Structural evidence for non-canonical binding of Ca2+ to a canonical EF-hand of a conventional myosin

We have previously identified a single inhibitory Ca2+-binding site in the first EF-hand of the essential light chain of Physarum conventional myosin (Farkas, L., Malnasi-Csizmadia, A., Nakamura, A., Kohama, K., and Nyitray, L. (2003) J. Biol. Chem. 278, 27399-27405). As a general rule, conformation of the EF-hand-containing domains in the calmodulin family is "closed" in the absence and "open" in the presence of bound cations; a notable exception is the unusual Ca2+-bound closed domain in the essential light chain of the Ca2+-activated scallop muscle myosin. Here we have reported the 1.8 A resolution structure of the regulatory domain (RD) of Physarum myosin II in which Ca2+ is bound to a canonical EF-hand that is also in a closed state. The 12th position of the EF-hand loop, which normally provides a bidentate ligand for Ca2+ in the open state, is too far in the structure to participate in coordination of the ion. The structure includes a second Ca2+ that only mediates crystal contacts. To reveal the mechanism behind the regulatory effect of Ca2+, we compared conformational flexibilities of the liganded and unliganded RD. Our working hypothesis, i.e. the modulatory effect of Ca2+ on conformational flexibility of RD, is in line with the observed suppression of hydrogen-deuterium exchange rate in the Ca2+-bound form, as well as with results of molecular dynamics calculations. Based on this evidence, we concluded that Ca2+-induced change in structural dynamics of RD is a major factor in Ca2+-mediated regulation of Physarum myosin II activity.     

A hydrophobic region on myosin light chains modulated by divalent cations

A hydrophobic region was detected on several types of myosin light chain by enhancement of the quantum yield of 1-anilino-8-naphthalenesulfonate (ANS) fluorescence. The character of this non-polar region was altered by the binding of Ca2+ or Mg2+ to the light chain, the quantum yield of the ANS being increased, and its emission maximum undergoing a blue-shift. These changes enabled the binding of divalent cations to the myosin light chains to be monitored. When Ca2+ was bound to gizzard regulatory light chain, a biphasic enhancement of light-chain-bound ANS fluorescence occurred, the first phase taking place in the micromolar range and the second in the millimolar range of free Ca2+ concentration. Enhancement of protein-bound ANS fluorescence as divalent cations were bound was also observed with other types of myosin light chain.     

Fluorescence studies on the nucleotide- and Ca2+-binding domains of molluscan myosin.

The effects of nucleotides and Ca2+ on the intrinsic tryptophan fluorescence of molluscan myosin and its proteolytic fragments were studied. By using these proteins from the scallop, Pecten maximus, the existence of two distinct tryptophan-containing domains was established, which respond independently to ATP and Ca2+-specific binding. The latter is located in the 'neck' region of the myosin, which constitutes the regulatory domain. Subfragment 1, lacking the regulatory domain, responded only to ATP binding. On the other hand a tryptic fragment comprising the regulatory domain responded only to Ca2+ binding. Subfragment 1, containing the regulatory domain, responded to both ATP and Ca2+, but its ATPase activity was Ca2+-insensitive. By contrast, the ATPase activity of HMM was Ca2+-sensitive. Increasing the ionic strength had a detrimental effect on Ca2+-sensitivity, and fluorescence studies on solubilized myosin were therefore of limited value. Myosin and its fragments from other molluscan species which were investigated produced similar changes to those of Pectan maximus.     

The C-terminal helix in subdomain 4 of the regulatory light chain is essential for myosin regulation.

In vertebrate smooth/non-muscle myosins, phosphorylation of the regulatory light chains by a specific calmodulin-activated kinase controls both myosin head interaction with actin and assembly of the myosin into filaments. Previous studies have shown that the C-terminal domain of the regulatory light chain is crucial for the regulation of these myosin functions. To further dissect the role of this region of the light chain in myosin regulation, a series of chicken smooth muscle myosin regulatory light chain mutants has been constructed with successive C-terminal deletions. These mutants were synthesized in Escherichia coli and analysed by their ability to restore Ca2+ regulation to scallop myosin that had been stripped of its native regulatory light chains ('desensitized'). The results show that regulatory light chain mutants with deletions in the C-terminal helix in subdomain 4 were able to reform the regulatory Ca2+ binding site on the scallop myosin head, but had lost the ability to suppress scallop myosin filament assembly and interaction with actin in the absence of Ca2+. Further deletions in the C-terminal domain led to a gradual loss of ability to restore the regulatory Ca2+ binding site. Thus, the regions in the C-terminal half of the regulatory light chain responsible for myosin regulation can be identified.     

