Conformational substates of calmodulin revealed by single-pair fluorescence resonance energy transfer: influence of solution conditions and oxidative modification

A calmodulin (CaM) mutant (T34,110C-CaM) doubly labeled with fluorescence probes AlexaFluor 488 and Texas Red in opposing domains (CaM-DA) has been used to examine conformational heterogeneity in CaM by single-pair fluorescence resonance energy transfer (spFRET). Burst-integrated FRET efficiencies of freely diffusing CaM-DA single molecules yielded distributions of distance between domains of CaM-DA. We recently reported distinct conformational substates of Ca(2+)-CaM-DA and apoCaM-DA, with peaks in the distance distributions centered at approximately 28 A, 34-38 A, and 55 A [Slaughter et al. (2004) J. Phys. Chem. B 108, 10388-10397]. In the present study, shifts in the amplitudes and center distances of the conformational substates were detected with variation in solution conditions. The amplitude of an extended conformation was observed to change as a function of Ca(2+) over a free Ca(2+) range that is consistent with binding to the high affinity, C-terminal Ca(2+) binding sites, suggesting the existence of communication between lobes of CaM. Lowering pH shifted the relative amplitudes of the conformations, with a marked increase in the presence of the compact conformations and an almost complete absence of the extended conformation. In addition, the single-molecule distance distribution of apoCaM-DA at reduced ionic strength was shifted to longer distance and showed evidence of an increase in conformational heterogeneity relative to apoCaM-DA at physiological ionic strength. Oxidation of methionine residues in CaM-DA produced a substantial increase in the amplitude of the extended conformation relative to the more compact conformation. The results are considered in light of a hypothesis that suggests that electrostatic interactions between charged amino acid side chains play an important role in determining the most stable CaM conformation under varying solution conditions.     

Ca2+-induced structural changes in phosphorylase kinase detected by small-angle X-ray scattering

Phosphorylase kinase (PhK), a 1.3-MDa (alphabetagammadelta)(4) hexadecameric complex, is a Ca(2+)-dependent regulatory enzyme in the cascade activation of glycogenolysis. PhK comprises two arched (alphabetagammadelta)(2) octameric lobes that are oriented back-to-back with overall D(2) symmetry and joined by connecting bridges. From chemical cross-linking and electron microscopy, it is known that the binding of Ca(2+) by PhK perturbs the structure of all its subunits and promotes redistribution of density throughout both its lobes and bridges; however, little is known concerning the interrelationship of these effects. To measure structural changes induced by Ca(2+) in the PhK complex in solution, small-angle X-ray scattering was performed on nonactivated and Ca(2+)-activated PhK. Although the overall dimensions of the complex were not affected by Ca(2+), the cation did promote a shift in the distribution of the scattering density within the hydrated volume occupied by the PhK molecule, indicating a Ca(2+)-induced conformational change. Computer-generated models, based on elements of the known structure of PhK from electron microscopy, were constructed to aid in the interpretation of the scattering data. Models containing two ellipsoids and four cylinders to represent, respectively, the lobes and bridges of the PhK complex provided theoretical scattering profiles that accurately fit the experimental data. Structural differences between the models representing the nonactivated and Ca(2+)-activated conformers of PhK are consistent with Ca(2+)-induced conformational changes in both the lobes and the interlobal bridges.     

Electrostatic changes in phosphorylase kinase induced by its obligatory allosteric activator Ca2+

