Tryptophan fluorescence of calmodulin binding domain peptides interacting with calmodulin containing unnatural methionine analogues

The interactions between the abundant methionine residues ofthe calcium regulatory protein calmodulin (CaM) and severalof its binding targets were probed using fluorescence spectroscopy.Tryptophan steady-state fluorescence from peptides encompassingthe CaM-binding domains of the target proteins myosin lightchain kinase (MLCK), cyclic nucleotide phosphodiesterase (PDE)and caldesmon site A and B (CaD A, CaD B), and the model peptidemelittin showed Ca²⁺-dependent blue-shifts in their maximumemission wavelength when complexed with wild-type CaM. Blue-shiftswere also observed for complexes in which the CaM methionineresidues were replaced by selenomethionine, norleucine and ethionine,and when a quadruple methionine to leucine C-terminal mutantof CaM was studied. Quenching of the tryptophan fluorescenceintensity was observed with selenomethionine, but not with norleucineor ethionine substituted protein. Fluorescence quenching studieswith added potassium iodide (KI) demonstrate that the non-nativeproteins limit the solvent accessibility of the Trp in the MLCKpeptide to levels close to that of the wild-type CaM–MLCKinteraction. Our results show that the methionine residues fromCaM are highly sensitive to the target peptide in question,confirming the importance of their role in binding interactions.In addition, we provide evidence that the nature of bindingin the CaM–CaD B complex is unique compared with the othercomplexes studied, as the Trp residue of this peptide remainspartially solvent exposed upon binding to CaM.     

Calmodulin as Ca2+-Dependent Interactor of FTO Dioxygenase

FTO is an N⁶-methyladenosine demethylase removing methyl groups from nucleic acids. Several studies indicate the creation of FTO complexes with other proteins. Here, we looked for regulatory proteins recognizing parts of the FTO dioxygenase region. In the Calmodulin (CaM) Target Database, we found the FTO C-domain potentially binding CaM, and we proved this finding experimentally. The interaction was Ca²⁺-dependent but independent on FTO phosphorylation. We found that FTO–CaM interaction essentially influences calcium-binding loops in CaM, indicating the presence of two peptide populations—exchanging as CaM alone and differently, suggesting that only one part of CaM interacts with FTO, and the other one reminds free. The modeling of FTO–CaM interaction showed its stable structure when the half of the CaM molecule saturated with Ca²⁺ interacts with the FTO C-domain, whereas the other part is disconnected. The presented data indicate calmodulin as a new FTO interactor and support engagement of the FTO protein in calcium signaling pathways.     

Amino Acid-Mediated Intracellular Ca2+ Rise Modulates mTORC1 by Regulating the TSC2-Rheb Axis through Ca2+/Calmodulin

Mechanistic target of rapamycin complex 1 (mTORC1) is a master growth regulator by controlling protein synthesis and autophagy in response to environmental cues. Amino acids, especially leucine and arginine, are known to be important activators of mTORC1 and to promote lysosomal translocation of mTORC1, where mTORC1 is thought to make contact with its activator Rheb GTPase. Although amino acids are believed to exclusively regulate lysosomal translocation of mTORC1 by Rag GTPases, how amino acids increase mTORC1 activity besides regulation of mTORC1 subcellular localization remains largely unclear. Here we report that amino acids also converge on regulation of the TSC2-Rheb GTPase axis via Ca²⁺/calmodulin (CaM). We showed that the amino acid-mediated increase of intracellular Ca²⁺ is important for mTORC1 activation and thereby contributes to the promotion of nascent protein synthesis. We found that Ca²⁺/CaM interacted with TSC2 at its GTPase activating protein (GAP) domain and that a CaM inhibitor reduced binding of CaM with TSC2. The inhibitory effect of a CaM inhibitor on mTORC1 activity was prevented by loss of TSC2 or by an active mutant of Rheb GTPase, suggesting that a CaM inhibitor acts through the TSC2-Rheb axis to inhibit mTORC1 activity. Taken together, in response to amino acids, Ca²⁺/CaM-mediated regulation of the TSC2-Rheb axis contributes to proper mTORC1 activation, in addition to the well-known lysosomal translocation of mTORC1 by Rag GTPases.     

