Interactions between troponin C (TnC) and troponin I (TnI) play an important role in the Ca(2+)-dependent regulation of vertebrate
striated muscle contraction. In the present study, we investigated ...the sites of interaction between the N-terminal regulatory
domain of TnC and the inhibitory region (residues 96-116) of TnI, using a mutant rabbit skeletal TnC (designated as TnC57)
that contains a single Cys at residue 57 in the C-helix. TnC57 was modified with the photoreactive cross-linker 4-maleimidobenzophenone
(BP-Mal), and, after formation of a binary complex with TnI, cross-linking between the proteins was induced by photolysis.
The resulting product was cleaved with CNBr and several proteases, and peptides containing cross-links were purified and subjected
to amino acid sequencing. The results show that Cys-57 of TnC57 is cross-linked to the segment of TnI spanning residues 113-121.
Previously, we showed that Cys-98 of TnC can be cross-linked via BP-Mal to TnI residues 103-110 (Leszyk, J., Collins, J.H.,
Leavis, P.C., and Tao, T. (1987) Biochemistry 26, 7042-7047). Taken together, these results demonstrate that both the C- and
the N-terminal domains of TnC interact with the inhibitory region of TnI and are consistent with the hypothesis that, in a
complex with TnI, TnC adopts a more compact conformation than in the crystal structure.
CaMKII (Ca super(2+)/calmodulin-dependent kinase II) is a serine/threonine phosphotransferase that is capable of long-term retention of activity due to autophosphorylation at a specific threonine ...residue within each subunit of its oligomeric structure. The gamma iso form of CaMKII is a significant regulator of vascular contractility. Here, we show that phosphorylation of CaMKII gamma at Ser super(26), a residue located within the ATP-binding site, terminates the sustained activity of the enzyme. To test the physiological importance of phosphorylation at Ser super(26), we generated a phosphospecific Ser super(26) antibody and demonstrated an increase in Ser super(26) phosphorylation upon depolarization and contraction of blood vessels. To determine if the phosphorylation of Ser super(26) affects the kinase activity, we mutated Ser super(26) to alanine or aspartic acid. The S26D mutation mimicking the phosphorylated state of CaMKII causes a dramatic decrease in Thr super(287) autophosphorylation levels and greatly reduces the catalytic activity towards an exogenous substrate (autocamtide-3), whereas the S26A mutation has no effect. These data combined with molecular modelling indicate that a negative charge at Ser super(26) of CaMKII gamma inhibits the catalytic activity of the enzyme towards its autophosphorylation site at Thr super(287) most probably by blocking ATP binding. We propose that Ser super(26) phosphorylation constitutes an important mechanism for switching off CaMKII activity.
Calmodulin (CaM) is a ubiquitous transducer of intracellular Ca²⁺ signals and plays a key role in the regulation of the function of all cells. The interaction of CaM with a specific target is ...determined not only by the Ca²⁺-dependent affinity of calmodulin but also by the proximity to that target in the cellular environment. Although a few reports of stimulus-dependent nuclear targeting of CaM have appeared, the mechanisms by which CaM is targeted to non-nuclear sites are less clear. Here, we investigate the hypothesis that MARCKS is a regulator of the spatial distribution of CaM within the cytoplasm of differentiated smooth-muscle cells. In overlay assays with portal-vein homogenates, CaM binds predominantly to the MARCKS-containing band. MARCKS is abundant in portal-vein smooth muscle (approximately16 microM) in comparison to total CaM (approximately40 microM). Confocal images indicate that calmodulin and MARCKS co-distribute in unstimulated freshly dissociated smooth-muscle cells and are co-targeted simultaneously to the cell interior upon depolarization. Protein-kinase-C (PKC) activation triggers a translocation of CaM that precedes that of MARCKS and causes multisite, sequential MARCKS phosphorylation. MARCKS immunoprecipitates with CaM in a stimulus-dependent manner. A synthetic MARCKS effector domain (ED) peptide labelled with a photoaffinity probe cross-links CaM in smooth-muscle tissue in a stimulus-dependent manner. Both cross-linking and immunoprecipitation increase with increased Ca²⁺ concentration, but decrease with PKC activation. Introduction of a nonphosphorylatable MARCKS decoy peptide blocks the PKC-mediated targeting of CaM. These results indicate that MARCKS is a significant, PKC-releasable reservoir of CaM in differentiated smooth muscle and that it contributes to CaM signalling by modulating the intracellular distribution of CaM.