Skeletal muscle phosphorylase kinase (PhK) is a 1.3-MDa hexadecameric complex that catalyzes the phosphorylation and activation of glycogen phosphorylase b. PhK has an absolute requirement for Ca(2+) ions, which couples the cascade activation of glycogenolysis with muscle contraction. Ca(2+) activates PhK by binding to its nondissociable calmodulin subunits; however, specific changes in the structure of the PhK complex associated with its activation by Ca(2+) have been poorly understood. We present herein the first comparative investigation of the physical characteristics of highly purified hexadecameric PhK in the absence and presence of Ca(2+) ions using a battery of biophysical probes as a function of temperature. Ca(2+)-induced differences in the tertiary and secondary structure of PhK measured by fluorescence, UV absorption, FTIR, and CD spectroscopies as low resolution probes of PhK's structure were subtle. In contrast, the surface electrostatic properties of solvent accessible charged and polar groups were altered upon the binding of Ca(2+) ions to PhK, which substantially affected both its diffusion rate and electrophoretic mobility, as measured by dynamic light scattering and zeta potential analyses, respectively. Overall, the observed physicochemical effects of Ca(2+) binding to PhK were numerous, including a decrease in its electrostatic surface charge that reduced particle mobility without inducing a large alteration in secondary structure content or hydrophobic tertiary interactions. Without exception, for all analyses in which the temperature was varied, the presence of Ca(2+) rendered the enzyme increasingly labile to thermal perturbation.     

A Ca(2+)-dependent global conformational change in the 3D structure of phosphorylase kinase obtained from electron microscopy.

Phosphorylase kinase (PhK), a Ca(2+)-dependent regulatory enzyme of the glycogenolytic cascade in skeletal muscle, is a 1.3 MDa hexadecameric oligomer comprising four copies of four distinct subunits, termed alpha, beta, gamma, and delta, the last being endogenous calmodulin. The structures of both nonactivated and Ca(2+)-activated PhK were determined to elucidate Ca(2+)-induced structural changes associated with PhK's activation. Reconstructions of both conformers of the kinase, each including over 11,000 particles, yielded bridged, bilobal structures with resolutions estimated by Fourier shell correlation at 24 A using a 0.5 correlation cutoff, or at 18 A by the 3sigma (corrected for D(2) symmetry) threshold curve. Extensive Ca(2+)-induced structural changes were observed in regions encompassing both the lobes and bridges, consistent with changes in subunit interactions upon activation. The relative placement of the alpha, beta, gamma, and delta subunits in the nonactivated three-dimensional structure, relying upon previous two-dimensional localizations, is in agreement with the known effects of Ca(2+) on subunit conformations and interactions in the PhK complex.     

Preassociation of calmodulin with voltage-gated Ca(2+) channels revealed by FRET in single living cells

Among the most intriguing forms of Ca(2+) channel modulation is the regulation of L-type and P/Q-type channels by intracellular Ca(2+), acting via unconventional channel-calmodulin (CaM) interactions. In particular, overexpressing Ca(2+)-insensitive mutant CaM abolishes Ca(2+)-dependent modulation, hinting that Ca(2+)-free CaM may "preassociate" with these channels to enhance detection of local Ca(2+). Despite the far-reaching consequences of this proposal, in vitro experiments testing for preassociation provide conflicting results. Here, we develop a three filter-cube fluorescence resonance energy transfer method (three-cube FRET) to directly probe for constitutive associations between channel subunits and CaM in single living cells. This FRET assay detects Ca(2+)-independent associations between CaM and the pore-forming alpha(1) subunit of L-type, P/Q-type, and, surprisingly, R-type channels. These results now definitively demonstrate channel-CaM preassociation in resting cells and underscore the potential of three-cube FRET for probing protein-protein interactions.     

Physicochemical changes in phosphorylase kinase associated with its activation

Phosphorylase kinase (PhK) regulates glycogenolysis through its Ca(2+)-dependent phosphorylation and activation of glycogen phosphorylase. The activity of PhK increases dramatically as the pH is raised from 6.8 to 8.2 (denoted as upward arrow pH), but Ca(2+) dependence is retained. Little is known about the structural changes associated with PhK's activation by upward arrow pH and Ca(2+), but activation by both mechanisms is mediated through regulatory subunits of the (alphabetagammadelta)(4) PhK complex. In this study, changes in the structure of PhK induced by upward arrow pH and Ca(2+) were investigated using second derivative UV absorption, synchronous fluorescence, circular dichroism spectroscopy, and zeta potential analyses. The joint effects of Ca(2+) and upward arrow pH on the physicochemical properties of PhK were found to be interdependent, with their effects showing a strong inflection point at pH approximately 7.6. Comparing the properties of the conformers of PhK present under the condition where it would be least active (pH 6.8 - Ca(2+)) versus that where it would be most active (pH 8.2 + Ca(2+)), the joint activation by upward arrow pH and Ca(2+) is characterized by a relatively large increase in the content of sheet structure, a decrease in interactions between helix and sheet structures, and a dramatically less negative electrostatic surface charge. A model is presented that accounts for the interdependent activating effects of upward arrow pH and Ca(2+) in terms of the overall physicochemical properties of the four PhK conformers described herein, and published data corroborating the transitions between these conformers are tabulated.     

