Conformational Changes Of Calmodulin Research Articles

Calmodulin Release from the IQ Domain of Cav1.3 Channels During Calcium Dependent Inactivation?

Calmodulin (CaM) regulation of CaV channels has long fascinated biophysicists, with structure-function analysis mostly focused on an IQ domain in the carboxy-terminus of channels. It is clear that Ca2+-free CaM (apoCaM) preassociates with the IQ domain before Ca2+ entry through channels (see Liu X et al this meeting), and that Ca2+/CaM has the potential to bind the IQ. Hence, Ca2+-dependent inactivation (CDI) of channels has been thought to result by transducing Ca2+-dependent conformational changes of CaM, all while bound to the IQ. By contrast, functional analysis of our structure of Ca2+/CaM complexed with the IQ domain of CaV2.1 (Structure 16:607) hints that Ca2+/CaM may depart from the IQ domain during channel regulation. To generalize this hypothesis, we here alanine scanned the entire IQ domain of CaV1.3, an exemplary prototype of CaV1 channels with high homology to long-studied CaV1.2. Importantly, alanine substitution likely preserves backbone fold, whereas prior studies often used more disruptive mutations targeting specific loci. As before, alanine substitution of the signature isoleucine strongly suppressed CDI. Suprisingly, however, CDI was strongly diminished only at one other residue, upstream of the central isoleucine. Altering many other sites, presumed important for Ca2+/CaM-IQ binding in crystal structures, left CDI unscathed. Moreover, we homology modeled Ca2+/CaM bound to the CaV1.3 IQ domain, based on x-ray structures of CaV1.2. We then computed (Robetta) the energetic cost of alanine mutations (ΔΔG for binding). If Ca2+/CaM-IQ binding begets CDI, plots of CDI versus ΔΔG should define a Boltzmann function. Instead, a highly scattered relationship was produced. Thus, CDI may involve (partial) departure of Ca2+/CaM from the IQ domain of CaV1.3, to interact with alternative sites (Yang, Ben Johny, & Yue, this meeting). This departure would fundamentally transform our understanding of CaM/channel regulation.

Effect of Trifluoperazine on Ca2+ -Bound Calmodulin binding to Fas Death Domain for DISC Formation

Fas death receptor-activated signaling pathway is one important regulating mechanism of apoptosis in a variety of cells. The formation of the death inducing signaling complex (DISC) is a critical step for Fas-mediated signaling of apoptosis. Recent experimental studies showed that calmodulin (CaM) binds to Fas and regulates Fas-mediated DISC formation and the binding of CaM to Fas is inhibited by CaM antagonist, trifluoperazine (TFP). However, the exact molecular mechanisms for the effect of TFP on Fas-mediated DISC formation are still unknown. Knowledge about these is important for identifying new drug candidate to regulate Fas-mediated signaling pathway for apoptosis. In this study, we investigated the effect of TFP on CaM/Fas binding with molecular dynamics simulations. Conformation and binding free energy analyses were performed to examine the connections between the conformational changes of CaM by TFP and CaM/Fas binding affinity. Conformational characteristics of Fas by TFP were also examined for the further determining TFP effects on Fas recruiting FADD to form DISC. Binding free energy analyses showed that CaM antagonist, TFP inhibited CaM binding to Fas. The results are consistent with experimental results. The further conformational analyses showed that TFP significantly changed the CaM conformation, resulted in the increased Fas conformational fluctuations and the degree of correlation between motions of the residues in Fas, which provides structural insight for Fas further binding to FADD for DISC formation. Understanding the molecular mechanisms of CaM antagonist TFP in CaM/Fas binding for Fas-mediated DISC formation should provide important insight into the function of CaM antagonists in regulating Fas-mediated apoptosis.Keywords: CaM antagonist TFP; CaM/Fas binding; DISC; binding free energy, conformational analysis

In Situ Monitoring of Conformational Changes of and Peptide Bindings to Calmodulin on a 27 MHz Quartz-Crystal Microbalance