Multilevel Changes in Protein Dynamics upon Complex Formation of the Calcium‐Loaded S100A4 with a Nonmuscle Myosin IIA Tail Fragment

Dysregulation of Ca²⁺‐binding S100 proteins plays important role in various diseases. The asymmetric complex of Ca²⁺‐bound S100A4 with nonmuscle myosin IIA has high stability and highly increased Ca²⁺ affinity. Here we investigated the possible causes of this allosteric effect by NMR spectroscopy. Chemical shift‐based secondary‐structure analysis did not show substantial changes for the complex. Backbone dynamics revealed slow‐timescale local motions in the H1 helices of homodimeric S100A4; these were less pronounced in the complex form and might be accompanied by an increase in dimer stability. Different mobilities in the Ca²⁺‐coordinating EF‐hand sites indicate that they communicate by an allosteric mechanism operating through changes in protein dynamics; this must be responsible for the elevated Ca²⁺ affinity. These multilevel changes in protein dynamics as conformational adaptation allow S100A4 fine‐tuning of its protein–protein interactions inside the cell during Ca²⁺ signaling.     

In Search of Monocot Phosphodiesterases: Identification of a Calmodulin Stimulated Phosphodiesterase from Brachypodium distachyon

In plants, rapid and reversible biological responses to environmental cues may require complex cellular reprograming. This is enabled by signaling molecules such as the cyclic nucleotide monophosphates (cNMPs) cAMP and cGMP, as well as Ca²⁺. While the roles and synthesis of cAMP and cGMP in plants are increasingly well-characterized, the “off signal” afforded by cNMP-degrading enzymes, the phosphodiesterases (PDEs), is, however, poorly understood, particularly so in monocots. Here, we identified a candidate PDE from the monocot Brachypodium distachyon (BDPDE1) and showed that it can hydrolyze cNMPs to 5′NMPs but with a preference for cAMP over cGMP in vitro. Notably, the PDE activity was significantly enhanced by Ca²⁺ only in the presence of calmodulin (CaM), which interacts with BDPDE1, most likely at a predicted CaM-binding site. Finally, based on our biochemical, mutagenesis and structural analyses, we constructed a comprehensive amino acid consensus sequence extracted from the catalytic centers of annotated and/or experimentally validated PDEs across species to enable a broad application of this search motif for the identification of similar active sites in eukaryotes and prokaryotes.     

Multifaceted Roles of ALG-2 in Ca2+-Regulated Membrane Trafficking

ALG-2 (gene name: PDCD6) is a penta-EF-hand Ca²⁺-binding protein and interacts with a variety of proteins in a Ca²⁺-dependent fashion. ALG-2 recognizes different types of identified motifs in Pro-rich regions by using different hydrophobic pockets, but other unknown modes of binding are also used for non-Pro-rich proteins. Most ALG-2-interacting proteins associate directly or indirectly with the plasma membrane or organelle membranes involving the endosomal sorting complex required for transport (ESCRT) system, coat protein complex II (COPII)-dependent ER-to-Golgi vesicular transport, and signal transduction from membrane receptors to downstream players. Binding of ALG-2 to targets may induce conformational change of the proteins. The ALG-2 dimer may also function as a Ca²⁺-dependent adaptor to bridge different partners and connect the subnetwork of interacting proteins.     

Calmodulin Interactions with Voltage-Gated Sodium Channels

Calmodulin (CaM) is a small protein that acts as a ubiquitous signal transducer and regulates neuronal plasticity, muscle contraction, and immune response. It interacts with ion channels and plays regulatory roles in cellular electrophysiology. CaM modulates the voltage-gated sodium channel gating process, alters sodium current density, and regulates sodium channel protein trafficking and expression. Many mutations in the CaM-binding IQ domain give rise to diseases including epilepsy, autism, and arrhythmias by interfering with CaM interaction with the channel. In the present review, we discuss CaM interactions with the voltage-gated sodium channel and modulators involved in CaM regulation, as well as summarize CaM-binding IQ domain mutations associated with human diseases in the voltage-gated sodium channel family.     

