calcium-binding protein calmodulin (CaM) directly binds to membrane transport proteins to modulate their function in response to changes in intracellular calcium concentrations. Because CaM recognizes and binds to a wide variety of target sequences, identifying CaM-binding sites is difficult, requiring intensive sequence gazing and extensive biochemical analysis. Here, we describe a straightforward computational script that rapidly identifies canonical CaM-binding motifs within an amino acid sequence. Analysis of the target sequences from high resolution CaM-peptide structures using this script revealed that CaM often binds to sequences that have multiple overlapping canonical CaM-binding motifs. The addition of a positive charge discriminator to this meta-analysis resulted in a tool that identifies potential CaM-binding domains within a given sequence. To allow users to search for CaM-binding motifs within a protein of interest, perform the meta-analysis, and then compare the results to target peptide-CaM structures deposited in the Protein Data Bank, we created a website and online database. The availability of these tools and analyses will facilitate the design of CaM-related studies of ion channels and membrane transport proteins.

Structural insights into the mechanism of calmodulin binding to death receptors.

The death receptors Fas, p75(NTR) and DR6 are key components of extrinsically activated apoptosis. Characterization of how they interact with the adaptors is crucial in order to unravel the signalling mechanisms. However, the exact conformation that their intracellular death domain adopts upon binding downstream partners remains unclear. One model suggests that it adopts a typical compact fold, whilst a second model proposed an open conformation. Calmodulin (CaM), a major calcium sensor, has previously been reported to be one of the Fas adaptors that modulate apoptosis. This work reports that CaM also binds directly to the death domains of p75(NTR) and DR6, indicating that it serves as a common modulator of the death receptors. Two crystal structures of CaM in complexes with the corresponding binding regions of Fas and p75(NTR) are also reported. Interestingly, the precise CaM-binding sites were mapped to different regions: helix 1 in Fas and helix 5 in p75(NTR) and DR6. A novel 1-11 motif for CaM binding was observed in p75(NTR). Modelling the complexes of CaM with full-length receptors reveals that the opening of the death domains would be essential in order to expose their binding sites for CaM. These results may facilitate understanding of the diverse functional repertoire of death receptors and CaM and provide further insights necessary for the design of potential therapeutic peptide agents.

Structural insights into calmodulin/Munc13 interaction.

Munc13 proteins are essential presynaptic regulators that mediate synaptic vesicle priming and play a role in the regulation of neuronal short-term synaptic plasticity. All four Munc13 isoforms share a common domain structure, including a calmodulin (CaM) binding site in their otherwise divergent N-termini. Here, we summarize recent results on the investigation of the CaM/Munc13 interaction. By combining chemical cross-linking, photoaffinity labeling, and mass spectrometry, we showed that all neuronal Munc13 isoforms exhibit similar CaM binding modes. Moreover, we demonstrated that the 1-5-8-26 CaM binding motif discovered in Munc13-1 cannot be induced in the classical CaM target skMLCK, indicating unique features of the Munc13 CaM binding motif.

Capping of the N-terminus of PSD-95 by calmodulin triggers its postsynaptic release

Postsynaptic density protein-95 (PSD-95) is a central element of the postsynaptic architecture of glutamatergic synapses. PSD-95 mediates postsynaptic localization of AMPA receptors and NMDA receptors and plays an important role in synaptic plasticity. PSD-95 is released from postsynaptic membranes in response to Ca(2+) influx via NMDA receptors. Here, we show that Ca(2+)/calmodulin (CaM) binds at the N-terminus of PSD-95. Our NMR structure reveals that both lobes of CaM collapse onto a helical structure of PSD-95 formed at its N-terminus (residues 1-16). This N-terminal capping of PSD-95 by CaM blocks palmitoylation of C3 and C5, which is required for postsynaptic PSD-95 targeting and the binding of CDKL5, a kinase important for synapse stability. CaM forms extensive hydrophobic contacts with Y12 of PSD-95. The PSD-95 mutant Y12E strongly impairs binding to CaM and Ca(2+)-induced release of PSD-95 from the postsynaptic membrane in dendritic spines. Our data indicate that CaM binding to PSD-95 serves to block palmitoylation of PSD-95, which in turn promotes Ca(2+)-induced dissociation of PSD-95 from the postsynaptic membrane.