Structural characterization of Ca2+/CaM in complex with the phosphorylase kinase PhK5 peptide

Phosphorylase kinase (PhK) is a large hexadecameric enzyme consisting of four copies of four subunits: (alphabetagammadelta)4. An intrinsic calmodulin (CaM, the delta subunit) binds directly to the gamma protein kinase chain. The interaction site of CaM on gamma has been localized to a C-terminal extension of the kinase domain. Two 25-mer peptides derived from this region, PhK5 and PhK13, were identified previously as potential CaM-binding sites. Complex formation between Ca2+/CaM with these two peptides was characterized using analytical gel filtration and NMR methods. NMR chemical shift perturbation studies showed that while PhK5 forms a robust complex with Ca2+/CaM, no interactions with PhK13 were observed. 15N relaxation characteristics of Ca2+/CaM and Ca2+/CaM/PhK5 complexes were compared with the experimentally determined structures of several Ca2+/CaM/peptide complexes. Good fits were observed between Ca2+/CaM/PhK5 and three structures: Ca2+/CaM complexes with peptides from endothelial nitric oxide synthase, with smooth muscle myosin light chain kinase and CaM kinase I. We conclude that the PhK5 site is likely to have a direct role in Ca2+-regulated control of PhK activity through the formation of a classical 'compact' CaM complex.     

Conformational changes of the Ca(2+) regulatory site of the Na(+)-Ca(2+) exchanger detected by FRET

The Na(+)-Ca(2+) exchanger is a plasma membrane protein expressed at high levels in cardiomyocytes. It extrudes 1 Ca(2+) for 3 Na(+) ions entering the cell, regulating intracellular Ca(2+) levels and thereby contractility. Na(+)-Ca(2+) exchanger activity is regulated by intracellular Ca(2+), which binds to a region (amino acids 371-508) within the large cytoplasmic loop between transmembrane segments 5 and 6. Regulatory Ca(2+) activates the exchanger and removes Na(+)-dependent inactivation. The physiological role of intracellular Ca(2+) regulation of the exchanger is not yet established. Yellow (YFP) and cyan (CFP) fluorescent proteins were linked to the NH(2)- and CO(2)H-termini of the exchanger Ca(2+) binding domain (CBD) to generate a construct (YFP-CBD-CFP) capable of responding to changes in intracellular Ca(2+) concentrations by FRET efficiency measurements. The two fluorophores linked to the CBD are sufficiently close to generate FRET. FRET efficiency was reduced with increasing Ca(2+) concentrations. Titrations of Ca(2+) concentration versus FRET efficiency indicate a K(D) for Ca(2+) of approximately 140 nM, which increased to approximately 400 nM in the presence of 1 mM Mg(2+). Expression of YFP-CBD-CFP in myocytes, generated changes in FRET associated with contraction, suggesting that NCX is regulated by Ca(2+) on a beat-to-beat basis during excitation-contraction coupling.     