Calponin is a thin-filament-associated protein that has been implicated in the regulation of smooth-muscle contractility. It binds to F-actin and inhibits the MgATPase activity of actomyosin. In the ...present work we have examined the effect of recombinant chicken gizzard alpha-calponin (R alpha CaP) on the binding of rabbit skeletal-muscle myosin subfragment 1 (S1) to F-actin and on the inhibition of its actin-activated MgATPase. We have found that binding of one R alpha CaP molecule to every three to four actin monomers is sufficient for maximal inhibition of acto-S1 ATPase. At this R alpha CaP/actin ratio R alpha CaP does not interfere with S1 binding to F-actin. At higher concentrations, R alpha CaP displaces S1 from F-actin and a 1:1 R alpha CaP-actin monomer complex is formed. R alpha CaP is also able to displace troponin I from its complex with F-actin which may reflect the amino acid sequence similarity between R alpha CaP and troponin I in their actin-binding regions.
Edema factor (EF) and CyaA are calmodulin (CaM)‐activated adenylyl cyclase exotoxins involved in the pathogenesis of anthrax and whooping cough, respectively. Using spectroscopic, enzyme kinetic and ...surface plasmon resonance spectroscopy analyses, we show that low Ca2+ concentrations increase the affinity of CaM for EF and CyaA causing their activation, but higher Ca2+ concentrations directly inhibit catalysis. Both events occur in a physiologically relevant range of Ca2+ concentrations. Despite the similarity in Ca2+ sensitivity, EF and CyaA have substantial differences in CaM binding and activation. CyaA has 100‐fold higher affinity for CaM than EF. CaM has N‐ and C‐terminal globular domains, each binding two Ca2+ ions. CyaA can be fully activated by CaM mutants with one defective C‐terminal Ca2+‐binding site or by either terminal domain of CaM while EF cannot. EF consists of a catalytic core and a helical domain, and both are required for CaM activation of EF. Mutations that decrease the interaction of the helical domain with the catalytic core create an enzyme with higher sensitivity to Ca2+–CaM activation. However, CyaA is fully activated by CaM without the domain corresponding to the helical domain of EF.
The activation mechanism of Ca2+/calmodulin-dependent protein kinase II (αCaMKII) is investigated by steady-state and stopped-flow fluorescence spectroscopies. Lys75-labeled TA-cal Török, K., and ...Trentham, D. R. (1994) Biochemistry 33, 12807−12820 is used to measure binding events, and double-labeled AEDANS,DDP-T34C/T110/C-calmodulin Drum et al. (2000) J. Biol. Chem. 275, 36334−36340 (DA-cal) is used to detect changes in calmodulin conformation. Fluorescence quenching of DA-cal attributed to resonance energy transfer is related to the compactness of the calmodulin molecule. Interprobe distances are estimated by lifetime measurements of Ca2+/DA-cal in complexes with unphosphorylated nucleotide-free, nucleotide-bound, and Thr286-phospho-αCaMKII as well as with αCaMKII-derived calmodulin-binding peptides in the presence of Ca2+. These measurements show that calmodulin can assume at least two spectrally distinct conformations when bound to αCaMKII with estimated interprobe distances of 40 and 22−26 Å. Incubation with ATP facilitates the assumption of the most compact conformation. Nonhydrolyzable ATP analogues partially replicate the effects of ATP, suggesting that while the binding of ATP induces a conformational change, Thr286-autophosphorylation is probably required for the transition of calmodulin into its most compact conformer. The rate constant for the association of Ca2+/TA-cal with αCaMKII is estimated as 2 × 107 M-1 s-1 and is not substantially affected by the presence of ATP. The rate of net calmodulin compaction measured by Ca2+/DA-cal is markedly slower, occurring with a rate constant of 2.5 × 106 M-1 s-1, suggesting that unproductive complexes may play a role in the activation mechanism.
Residues 89-100 of troponin C (C89-100) and 96-116 of troponin I (I96-116) interact with each other in the troponin complex (Dalgarno, D.C., Grand, R.J.A., Levine, B.A. Moir, A., J.G., Scott, G.M.M., ...and Perry, S.V. (1982) FEBS Lett. 150, 54-58) and are necessary for the Ca2+ sensitivity of actomyosin ATPase (Syska, H., Wilkinson, J.M., Grand, R.J.A., and Perry, S.V. (1976) Biochem. J. 153, 375-387 and Grabarek, Z., Drabikowski, W., Leavis, P.C., Rosenfeld, S.S., and Gergely, J. (1981) J. Biol. Chem. 256, 13121-13127). We have studied Ca2+-induced changes in the region C89-100 by monitoring the fluorescence of troponin C (TnC) labeled at Cys-98 with 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid. Equilibrium titration of the labeled TnC with Ca2+ indicates that the probe is sensitive to binding to both classes of sites in free TnC as well as in its complex with TnI. When Mg2 X TnC is mixed with Ca2+ in a stopped flow apparatus, there is a rapid fluorescence increase related to Ca2+ binding to the unoccupied sites I and II followed by a slower increase (k = 9.9 s-1) that represents Mg2+-Ca2+ exchange at sites III and IV. In the TnC X TnI complex, the fast phase is much larger and the Mg2+-Ca2+ exchange at sites III and IV results in a small decrease rather than an increase in the fluorescence of the probe. The possibility is discussed that the fast change in the environment of Cys-98 upon Ca2+ binding to sites I and II may be instrumental in triggering activation of the thin filament by facilitating a contact between C89-100 and I96-116.