Conformational changes of calmodulin (CaM) by additions of Ca(2+) ions and bindings of CaM-binding peptides to Ca(2+)/CaM followed by conformational changes were monitored by a CaM-immobilized 27 MHz quartz-crystal microbalance (QCM) with an admittance analysis. Both the binding and the conformational change events could be detected from the time-dependence of frequency decreases (mass increases) and energy dissipation decreases (elasticity increases), respectively. When Ca(2+) ions were injected to a QCM cell on which biotinylated CaM was immobilized with avidin-biotin interactions, a frequency increase (a mass decrease) and an energy dissipation decrease (an elasticity increase) were observed because of the dehydration and the elasticity increase caused by conformational changes from the flexible CaM to the rigid Ca(2+)/CaM exposing the hydrophobic surface. In the case of the addition of CaMKII-peptide in the Ca(2+)/CaM-immobilized QCM, the immediate frequency decrease (the mass increase) due to the binding of the peptide to Ca(2+)/CaM and the following energy dissipation decrease (the elasticity increase) with a time lag were observed. This suggests that the interaction of the CaMKII-peptide to Ca(2+)/CaM follows an allosteric binding mode. Binding kinetics of the peptide to Ca(2+)/CaM (k(1) and k(-1)) and kinetics of the following conformational change of Ca(2+)/CaM (k(2) and k(-2)) could be obtained. This technique is useful to investigate biomolecular interactions involving the conformational and/or viscoelastic property changes that are biologically important.

Commentary: The Year in Basic Science: Calmodulin Kinase Cascades

This article highlights studies published during the past year that represent significant scientific achievements in the world of calmodulin kinase cascades. Calmodulin is the primary receptor for calcium present in all cells. The binding of its calcium ligand results in a conformational change in calmodulin, which allows the calcium-calmodulin complex to interact with many different targets. In the studies to be summarized in this review, the particular calmodulin cascade involved is shown to be the pathway responsible for important biological responses, including long-term memory formation, dendritic cell survival, hypercapnia, neuronal migration, synapse formation, autophagy, fatty acid oxidation, and energy balance. In some cases, the pathway was previously unknown, and the identification of the calmodulin cascade represents the definition of roles. In other cases, manipulating the cascade has suggested therapeutic approaches to certain diseases, most significantly, type 2 diabetes and obesity.

A Calcium-Modulated Plasmonic Switch

A plasmonic switch based on the calcium-induced conformational changes of calmodulin is shown to exhibit reversible wavelength modulations in response to changing calcium concentration. The extinction maximum (lambdamax) of a localized surface plasmon resonance (LSPR) sensor functionalized with a novel calmodulin construct, cutinase-calmodulin-cutinase (CutCaMCut), reversibly shifts by 2-3 nm. A high-resolution (HR) LSPR spectrometer with a wavelength resolution (3sigma) of 1.5 x 10-2 nm was developed to detect these wavelength modulations in real-time, providing information about the dynamics and structure of the protein. The rate of conversion from open (Ca2+-bound) to closed (Ca2+-free) calmodulin is shown to be 4-fold faster than the reverse process, with a closing rate of 0.127 s-1 and opening rate of 0.034 s-1. As far as we are aware, this plasmonic switch marks the first use of LSPR spectroscopy to detect reversible conformational changes in an unlabeled protein.

Dynamic Conformational Changes of Calmodulin when Bound to Nitric Oxide Synthase using FRET

Calmodulin (CaM) is a ubiquitous Ca2+‐sensor protein that binds and activates the Nitric Oxide Synthase (NOS) enzymes (E.C. ). Although the structure of CaM has been determined when bound to a peptide derived from the CaM‐binding domain of endothelial NOS, the conformation of CaM when bound to the three mammalian NOS holoenzymes has yet to be determined. The objective of this study is to determine the dynamic conformational changes of CaM when bound to all three NOS isoforms through the use of Förster Resonance Energy Transfer (FRET). We have produced a double cysteine CaM mutant (T34C/T110C) using site‐directed mutagenesis for the performance of FRET measurements. Far‐red shifted fluorescent dyes were employed so that they could be selectively excited in the presence of the NOS cofactors that absorb light strongly between 250 and 500 nm. FRET studies were performed in the presence of a range of physiologically relevant free Ca2+ concentrations for CaM binding to the constitutive NOS enzymes. These studies will provide a better understanding of the differences between the Ca2+‐dependent and Ca2+‐independent binding of CaM to the NOS isozymes. This work is supported by NSERC (J.G.G.).

Conformational changes of calmodulin upon Ca 2+ binding studied with a microfluidic mixer

A microfluidic mixer is applied to study the kinetics of calmodulin conformational changes upon Ca2+ binding. The device facilitates rapid, uniform mixing by decoupling hydrodynamic focusing from diffusive mixing and accesses time scales of tens of microseconds. The mixer is used in conjunction with multiphoton microscopy to examine the fast Ca2+-induced transitions of acrylodan-labeled calmodulin. We find that the kinetic rates of the conformational changes in two homologous globular domains differ by more than an order of magnitude. The characteristic time constants are approximately 490 micros for the transitions in the C-terminal domain and approximately 20 ms for those in the N-terminal domain of the protein. We discuss possible mechanisms for the two distinct events and the biological role of the stable intermediate, half-saturated calmodulin.