X-ray Structures of Magnesium and Manganese Complexes with the N-Terminal Domain of Calmodulin: Insights into the Mechanism and Specificity of Metal Ion Binding to an EF-Hand

Calmodulin (CaM), a member of the EF-hand superfamily, regulates many aspects of cell function by responding specifically to micromolar concentrations of Ca(2+) in the presence of an ～1000-fold higher concentration of cellular Mg(2+). To explain the structural basis of metal ion binding specificity, we have determined the X-ray structures of the N-terminal domain of calmodulin (N-CaM) in complexes with Mg(2+), Mn(2+), and Zn(2+). In contrast to Ca(2+), which induces domain opening in CaM, octahedrally coordinated Mg(2+) and Mn(2+) stabilize the closed-domain, apo-like conformation, while tetrahedrally coordinated Zn(2+) ions bind at the protein surface and do not compete with Ca(2+). The relative positions of bound Mg(2+) and Mn(2+) within the EF-hand loops are similar to those of Ca(2+); however, the Glu side chain at position 12 of the loop, whose bidentate interaction with Ca(2+) is critical for domain opening, does not bind directly to either Mn(2+) or Mg(2+), and the vacant ligand position is occupied by a water molecule. We conclude that this critical interaction is prevented by specific stereochemical constraints imposed on the ligands by the EF-hand β-scaffold. The structures suggest that Mg(2+) contributes to the switching off of calmodulin activity and possibly other EF-hand proteins at the resting levels of Ca(2+). The Mg(2+)-bound N-CaM structure also provides a unique view of a transiently bound hydrated metal ion and suggests a role for the hydration water in the metal-induced conformational change.     

Al(3+) interaction sites of calmodulin and the Al(3+) effect on target binding of calmodulin

The interaction between calmodulin (CaM) and Al(3+) was studied by spectroscopic methods. Heteronuclear two-dimensional NMR data indicated that peaks related to the both lobes and middle of the central helix of CaM are largely affected by Al(3+). But chemical shift perturbation suggested that overall conformation of Ca(2+)-loaded CaM is not changed by Al(3+) binding. It is thought that Al(3+) interaction to the middle of the central helix is a key for the property of CaM's target recognition. If the structure and/or flexibility of the central helix are/is changed by Al(3+), target affinity to CaM must be influenced by Al(3+). Thus, we performed surface plasmon resonance experiments to observe the effect of Al(3+) on the target recognition by CaM. The data clearly indicated that target affinity to CaM is reduced by addition of Al(3+). All the results presented here support a hypothesis that Al(3+) may affect on the Ca(2+) signaling pathway in cells.     

The regulatory beta subunit of phosphorylase kinase interacts with glyceraldehyde-3-phosphate dehydrogenase

Skeletal muscle phosphorylase kinase (PhK) is an (alphabetagammadelta) 4 hetero-oligomeric enzyme complex that phosphorylates and activates glycogen phosphorylase b (GP b) in a Ca (2+)-dependent reaction that couples muscle contraction with glycogen breakdown. GP b is PhK's only known in vivo substrate; however, given the great size and multiple subunits of the PhK complex, we screened muscle extracts for other potential targets. Extracts of P/J (control) and I/lnJ (PhK deficient) mice were incubated with [gamma- (32)P]ATP with or without Ca (2+) and compared to identify potential substrates. Candidate targets were resolved by two-dimensional polyacrylamide gel electrophoresis, and phosphorylated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified by matrix-assisted laser desorption ionization mass spectroscopy. In vitro studies showed GAPDH to be a Ca (2+)-dependent substrate of PhK, although the rate of phosphorylation is very slow. GAPDH does, however, bind tightly to PhK, inhibiting at low concentrations (IC 50 approximately 0.45 microM) PhK's conversion of GP b. When a short synthetic peptide substrate was substituted for GP b, the inhibition was negligible, suggesting that GAPDH may inhibit predominantly by binding to the PhK complex at a locus distinct from its active site on the gamma subunit. To test this notion, the PhK-GAPDH complex was incubated with a chemical cross-linker, and a dimer between the regulatory beta subunit of PhK and GAPDH was formed. This interaction was confirmed by the fact that a subcomplex of PhK missing the beta subunit, specifically an alphagammadelta subcomplex, was unable to phosphorylate GAPDH, even though it is catalytically active toward GP b. Moreover, GAPDH had no effect on the conversion of GP b by the alphagammadelta subcomplex. The interactions described herein between the beta subunit of PhK and GAPDH provide a possible mechanism for the direct linkage of glycogenolysis and glycolysis in skeletal muscle.     