Cobalt Regulates Activation of Camk2α in Neurons by Influencing Fructose 1,6-Bisphosphatase 2 Quaternary Structure and Subcellular Localization

Fructose 1,6-bisphosphatase 2 (Fbp2) is a gluconeogenic enzyme and multifunctional protein modulating mitochondrial function and synaptic plasticity via protein-protein interactions. The ability of Fbp2 to bind to its cellular partners depends on a quaternary arrangement of the protein. NAD⁺ and AMP stabilize an inactive T-state of Fbp2 and thus, affect these interactions. However, more subtle structural changes evoked by the binding of catalytic cations may also change the affinity of Fbp2 to its cellular partners. In this report, we demonstrate that Fbp2 interacts with Co²⁺, a cation which in excessive concentrations, causes pathologies of the central nervous system and which has been shown to provoke the octal-like events in hippocampal slices. We describe for the first time the kinetics of Fbp2 in the presence of Co²⁺, and we provide a line of evidence that Co²⁺ blocks the AMP-induced transition of Fbp2 to the canonical T-state triggering instead of a new, non-canonical T-state. In such a state, Fbp2 is still partially active and may interact with its binding partners e.g., Ca²⁺/calmodulin-dependent protein kinase 2α (Camk2α). The Fbp2-Camk2α complex seems to be restricted to mitochondria membrane and it facilitates the Camk2α autoactivation and thus, synaptic plasticity.     

Characterization and Purification of Kinase Activities against Arabidopsis COP9 Signalosome Subunit 7

The COP9 signalosome (CSN) is a multisubunit protein complex that intersects the ubiquitin (Ub)–proteasome pathway at several junctions. The CSN associates with several protein kinases that target key regulatory proteins that themselves are targets for Ub-dependent degradation, and several subunits themselves are phosphoproteins. We previously reported that Arabidopsis CSN7 is a phosphoprotein. To identify the kinases responsible for CSN7 phosphorylation, we biochemically purified CSN7-kinsae activities from cauliflower through a four-step purification procedure that resulted in a ca. 300-fold enrichment. One of these activities is Ca²⁺ dependent, while the second is not. Peptide fingerprint analysis of the purified proteins identified three putative CSN7 kinases.     

A calmodulin-target peptide hybrid molecule with unique calcium-binding properties

This paper describes the production and properties of a hybridprotein comprising the full length of the Xenopus laevis cabnodulin(CaM) sequence, followed, through a gh/cylgh/dne linker, bythe 26-residue CaM-binding region of myosin light-chain kinase(M13). This hybrid molecule appears to have high thermal stability(T_m > 75°C in the presence of Ca²⁺) as well as unusualCa²⁺-binding properties: (i) a wide-range biphasic Ca²⁺-bindingresponse (extending over pCa 4.8-7.4) and (ii) a high apparentbinding constant (pCa_50% = 6.3, a 10-fold increase from thatof wild-type CaM). NMR and CD data indicate that the CaM-M13hybrid molecule exists in equilibrium in an approximate 1:1ratio between two major conformations, one of which is similarto the compact globular structure of the CaM-M13 complex [M.Daira,G.M.Clore, A.M.Gronenborn, G.Zhu, C.B.Klee and A.Bax (1992)Science, 256, 632-638] and the other to the dumbbell-like structureof the wild type CaM [Y.S.Babu, C.E.Bugg and W.J.Cook (1988)J. Mol. Biol., 204, 191-204]. The biphasic Ca²⁺-binding curvecan be interpreted using a linear combination of two Hill bindingcurves with significantly different dissociation constants (2x 10^-6 M and 8 x 10^-6 M), which can be attributed to the twoconformations in equilibrium. The present study has opened anavenue to engineer proteins with higher Ca²⁺-binding affinitiesusing the known CaM structures as a template Received April 23, 1993; revised August 5, 1993; accepted August 25, 1993.     