Simulating Protein Motions with Rigidity Analysis

Protein motions, ranging from molecular flexibility to large-scale conformational change, play an essential role in many biochemical processes. Despite the explosion in our knowledge of structural and functional data, our understanding of protein movement is still very limited. In previous work, we developed and validated a motion planning based method for mapping protein folding pathways from unstructured conformations to the native state. In this paper, we propose a novel method based on rigidity theory to sample conformation space more effectively, and we describe extensions of our framework to automate the process and to map transitions between specified conformations. Our results show that these additions both improve the accuracy of our maps and enable us to study a broader range of motions for larger proteins. For example, we show that rigidity-based sampling results in maps that capture subtle folding differences between protein G and its mutants, NuG1 and NuG2, and we illustrate how our technique can be used to study large-scale conformational changes in calmodulin, a 148 residue signaling protein known to undergo conformational changes when binding to Ca(2+). Finally, we announce our web-based protein folding server which includes a publicly available archive of protein motions: (http://parasol.tamu.edu/foldingserver/).

Comparison of negative and positive ion electrospray ionization mass spectra of calmodulin and its complex with trifluoperazine

The protein calmodulin (apoCaM) undergoes a conformational change when it binds calcium. This structure of the protein (Ca4CaM) is a dumbbell-shaped molecule that undergoes a further profound conformational change on binding of the antipsychotic drug trifluoperazine (TFP). Experimental conditions were developed to prepare samples of apoCaM, Ca4CaM and Ca4CaM/TFP that were substantially free of sodium. The effects of the conformational changes of calmodulin on the charge-state distributions observed in positive ion and negative ion electrospray ionization (ESI) mass spectra were examined. Conversion of apoCaM into Ca4CaM was concomitant with a change in the negative ion ESI mass spectrum whereby the 16- ion was the most abundant ion observed for the apo form and the 8- ion was the most abundant for the complex. In contrast, in the positive ion ESI mass spectra of apoCaM and Ca4CaM, the most abundant species in each case was the 8+ ion. When a complex of Ca4CaMwith TFP was prepared, the most abundant species was the 5+ ion. This is consistent with a conformational change of Ca4CaM that rendered some basic sites inaccessible to ionization in the ESI process. Using the same Ca4CaM/TFP mixture, no complex with TFP was observed in negative ion ESI mass spectra. These observations are discussed in the context of the structural changes that are known to occur in calmodulin, and suggestions are made to explain the apparently conflicting data. The results reported here reflect on the validity of using differences in charge-state distributions observed in ESI mass spectra to assess conformational changes in proteins.

Sites on Calmodulin That Interact with the C-terminal Tail of Cav1.2 Channel

Two fragments of the C-terminal tail of the alpha(1) subunit (CT1, amino acids 1538-1692 and CT2, amino acids 1596-1692) of human cardiac L-type calcium channel (Ca(V)1.2) have been expressed, refolded, and purified. A single Ca(2+)-calmodulin binds to each fragment, and this interaction with Ca(2+)-calmodulin is required for proper folding of the fragment. Ca(2+)-calmodulin, bound to these fragments, is in a more extended conformation than calmodulin bound to a synthetic peptide representing the IQ motif, suggesting that either the conformation of the IQ sequence is different in the context of the longer fragment, or other sequences within CT2 contribute to the binding of calmodulin. NMR amide chemical shift perturbation mapping shows the backbone conformation of calmodulin is nearly identical when bound to CT1 and CT2, suggesting that amino acids 1538-1595 do not contribute to or alter calmodulin binding to amino acids 1596-1692 of Ca(V)1.2. The interaction with CT2 produces the greatest changes in the backbone amides of hydrophobic residues in the N-lobe and hydrophilic residues in the C-lobe of calmodulin and has a greater effect on residues located in Ca(2+) binding loops I and II in the N-lobe relative to loops III and IV in the C-lobe. In conclusion, Ca(2+)-calmodulin assumes a novel conformation when part of a complex with the C-terminal tail of the Ca(V)1.2 alpha(1) subunit that is not duplicated by synthetic peptides corresponding to the putative binding motifs.