RC3/Neurogranin and Ca2+/calmodulin-dependent protein kinase II produce opposing effects on the affinity of calmodulin for calcium

The interaction of calmodulin with its target proteins is known to affect the kinetics and affinity of Ca(2+) binding to calmodulin. Based on thermodynamic principles, proteins that bind to Ca(2+)-calmodulin should increase the affinity of calmodulin for Ca(2+), while proteins that bind to apo-calmodulin should decrease its affinity for Ca(2+). We quantified the effects on Ca(2+)-calmodulin interaction of two neuronal calmodulin targets: RC3, which binds both Ca(2+)- and apo-calmodulin, and alphaCaM kinase II, which binds selectively to Ca(2+)-calmodulin. RC3 was found to decrease the affinity of calmodulin for Ca(2+), whereas CaM kinase II increases the calmodulin affinity for Ca(2+). Specifically, RC3 increases the rate of Ca(2+) dissociation from the C-terminal sites of calmodulin up to 60-fold while having little effect on the rate of Ca(2+) association. Conversely, CaM kinase II decreases the rates of dissociation of Ca(2+) from both lobes of calmodulin and autophosphorylation of CaM kinase II at Thr(286) induces a further decrease in the rates of Ca(2+) dissociation. RC3 dampens the effects of CaM kinase II on Ca(2+) dissociation by increasing the rate of dissociation from the C-terminal lobe of calmodulin when in the presence of CaM kinase II. This effect is not seen with phosphorylated CaM kinase II. The results are interpreted according to a kinetic scheme in which there are competing pathways for dissociation of the Ca(2+)-calmodulin target complex. This work indicates that the Ca(2+) binding properties of calmodulin are highly regulated and reveals a role for RC3 in accelerating the dissociation of Ca(2+)-calmodulin target complexes at the end of a Ca(2+) signal.     

Ca2+ activation of smooth muscle contraction: evidence for the involvement of calmodulin that is bound to the triton insoluble fraction even in the absence of Ca2+.

Smooth muscle contraction is activated by phosphorylation of the 20-kDa light chains of myosin catalyzed by Ca(2+)/calmodulin (CaM)-dependent myosin light chain kinase (MLCK). According to popular current theory, the CaM involved in MLCK regulation is Ca(2+)-free and dissociated from the kinase at resting cytosolic free Ca(2+) concentration ([Ca(2+)](i)). An increase in [Ca(2+)](i) saturates the four Ca(2+)-binding sites of CaM, which then binds to and activates actin-bound MLCK. The results of this study indicate that this theory requires revision. Sufficient CaM was retained after skinning (demembranation) of rat tail arterial smooth muscle in the presence of EGTA to support Ca(2+)-evoked contraction, as observed previously with other smooth muscle tissues. This tightly bound CaM was released by the CaM antagonist trifluoperazine (TFP) in the presence of Ca(2+). Following removal of the (Ca(2+))(4)-CaM-TFP(2) complex, Ca(2+) no longer induced contraction. The addition of exogenous CaM to TFP-treated tissue at a [Ca(2+)] subthreshold for contraction or even in the absence of Ca(2+) (presence of 5 mm EGTA), followed by washout of unbound CaM, restored Ca(2+)-induced contraction; this required MLCK activation, since it was blocked by the MLCK inhibitor ML-9. The data suggest, therefore, that a specific pool of cellular CaM, tightly bound to myofilaments at resting [Ca(2+)](i), or even in the absence of Ca(2+), is responsible for activation of contraction following a local increase in [Ca(2+)]. This mechanism would allow for localized changes in [Ca(2+)] in regions of the cell distant from the myofilaments to regulate distinct Ca(2+)-dependent processes without triggering a contractile response. Immobilized CaM, therefore, resembles troponin C, the Ca(2+)-binding regulatory protein of striated muscle, which is also bound to the thin filament in a Ca(2+)-independent manner.     