An interaction between extracellular regulated kinase 1 (ERK1) and calponin has previously been reported (Menice, Hulvershorn, Adam, Wang and Morgan (1997) J. Biol. Chem. 272 (40), 25157-25161) and ...has been suggested to reflect a function of calponin as a signalling molecule. We report in this study that calponin binds to both ERK1 and ERK2 under native conditions as well as in an overlay assay. Using chymotryptic fragments of calponin, the binding site of ERK on calponin was identified as the calponin homology (CH) domain, an N-terminal region of calponin found in other actin-binding proteins. ERK also bound, in a gel overlay assay, alpha-actinin, a protein with two tandem CH domains, as well as a 27 kDa thermolysin product of alpha-actinin containing the CH domains of alpha-actinin. The CH domain of calponin could compete with intact calponin or alpha-actinin for ERK binding. Titration of acrylodan-labelled calponin with ERK gave a K(a) of 6x10(6) M(-1) and titration of acrylodan-labelled calponin with a peptide from the alphaL16 helix of ERK gave a K(a) of 1x10(6) M(-1). Recombinant ERK was found to co-sediment with purified actin and induced a fluorescence change in pyrene-labelled F-actin (K(a)=5x10(6) M(-1)). The interaction of ERK with CH domains points to a new potential function for CH domains. The interaction of ERK with actin raises the possibility that actin may provide a scaffold for ERK signalling complexes in both muscle and non-muscle cells.
The regulatory activity of troponin C is reversibly inhibited by a disulfide bridge between cysteine residues introduced by
site-directed mutagenesis in positions 48 and 82 (TnC48/82) in the ...N-terminal domain of rabbit skeletal troponin C (sTnC;
Grabarek, Z., Tan, R.-Y., Tao, T., and Gergely, J. (1990) Nature 345, 132-135). In the present work we have investigated the
effects of the disulfide on structural properties of TnC48/82 monitored by CD spectroscopy and limited trypsinolysis. The
CD spectra of the mutant protein in the oxidized form (oxTnC48/82) with and without Ca2+ are similar to the corresponding
ones of the reduced and carboxamidomethylated form (CAMTnC48/82), indicating that the disulfide has essentially no effect
on the overall secondary structure. The N-terminal domain of oxTnC48/82 is resistant to thermal unfolding, but that of CAMTnC48/82
is only slightly more stable than the corresponding domain of sTnC. In the presence of Ca2+ oxTnC48/82 is more resistant to
trypsinolysis than sTnC whereas the rate of tryptic digestion of CAMTnC48/82 is the same as that of sTnC, indicating that
peptide bonds adjacent to lysine residues at position 84 and 88, the sites of tryptic attack, are protected by the disulfide.
The disulfide cross-linked N-terminal peptide of TnC48/82 does not bind TnI, unlike its reduced or carboxamidomethylated forms.
Our data indicate that the disulfide between Cys48 and Cys82 stabilizes the structure of the N-terminal domain of TnC and
blocks its ability to interact with TnI. The effects of the disulfide appear to be restricted to the N-terminal domain of
TnC.
We have used a highly environment-sensitive fluorescent probe 6-bromoacetyl-2-dimethylaminonaphthalene (badan) to study the interaction between calmodulin (CaM) and a CaM-binding peptide of the ...ryanodine receptor (CaMBP) and its sub-fragments F1 and F4. Badan was attached to the Thr34Cys mutant of CaM (CaM-badan). Ca2+ increase in a physiological range of Ca2+ (0.1–2μM) produced about 40 times increase in the badan fluorescence. Upon binding to CaMBP, the badan fluorescence of apo-CaM showed a small increase at a slow rate; whereas that of Ca–CaM showed a large decrease at a very fast rate. Upon binding of CaM to the badan-labeled CaMBP, the badan fluorescence showed a small and slow increase at low Ca2+, and a large and fast increase at high Ca2+. Thus, the badan probe attached to CaM Cys34 can be used to monitor conformational changes occurring not only in CaM, but also those in the CaM–CaMBP interface. Based on our results we propose that both the interaction interface and the global conformation of the CaM–CaMBP complex are altered by calcium.
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