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Optical Emission of a Conjugated Polyelectrolyte: Calcium-Induced Conformational Changes in Calmodulin and Calmodulin−Calcineurin Interactions

Electronic polymers in aqueous media offer bioelectronic detection of biomolecular processes. Here we report fluorometric detection of calcium-induced conformational changes in calmodulin based on noncovalent assembly of calmodulin to a water-soluble zwitterionic polythiophene derivative. Assembly with calmodulin will induce a planar geometry and aggregation of the polymer chains, detected as a decrease of the intensity and a red shift of the fluorescence. Upon addition of Ca2+ the intensity of the emitted light is increased and blue-shifted. The geometrical alteration of the polymer chains can further be utilized for recording of the binding of calcineurin to the calcium-activated POWT−calmodulin complex. This novel methodology, using a conformation-sensitive probe, allows fluorometric detection of conformational changes in biomolecules and protein−protein interactions without any covalent modifications of the biomolecules. The rapid and selective method is based on noncovalent interactions between a zwitterionic polythiophene derivative and the biomolecule of interest. This offers a novel way to create microarrays without using covalent attachment of the receptor or labeling of the analyte.

Ca 2+ binding sites in calmodulin and troponin C alter interhelical angle movements

Molecular dynamics analyses were performed to examine conformational changes in the C-domain of calmodulin and the N-domain of troponin C induced by binding of Ca 2+ ions. Analyses of conformational changes in calmodulin and troponin C indicated that the shortening of the distance between Ca 2+ ions and Ca 2+ binding sites of helices caused widening of the distance between Ca 2+ binding sites of helices on opposite sides, while the hydrophobic side chains in the center of helices hardly moved due to their steric hindrance. This conformational change acts as the clothespin mechanism.

Drug detection based on the conformational changes of calmodulin and the fluorescence of its enhanced green fluorescent protein fusion partner

A homogeneous phase protein-based assay for the high throughput screening of drugs was developed using enhanced green fluorescent protein (EGFP) as the reporter. For that, a fusion protein between calmodulin (CaM) and EGFP was constructed in order to monitor the conformational changes induced in CaM upon binding to tricyclic anti-depressant drugs. In the presence of Ca 2+, CaM undergoes a conformational change exposing a hydrophobic pocket that interacts with CaM-binding proteins, peptides, and drugs. Further, the conformational changes induced in CaM upon binding to Ca 2+ and the target analyte drug, leads to a change in the microenvironment of EGFP concomitant with a change in its fluorescence intensity. The observed change in fluorescence intensity of EGFP can be correlated to the concentration of the analyte present in the sample. Further, the response of CaM–EGFP fusion protein in the presence of Ca 2+ to increasing concentrations of phenothiazines and structurally related tricyclic anti-depressants was investigated. Dose-response curves for various tricyclic anti-depressants were prepared. Moreover, this assay can serve as a model system for other homogeneous binding assays for pharmaceuticals employing genetically fused binding proteins with reporter proteins and may find applications in the high throughput screening of tricyclic anti-depressants.

Drug detection based on the conformational changes of calmodulin and the fluorescence of its enhanced green fluorescent protein fusion partner

Drug detection based on the conformational changes of calmodulin and the fluorescence of its enhanced green fluorescent protein fusion partner

Dual Regulation of Mammalian Myosin VI Motor Function

Myosin VI is expressed in a variety of cell types and is thought to play a role in membrane trafficking and endocytosis, yet its motor function and regulation are not understood. The present study clarified mammalian myosin VI motor function and regulation at a molecular level. Myosin VI ATPase activity was highly activated by actin with K(actin) of 9 microm. A predominant amount of myosin VI bound to actin in the presence of ATP unlike conventional myosins. K(ATP) was much higher than those of other known myosins, suggesting that myosin VI has a weak affinity or slow binding for ATP. On the other hand, ADP markedly inhibited the actin-activated ATPase activity, suggesting a high affinity for ADP. These results suggested that myosin VI is predominantly in a strong actin binding state during the ATPase cycle. p21-activated kinase 3 phosphorylated myosin VI, and the site was identified as Thr(406). The phosphorylation of myosin VI significantly facilitated the actin-translocating activity of myosin VI. On the other hand, Ca(2+) diminished the actin-translocating activity of myosin VI although the actin-activated ATPase activity was not affected by Ca(2+). Calmodulin was not dissociated from the heavy chain at high Ca(2+), suggesting that a conformational change of calmodulin upon Ca(2+) binding, but not its physical dissociation, determines the inhibition of the motility activity. The present results revealed the dual regulation of myosin VI by phosphorylation and Ca(2+) binding to calmodulin light chain.