The crystal structures of semi-synthetic aequorins

The photoprotein aequorin emits light by an intramolecular reaction in the presence of a trace amount of Ca(2+). Semi-synthetic aequorins, produced by replacing the coelenterazine moiety in aequorin with the analogues of coelenterazine, show widely different sensitivities to Ca(2+). To understand the structural basis of the Ca(2+)-sensitivity, we determined the crystal structures of four semi-synthetic aequorins (cp-, i-, br- and n-aequorins) at resolutions of 1.6-1.8 A. In general, the protein structures of these semi-synthetic aequorins are almost identical to native aequorin. Of the four EF-hand domains in the molecule, EF-hand II does not bind Ca(2+), and the loop of EF-hand IV is clearly deformed. It is most likely that the binding of Ca(2+) with EF-hands I and III triggers luminescence. Although little difference was found in the overall structures of aequorins investigated, some significant differences were found in the interactions between the substituents of coelenterazine moiety and the amino acid residues in the binding pocket. The coelenterazine moieties in i-, br-, and n-aequorins have bulky 2-substitutions, which can interfere with the conformational changes of protein structure that follow the binding of Ca(2+) to aequorin. In cp-aequorin, the cyclopentylmethyl group that substitutes for the original 8-benzyl group does not interact hydrophobically with the protein part, giving the coelenterazine moiety more conformational freedom to promote the light-emitting reaction. The differences of various semi-synthetic aequorins in Ca(2+)-sensitivity and reaction rate are explained by the capability of the involved groups and structures to undergo conformational changes in response to the Ca(2+)-binding.     

Structural features contributing to complex formation between glycogen phosphorylase and phosphorylase kinase.

A polyclonal antibody was generated against a peptide corresponding to a region opposite the regulatory face of glycogen phosphorylase b (P-b), providing a probe for detecting and quantifying P-b when it is bound to its activating kinase, phosphorylase kinase (PhK). Using both direct and competition enzyme-linked immunosorbent assays (ELISAs), we have measured the extent of direct binding to PhK of various forms of phosphorylase, including different conformers induced by allosteric effectors as well as forms differing at the N-terminal site phosphorylated by PhK. Strong interactions with PhK were observed for both P-b', a truncated form lacking the site for phosphorylation, and P-a, the phosphorylated form of P-b. Further, the binding of P-b, P-b', and P-a was stimulated a similar amount by Mg(2+), or by Ca(2+) (both being activators of PhK). Our results suggest that the presence and conformation of P-b's N-terminal phosphorylation site do not fully account for the protein's affinity for PhK and that regions distinct from that site may also interact with PhK. Direct ELISAs detected the binding of P-b by a truncated form of the catalytic gamma subunit of PhK, consistent with the necessary interaction of PhK's catalytic subunit with its substrate P-b. In contrast, P-b' bound very poorly to the truncated gamma subunit, suggesting that the N-terminal phosphorylatable region of P-b may be critical in directing P-b to PhK's catalytic subunit and that the binding of P-b' by the PhK holoenzyme may involve more than just its catalytic core. The sum of our results suggests that structural features outside the catalytic domain of PhK and outside the phosphorylatable region of P-b may both be necessary for the maximal interaction of these two proteins.     