Structural Insights into Retinal Guanylate Cyclase Activator Proteins (GCAPs)

Retinal guanylate cyclases (RetGCs) promote the Ca²⁺-dependent synthesis of cGMP that coordinates the recovery phase of visual phototransduction in retinal rods and cones. The Ca²⁺-sensitive activation of RetGCs is controlled by a family of photoreceptor Ca²⁺ binding proteins known as guanylate cyclase activator proteins (GCAPs). The Mg²⁺-bound/Ca²⁺-free GCAPs bind to RetGCs and activate cGMP synthesis (cyclase activity) at low cytosolic Ca²⁺ levels in light-activated photoreceptors. By contrast, Ca²⁺-bound GCAPs bind to RetGCs and inactivate cyclase activity at high cytosolic Ca²⁺ levels found in dark-adapted photoreceptors. Mutations in both RetGCs and GCAPs that disrupt the Ca²⁺-dependent cyclase activity are genetically linked to various retinal diseases known as cone-rod dystrophies. In this review, I will provide an overview of the known atomic-level structures of various GCAP proteins to understand how protein dimerization and Ca²⁺-dependent conformational changes in GCAPs control the cyclase activity of RetGCs. This review will also summarize recent structural studies on a GCAP homolog from zebrafish (GCAP5) that binds to Fe²⁺ and may serve as a Fe²⁺ sensor in photoreceptors. The GCAP structures reveal an exposed hydrophobic surface that controls both GCAP1 dimerization and RetGC binding. This exposed site could be targeted by therapeutics designed to inhibit the GCAP1 disease mutants, which may serve to mitigate the onset of retinal cone-rod dystrophies.     

PGRS Domain of Rv0297 of Mycobacterium tuberculosis Functions in A Calcium Dependent Manner

Mycobacterium tuberculosis (M.tb), the pathogen causing tuberculosis, is a major threat to human health worldwide. Nearly 10% of M.tb genome encodes for a unique family of PE/PPE/PGRS proteins present exclusively in the genus Mycobacterium. The functions of most of these proteins are yet unexplored. The PGRS domains of these proteins have been hypothesized to consist of Ca²⁺ binding motifs that help these intrinsically disordered proteins to modulate the host cellular responses. Ca²⁺ is an important secondary messenger that is involved in the pathogenesis of tuberculosis in diverse ways. This study presents the calcium-dependent function of the PGRS domain of Rv0297 (PE_PGRS5) in M.tb virulence and pathogenesis. Tandem repeat search revealed the presence of repetitive Ca²⁺ binding motifs in the PGRS domain of the Rv0297 protein (Rv0297PGRS). Molecular Dynamics simulations and fluorescence spectroscopy revealed Ca²⁺ dependent stabilization of the Rv0297PGRS protein. Calcium stabilized Rv0297PGRS enhances the interaction of Rv0297PGRS with surface localized Toll like receptor 4 (TLR4) of macrophages. The Ca²⁺ stabilized binding of Rv0297PGRS with the surface receptor of macrophages enhances its downstream consequences in terms of Nitric Oxide (NO) production and cytokine release. Thus, this study points to hitherto unidentified roles of calcium-modulated PE_PGRS proteins in the virulence of M.tb. Understanding the pathogenic potential of Ca²⁺ dependent PE_PGRS proteins can aid in targeting these proteins for therapeutic interventions.     

Calmodulin and Its Binding Proteins in Parkinson’s Disease

Parkinson’s disease (PD) is a neurodegenerative disorder that manifests with rest tremor, muscle rigidity and movement disturbances. At the microscopic level it is characterized by formation of specific intraneuronal inclusions, called Lewy bodies (LBs), and by a progressive loss of dopaminergic neurons in the striatum and substantia nigra. All living cells, among them neurons, rely on Ca²⁺ as a universal carrier of extracellular and intracellular signals that can initiate and control various cellular processes. Disturbances in Ca²⁺ homeostasis and dysfunction of Ca²⁺ signaling pathways may have serious consequences on cells and even result in cell death. Dopaminergic neurons are particularly sensitive to any changes in intracellular Ca²⁺ level. The best known and studied Ca²⁺ sensor in eukaryotic cells is calmodulin. Calmodulin binds Ca²⁺ with high affinity and regulates the activity of a plethora of proteins. In the brain, calmodulin and its binding proteins play a crucial role in regulation of the activity of synaptic proteins and in the maintenance of neuronal plasticity. Thus, any changes in activity of these proteins might be linked to the development and progression of neurodegenerative disorders including PD. This review aims to summarize published results regarding the role of calmodulin and its binding proteins in pathology and pathogenesis of PD.     