Striatin: a novel regulator of cardiomyocyte calcium homeostasis and contraction (864.3)

Although cardiac arrhythmias are still a major health threat for human, there is paucity of information on the molecular mechanisms involved in this pathology. Striatin (STRN) is a multivalent protein with dynamic domains including caveolin (Cav), Calmodulin (CaM), and Protein Phosphatase 2A (PP2A) binding sites, all of which are important regulatory units of the L‐type Calcium Channels (LCC) in cardiomyocytes. Previous studies indicate that mutations/deletions of the striatin gene correlate with the development of cardiac arrhythmias in animal models. We aimed to investigate whether STRN regulates calcium homeostasis and cardiomyocyte contraction rate. Data indicate that STRN is highly expressed in atrial myocardium when compared to the ventricles. Overexpression of STRN elevated the contraction rate (~ 3 fold) and resulted in sustained levels of KCl‐induced increases in intracellular calcium in neonatal rat cardiomyocytes. Conversely, silencing (>80%) the STRN gene using shRNA was associated with decreased contraction rate (~50%) of cardiomyocytes. Interestingly, pharmacological challenge with isoproterenol (β‐adrenergic agonist) failed to improve the contraction rate of cardiomyocytes with low levels of striatin (shRNA‐STRN). Biochemical analysis (CaM pull down) revealed that the interaction between CaM/PP2A/Cav‐3/LCC was increased in shRNA‐STRN cardiomyocytes and reduced when overexpressing STRN. Collectively our data indicate that striatin is a novel regulator of cardiomyocyte calcium homeostasis and contraction rate by modulating the CaM/PP2A/Cav‐3/LCC complex and influencing the activity of the LCC in the myocardium.Grant Funding Source: King Faisal Specialist Hospital and Research Center

A new mechanism in the binding between Homer3 EVH1 domain and inositol 1,4,5 trisphosphate receptor suppressor domain.

The suppressor domain of inositol 1,4,5 trisphosphate receptor (IP3R) has critical roles in regulating the calcium channel by interacting with many binding partners. The residue 49-53 (PPKKF) of the suppressor domain was suggested to be a canonical Homer EVH1 domain binding site and is also the first a part of calmodulin (CaM) binding site. As CaM-binding of the suppressor domain has been shown to involve large-scale conformational changes, we studied the binding characteristics of the Homer EVH1-suppressor domain with NMR spectroscopy and biochemical pull-down assays for mutants. Our data show that the suppressor domain employs the PPKKF motif in a similar but subtly different way compared to previously characterized interactions, and that the suppressor domain does not undergo large-scale conformational changes. Chemical shift assignments of the Homer3 EVH1 domain found that a new set of residues, located at the opposite side of the previously reported binding site, is also involved in binding, which was confirmed by mutant binding assays. Further analysis suggests that F40 in the new binding sites may have a critical role as a conformational lock-switch in Homer-target binding. The proposed mechanism is implicated in the signaling network involving calcium channels.

Kinase activity and calmodulin binding are essential for growth signaling by the phytosulfokine receptor PSKR1

The cell growth-promoting peptide phytosulfokine (PSK) is perceived by leucine-rich repeat (LRR) receptor kinases. To elucidate PSK receptor function we analyzed PSKR1 kinase activity and binding to Ca(2+) sensors and evaluated the contribution of these activities to growth control in planta. Ectopically expressed PSKR1 was capable of auto- and transphosphorylation. Replacement of a conserved lysine within the ATP-binding region by a glutamate resulted in the inhibition of auto- and transphosphorylation kinase activities. Expression of the kinase-inactive PSKR1(K762E) receptor in the pskr null background did not restore root or shoot growth. Instead, the mutant phenotype was enhanced suggesting that the inactive receptor protein exerts growth-inhibitory activity. Bioinformatic analysis predicted a putative calmodulin (CaM)-binding site within PSKR1 kinase subdomain VIa. Bimolecular fluorescence complementation analysis demonstrated that PSKR1 binds to all isoforms of CaM, more weakly to the CaM-like protein CML8 but apparently not to CML9. Mutation of a conserved tryptophan (W831S) within the predicted CaM-binding site strongly reduced CaM binding. Expression of PSKR1(W831S) in the pskr null background resulted in growth inhibition that was similar to that of the kinase-inactive receptor. We conclude that PSK signaling requires Ca(2+) /CaM binding and kinase activity of PSKR1 in planta. We further propose that the inactivated kinase interferes with other growth-promoting signaling pathway(s).