The calmodulin-binding domain of the catalytic gamma subunit of phosphorylase kinase interacts with its inhibitory alpha subunit: evidence for a Ca2+ sensitive network of quaternary interactions

Chemical cross-linking as a probe of conformation has consistently shown that activators, including Ca(2+) ions, of the (alphabetagammadelta)(4) phosphorylase kinase holoenzyme (PhK) alter the interactions between its regulatory alpha and catalytic gamma subunits. The gamma subunit is also known to interact with the delta subunit, an endogenous molecule of calmodulin that mediates the activation of PhK by Ca(2+) ions. In this study, we have used two-hybrid screening and chemical cross-linking to dissect the regulatory quaternary interactions involving these subunits. The yeast two-hybrid system indicated that regions near the C termini of the gamma (residues 343-386) and alpha (residues 1060-1237) subunits interact. The association of this region of alpha with gamma was corroborated by the isolation of a cross-linked fragment of alpha containing residues 1015-1237 from an alpha-gamma dimer that had been formed within the PhK holoenzyme by formaldehyde, a nearly zero-length cross-linker. Because the region of gamma that we found to interact with alpha has previously been shown to contain a high affinity binding site for calmodulin (Dasgupta, M., Honeycutt, T., and Blumenthal, D. K. (1989) J. Biol. Chem. 264, 17156-17163), we tested the influence of Ca(2+) on the conformation of the alpha subunit and found that the region of alpha that interacts with gamma was, in fact, perturbed by Ca(2+). The results herein support the existence of a Ca(2+)-sensitive communication network among the delta, gamma, and alpha subunits, with the regulatory domain of gamma being the primary mediator. The similarity of such a Ca(2+)-dependent network to the interactions among troponin C, troponin I, and actin is discussed in light of the known structural and functional similarities between troponin I and the gamma subunit of PhK.     

Novel interaction of the voltage-dependent sodium channel (VDSC) with calmodulin: does VDSC acquire calmodulin-mediated Ca2+-sensitivity?

The voltage-dependent sodium channel (VDSC) interacts with intracellular molecules to modulate channel properties and localizations in neuronal cells. To study protein interactions, we applied yeast two-hybrid screening to the cytoplasmic C-terminal domain of the main pore-forming alpha-subunit. We found a novel interaction between the C-terminal domain and calmodulin (CaM). By two-hybrid interaction assays, we specified the interaction site of VDSC in a C-terminal region, which is composed of 38 amino acid residues and contains both IQ-like and Baa motifs. Using a fusion protein of the C-terminal domain, we showed that interaction with CaM occurred in the presence and absence of Ca(2+). Two synthetic peptides, each covering the IQ-like (NaIQ) or the Baa motifs (NaBaa), were used to examine the binding property by a gel mobility shift assay. Although the NaIQ and NaBaa sequences are overlapped, NaBaa binds only to Ca(2+)-bound Ca(2+)CaM, whereas NaIQ binds to both Ca(2+)CaM and Ca(2+)-free apoCaM. Fluorescence spectroscopy of dansylated CaM showed Ca(2+)-dependent spectral changes not only for NaBaa.CaM but also for NaIQ.CaM. The results, taken together with other results, indicate that whereas the NaBaa.CaM complex is formed in a Ca(2+)-dependent manner, the NaIQ.CaM complex has two conformational states, distinct with respect to the peptide binding site and the CaM conformation, depending on the Ca(2+) concentration. These observations suggest the possibility that VDSC is functionally modulated through the direct CaM interaction and the Ca(2+)-dependent conformational transition of the complex.     

Binding of La3+ to calmodulin and its effects on the interaction between calmodulin and calmodulin binding peptide, polistes mastoparan

Binding of La(3+) to calmodulin (CaM) and its effects on the complexes of CaM and CaM-binding peptide, polistes mastoparan (Mas), were investigated by nuclear magnetic resonance (NMR) spectroscopy, fluorescence and circular dichroism spectroscopy, and by the fluorescence stopped-flow method. The four binding sites of La(3+) on CaM were identified as the same as the binding sites of Ca(2+) on CaM through NMR titration of La(3+) to uniformly (15)N-labeled CaM. La(3+) showed a slightly higher affinity to the binding sites on the N-terminal domain of CaM than that to the C-terminal. Large differences between the (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra of Ca(4)CaM and La(4)CaM suggest conformational differences between the two complexes. Fluorescence and CD spectra also exhibited structural differences. In the presence of Ca(2+) and La(3+), a hybrid complex, Ca(2)La(2)CaM, was formed, and the binding of La(3+) to the N-terminal domain of CaM seemed preferable over binding to the C-terminal domain. Through fluorescence titration, it was shown that La(4)CaM and Ca(2)La(2)CaM had similar affinities to Mas as Ca(4)CaM. Fluorescence stopped-flow experiments showed that the dissociation rate of La(3+) from the C-terminal domain of CaM was higher than that from the N-terminal. However, in the presence of Mas, the dissociation rate of La(3+) decreased and the dissociation processes from both global domains were indistinguishable. In addition, compared with the case of Ca(4)CaM-Mas, the slower dissociations of Mas from La(4)CaM-Mas and Ca(2)La(2)CaM-Mas complexes indicate that in the presence of La(3+), the CaM-Mas complex became kinetically inert. A possible role of La(3+) in the Ca(2+)-CaM-dependent pathway is discussed.     