Crosstalk among Calcium ATPases: PMCA,SERCA and SPCA in Mental Diseases

Calcium in mammalian neurons is essential for developmental processes, neurotransmitter release, apoptosis, and signal transduction. Incorrectly processed Ca²⁺ signal is well-known to trigger a cascade of events leading to altered response to variety of stimuli and persistent accumulation of pathological changes at the molecular level. To counterbalance potentially detrimental consequences of Ca²⁺, neurons are equipped with sophisticated mechanisms that function to keep its concentration in a tightly regulated range. Calcium pumps belonging to the P-type family of ATPases: plasma membrane Ca²⁺-ATPase (PMCA), sarco/endoplasmic Ca²⁺-ATPase (SERCA) and secretory pathway Ca²⁺-ATPase (SPCA) are considered efficient line of defense against abnormal Ca²⁺ rises. However, their role is not limited only to Ca²⁺ transport, as they present tissue-specific functionality and unique sensitive to the regulation by the main calcium signal decoding protein—calmodulin (CaM). Based on the available literature, in this review we analyze the contribution of these three types of Ca²⁺-ATPases to neuropathology, with a special emphasis on mental diseases.     

Integrated Self-Assembly of the Mms6 Magnetosome Protein to Form an Iron-Responsive Structure

A common feature of biomineralization proteins is their self-assembly to produce a surface consistent in size with the inorganic crystals that they produce. Mms6, a small protein of 60 amino acids from Magnetospirillum magneticum strain AMB-1 that promotes the in vitro growth of superparamagnetic magnetite nanocrystals, assembles in aqueous solution to form spherical micelles that could be visualized by TEM and AFM. The results reported here are consistent with the view that the N and C-terminal domains interact with each other within one polypeptide chain and across protein units in the assembly. From studies to determine the amino acid residues important for self-assembly, we identified the unique GL repeat in the N-terminal domain with additional contributions from amino acids in other positions, throughout the molecule. Analysis by CD spectroscopy identified a structural change in the iron-binding C-terminal domain in the presence of Fe³⁺. A change in the intrinsic fluorescence of tryptophan in the N-terminal domain showed that this structural change is transmitted through the protein. Thus, self-assembly of Mms6 involves an interlaced structure of intra- and inter-molecular interactions that results in a coordinated structural change in the protein assembly with iron binding.     

Decreased Interactions between Calmodulin and a Mutant Huntingtin Model Might Reduce the Cytotoxic Level of Intracellular Ca2+: A Molecular Dynamics Study

Mutant huntingtin (m-HTT) proteins and calmodulin (CaM) co-localize in the cerebral cortex with significant effects on the intracellular calcium levels by altering the specific calcium-mediated signals. Furthermore, the mutant huntingtin proteins show great affinity for CaM that can lead to a further stabilization of the mutant huntingtin aggregates. In this context, the present study focuses on describing the interactions between CaM and two huntingtin mutants from a biophysical point of view, by using classical Molecular Dynamics techniques. The huntingtin models consist of a wild-type structure, one mutant with 45 glutamine residues and the second mutant with nine additional key-point mutations from glutamine residues into proline residues (9P(EM) model). Our docking scores and binding free energy calculations show higher binding affinities of all HTT models for the C-lobe end of the CaM protein. In terms of dynamic evolution, the 9P(EM) model triggered great structural changes into the CaM protein’s structure and shows the highest fluctuation rates due to its structural transitions at the helical level from α-helices to turns and random coils. Moreover, our proposed 9P(EM) model suggests much lower interaction energies when compared to the 45Qs-HTT mutant model, this finding being in good agreement with the 9P(EM)’s antagonistic effect hypothesis on highly toxic protein–protein interactions.     