Ca2+-dependent Regulation of the Motor Activity of Myosin V

Mouse myosin V constructs were produced that consisted of the myosin motor domain plus either one IQ motif (M5IQ1), two IQ motifs (M5IQ2), a complete set of six IQ motifs (SHM5), or the complete IQ motifs plus the coiled-coil domain (thus permitting formation of a double-headed structure, DHM5) and expressed in Sf9 cells. The actin-activated ATPase activity of all constructs except M5IQ1 was inhibited above pCa 5, but this inhibition was completely reversed by addition of exogenous calmodulin. At the same Ca(2+) concentration, 2 mol of calmodulin from SHM5 and DHM5 or 1 mol of calmodulin from M5IQ2 were dissociated, suggesting that the inhibition of the ATPase activity is due to dissociation of calmodulin from the heavy chain. However, the motility activity of DHM5 and M5IQ2 was completely inhibited at pCa 6, where no dissociation of calmodulin was detected. Inhibition of the motility activity was not reversed by the addition of exogenous calmodulin. These results indicate that inhibition of the motility is due to conformational changes of calmodulin upon the Ca(2+) binding to the high affinity site but is not due to dissociation of calmodulin from the heavy chain.

Spectroscopic Characterization of the Interaction between Calmodulin-Dependent Protein Kinase I and Calmodulin

Calmodulin-dependent protein kinase I (CaM kinase I) is a member of the expanding class of protein kinases that are regulated by calmodulin (CaM). Its putative CaM-binding region is believed to occur within a 22-residue sequence (amino acids 299–320). This sequence was chemically synthesized and utilized for CaM interaction studies. Gel band shift assays and densitometry experiments with intact CaM kinase I and the CaM-binding domain peptide (CaMKIp) reveal that they bind in an analogous manner, giving rise to 1:1 complexes. Fluorescence analysis using dansyl-CaM showed that conformational changes in CaM on binding CaM kinase I or CaMKIp were nearly identical, suggesting that the peptide mimicked the CaM-binding ability of the intact protein. In the presence of CaM, the peptide displays an enhancement of its unique Trp fluorescence as well as a marked blue shift of the emission maximum, reflecting a transfer to a more rigid, less polar environment. Quenching studies, using acrylamide, confirmed that the Trp in the peptide on binding CaM is no longer freely exposed to solvent as is the case for the free peptide. Studies with a series of Met mutants of CaM showed that the Trp-containing N-terminal end of CaMKIp was bound to the C-terminal lobe of CaM. Near-UV CD spectra also indicate that the Trp of the peptide and Phe residues of the protein are involved in the binding. These results show that the CaM-binding domain of CaM kinase I binds to CaM in a manner analogous to that of myosin light chain kinase.

Ca2+-bound calmodulin forms a compact globular structure on binding four trifluoperazine molecules in solution

Small-angle X-ray scattering (SAXS), which determines the radius of gyration, Rg, and the pair distance distribution function, was used to investigate the conformational changes of calmodulin (CaM) on binding to an antagonist, trifluoperazine (TFP), with or without Ca2+ in solution. We previously applied this SAXS method to CaM complexed with N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7) [Osawa, Kuwamoto, Izumi, Yap, Ikura, Shibanuma, Yokokura, Hidaka and Matsushima (1999) FEBS Lett. 442, 173-177] and found that the binding of two W-7 TFP molecules to one Ca2+-saturated CaM molecule induces structural changes from a ‘dumb-bell’ shape to a compact globular shape. We report here that the most compact globular shape whose size is consistent with that of the 1:2 Ca2+-saturated CaM-W-7 complex is formed by the binding of four TFP molecules to one Ca2+-saturated CaM molecule. Even in the absence of Ca2+, the conformational changes of CaM occur on TFP binding, giving a slightly smaller Rg than Ca2+-free CaM alone.

Ca2+-bound calmodulin forms a compact globular structure on binding four trifluoperazine molecules in solution

Small-angle X-ray scattering (SAXS), which determines the radius of gyration, R(g), and the pair distance distribution function, was used to investigate the conformational changes of calmodulin (CaM) on binding to an antagonist, trifluoperazine (TFP), with or without Ca(2+) in solution. We previously applied this SAXS method to CaM complexed with N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7) [Osawa, Kuwamoto, Izumi, Yap, Ikura, Shibanuma, Yokokura, Hidaka and Matsushima (1999) FEBS Lett. 442, 173-177] and found that the binding of two W-7 TFP molecules to one Ca(2+)-saturated CaM molecule induces structural changes from a 'dumb-bell' shape to a compact globular shape. We report here that the most compact globular shape whose size is consistent with that of the 1:2 Ca(2+)-saturated CaM-W-7 complex is formed by the binding of four TFP molecules to one Ca(2+)-saturated CaM molecule. Even in the absence of Ca(2+), the conformational changes of CaM occur on TFP binding, giving a slightly smaller R(g) than Ca(2+)-free CaM alone.