Metal ion selectivity for formation of the calmodulin-metal-target peptide ternary complex studied by surface plasmon resonance spectroscopy.

Ion selectivities for Ca(2+) signaling pathways of 33 metal ions were examined based on the Ca(2+)-dependent on/off switching mechanism of calmodulin (CaM): Ca(2+) ion-induced selective binding of CaM-Ca(2+) ion complex to the target peptide was observed as an increase in surface plasmon resonance (SPR) signals. As the target peptide, M13 of 26-amino-acid residues derived from skeletal muscle myosin light-chain kinase was immobilized in the dextran matrix, over which sample solutions containing CaM and each metal ion were injected in a flow system. Large changes in SPR signals were also observed for Sr(2+), Ba(2+), Cd(2+), Pb(2+), Y(3+) and trivalent lanthanide ions, thereby indicating that not only Ca(2+) but also these metal ions induce the formation of CaM-M13-metal ion ternary complex. No SPR signal was, however, induced by Mg(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+) and all monovalent metal ions examined. The latter silent SPR signal indicates that these ions, even if they bind to CaM, are incapable of forming the CaM-M13-metal ion ternary complex. Comparing the obtained SPR results with ionic radii of those metal ions, it was found that all cations examined with ionic radii close to or greater than that of Ca(2+) induced the formation of the CaM-metal-M13 ternary complex, whereas those with smaller ionic radii were not effective, or much less so. Since these results are so consistent with earlier systematic data for the effects of various metal ions on the conformational changes of CaM, it is concluded that the present SPR analysis may be used for a simple screening and evaluating method for physiologically relevant metal ion selectivity for the Ca(2+) signaling via CaM based on CaM/peptide interactions.     

Conformation of the calmodulin-binding domain of metabotropic glutamate receptor subtype 7 and its interaction with calmodulin

Calmodulin (CaM), a Ca(2+)-binding protein, is a well-known regulator of various cellular functions. One of the targets of CaM is metabotropic glutamate receptor 7 (mGluR7), which serves as a low-pass filter for glutamate in the pre-synaptic terminal to regulate neurotransmission. Surface plasmon resonance (SPR), circular dichroism (CD) spectroscopy and nuclear magnetic spectroscopy (NMR) were performed to study the structure of the peptides corresponding to the CaM-binding domain of mGluR7 and their interaction with CaM. Unlike well-known CaM-binding peptides, mGluR7 has a random coil structure even in the presence of trifluoroethanol. Moreover, NMR data suggested that the complex between Ca(2+)/CaM and the mGluR7 peptide has multiple conformations. The mGluR7 peptide has been found to interact with CaM even in the absence of Ca(2+), and the binding is directed toward the C-domain of apo-CaM rather than the N-domain. We propose a possible mechanism for the activation of mGluR7 by CaM. A pre-binding occurs between apo-CaM and mGluR7 in the resting state of cells. Then, the Ca(2+)/CaM-mGluR7 complex is formed once Ca(2+) influx occurs. The weak interaction at lower Ca(2+) concentrations is likely to bind CaM to mGluR7 for the fast complex formation in response to the elevation of Ca(2+) concentration.