Sorcin Activates the Brain PMCA and Blocks the Inhibitory Effects of Molecular Markers of Alzheimer’s Disease on the Pump Activity

Since dysregulation of intracellular calcium (Ca²⁺) levels is a common occurrence in neurodegenerative diseases, including Alzheimer’s disease (AD), the study of proteins that can correct neuronal Ca²⁺ dysregulation is of great interest. In previous work, we have shown that plasma membrane Ca²⁺-ATPase (PMCA), a high-affinity Ca²⁺ pump, is functionally impaired in AD and is inhibited by amyloid-β peptide (Aβ) and tau, two key components of pathological AD hallmarks. On the other hand, sorcin is a Ca²⁺-binding protein highly expressed in the brain, although its mechanism of action is far from being clear. Sorcin has been shown to interact with the intracellular sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA), and other modulators of intracellular Ca²⁺ signaling, such as the ryanodine receptor or presenilin 2, which is closely associated with AD. The present work focuses on sorcin in search of new regulators of PMCA and antagonists of Aβ and tau toxicity. Results show sorcin as an activator of PMCA, which also prevents the inhibitory effects of Aβ and tau on the pump, and counteracts the neurotoxicity of Aβ and tau by interacting with them.     

E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein

The SPL2 protein is an E3 ubiquitin ligase of unknown function. It is one of only three types of E3 ligases found in the outer membrane of plant chloroplasts. In this study, we show that the cytosolic fragment of SPL2 binds lanthanide ions, as evidenced by fluorescence measurements and circular dichroism spectroscopy. We also report that SPL2 undergoes conformational changes upon binding of both Ca²⁺ and La³⁺, as evidenced by its partial unfolding. However, these structural rearrangements do not interfere with SPL2 enzymatic activity, as the protein retains its ability to auto-ubiquitinate in vitro. The possible applications of lanthanide-based probes to identify protein interactions in vivo are also discussed. Taken together, the results of this study reveal that the SPL2 protein contains a lanthanide-binding site, showing for the first time that at least some E3 ubiquitin ligases are also capable of binding lanthanide ions.     

Rutaecarpine Increases Nitric Oxide Synthesis via eNOS Phosphorylation by TRPV1-Dependent CaMKII and CaMKKβ/AMPK Signaling Pathway in Human Endothelial Cells

Rutaecarpine (RUT) is a bioactive alkaloid isolated from the fruit of Evodia rutaecarpa that exerts a cellular protective effect. However, its protective effects on endothelial cells and its mechanism of action are still unclear. In this study, we demonstrated the effects of RUT on nitric oxide (NO) synthesis via endothelial nitric oxide synthase (eNOS) phosphorylation in endothelial cells and the underlying molecular mechanisms. RUT treatment promoted NO generation by increasing eNOS phosphorylation. Additionally, RUT induced an increase in intracellular Ca²⁺ concentration and phosphorylation of Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ), AMP-activated protein kinase (AMPK), and Ca²⁺/calmodulin-dependent kinase II (CaMKII). Inhibition of transient receptor potential vanilloid type 1 (TRPV1) attenuated RUT-induced intracellular Ca²⁺ concentration and phosphorylation of CaMKII, CaMKKβ, AMPK, and eNOS. Treatment with KN-62 (a CaMKII inhibitor), Compound C (an AMPK inhibitor), and STO-609 (a CaMKKβ inhibitor) suppressed RUT-induced eNOS phosphorylation and NO generation. Interestingly, RUT attenuated the expression of ICAM-1 and VCAM-1 induced by TNF-α and inhibited the inflammation-related NF-κB signaling pathway. Taken together, these results suggest that RUT promotes NO synthesis and eNOS phosphorylation via the Ca²⁺/CaMKII and CaM/CaMKKβ/AMPK signaling pathways through TRPV1. These findings provide evidence that RUT prevents endothelial dysfunction and benefit cardiovascular health.