Conformational changes induced by binding of divalent cations to calregulin

Scatchard analysis of equilibrium dialysis studies have revealed that in the presence of 3.0 mM MgCl2 and 150 mM KCl, calregulin has a single binding site for Ca2+ with an apparent dissociation constant (apparent Kd) of 0.05 microM and 14 binding sites for Zn2+ with apparent Kd(Zn2+) of 310 microM. Ca2+ binding to calregulin induces a 5% increase in the intensity of intrinsic fluorescence and a 2-3-nm blue shift in emission maximum. Zn2+ binding to calregulin causes a dose-dependent increase of about 250% in its intrinsic fluorescence intensity and a red shift in the emission maximum of about 11 nm. Half-maximal wavelength shift occurs at 0.4 mol of Zn2+/mol of calregulin, and 100% of the wavelength shift is complete at 2 mol of Zn2+/mol of calregulin. In the presence of Zn2+ and calregulin the fluorescence intensity of the hydrophobic fluorescent probe 8-anilino-1-napthalenesulfonate (ANS) was enhanced 300-400% with a shift in emission maximum from 500 to 480 nm. Half-maximal Zn2+-induced shift in ANS emission maximum occurred at 1.2 mol of Zn2+/mol of calregulin, and 100% of this shift occurred at 6 mol of Zn2+/mol of calregulin. Of 12 cations tested, only Zn2+ and Ca2+ produced changes in calregulin intrinsic fluorescence, and none of these metal ions could inhibit the Zn2+-induced red shift in intrinsic fluorescence emission maximum. Furthermore, none of these cations could inhibit or mimic the Zn2+-induced blue shift in ANS emission maximum. These results suggest that calregulin contains distinct and specific ligand-binding sites for Ca2+ and Zn2+. While Ca2+ binding results in the movement of tryptophan away from the solvent, Zn2+ causes a movement of tryptophan into the solvent and the exposure of a domain with considerable hydrophobic character.     

Localization and characterization of the inhibitory Ca2+-binding site of Physarum polycephalum myosin II

A myosin II is thought to be the driving force of the fast cytoplasmic streaming in the plasmodium of Physarum polycephalum. This regulated myosin, unique among conventional myosins, is inhibited by direct Ca2+ binding. Here we report that Ca2+ binds to the first EF-hand of the essential light chain (ELC) subunit of Physarum myosin. Flow dialysis experiments of wild-type and mutant light chains and the regulatory domain revealed a single binding site that shows moderate specificity for Ca2+. The regulatory light chain, in contrast to regulatory light chains of higher eukaryotes, is unable to bind divalent cations. Although the Ca2+-binding loop of ELC has a canonical sequence, replacement of glutamic acid to alanine in the -z coordinating position only slightly decreased the Ca2+ affinity of the site, suggesting that the Ca2+ coordination is different from classical EF-hands; namely, the specific "closed-to-open" conformational transition does not occur in the ELC in response to Ca2+. Ca2+- and Mg2+-dependent conformational changes in the microenvironment of the binding site were detected by fluorescence experiments. Transient kinetic experiments showed that the displacement of Mg2+ by Ca2+ is faster than the change in direction of cytoplasmic streaming; therefore, we conclude that Ca2+ inhibition could operate in physiological conditions. By comparing the Physarum Ca2+ site with the well studied Ca2+ switch of scallop myosin, we surmise that despite the opposite effect of Ca2+ binding on the motor activity, the two conventional myosins could have a common structural basis for Ca2+ regulation.     

Regulation of scallop myosin by mutant regulatory light chains

Scallop adductor myosin is regulated by its subunits; the regulatory light chain (R-LC) and essential light chain (E-LC). Myosin light chains suppress muscle activity in the absence of calcium and are responsible for relaxation. The binding of Ca2+ to the myosin triggers contraction by releasing the inhibition imposed on myosin by the light chains. To map the functional domains of the R-LC, we have carried out mutagenesis followed by bacterial expression. Both wild-type and mutant proteins were hybridized to scallop myosin heavy chain/E-LC to map the regions of the light chain that are responsible for the binding to the myosin heavy chain/E-LC, for restoring the specific calcium-binding site, and controlling the myosin ATPase activity. The R-LC is expressed in Escherichia coli using the pKK223-3 (Pharmacia) expression vector and has been purified to greater than 90% purity. E. coli-expressed wild-type R-LC differs from the native R-LC by having the initiating methionine residue and an unblocked NH2 terminus. The wild-type R-LC restores Ca2+ binding and Ca2+ sensitivity when hybridized to scallop myosin. A point mutation of the sixth Ca2(+)-liganding position of domain I (Asp39----Ala39) results in a R-LC that binds more weakly to the heavy chain/E-LC and restores the specific Ca2(+)-binding site but not regulation of the actin-activated Mg2+ ATPase. A second mutation was produced by substituting the last 11 residues of the COOH terminus with 15 different residues. This mutant restores the specific Ca2(+)-binding site, but does not restore Ca2+ regulation to the actin-activated ATPase activity. Several other point mutations do not alter light chain function. The experiments directly establish that the divalent cation-binding site of domain I is functionally distinct from the specific Ca2(+)-binding site. The results indicate that an intact domain I and the COOH terminus are required to suppress the myosin ATPase activity. The fact that the domain I mutation and the COOH-terminal mutation disrupt regulation but do not affect Ca2(+)-binding indicates that these two aspects of regulation are separable and, therefore, the R-LC has distinct functional regions.     

Segmental flexibility and head-head interaction in scallop myosin. A study using saturation transfer electron paramagnetic resonance spectroscopy

Saturation transfer electron paramagnetic resonance spectroscopy was used to investigate the rotational motion of the head domains of native and desensitized scallop myosin and its proteolytic subfragments. Scallop myosin was spin-labelled with 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidinooxyl, which reacted with a heavy chain residue in the subfragment 1 domain. As previously shown for rabbit skeletal muscle myosin (Thomas et al., 1975), the two head domains of native scallop myosin appear to have independent motion (rotational correlation time, pi, = 0.8 X 10(-7) s for subfragment 1; 1.4 X 10(-7) s for myosin). However, removal of a regulatory light chain, to effect desensitization of the actin-activated ATPase, was associated with an increase in pi for myosin to a value of 2.4 X 10(-6) s. The Ca2+ sensitivity and initial correlation time were restored on recombination of the regulatory light chain in the presence of Mg2+. Sedimentation velocity profiles in an analytical ultracentrifuge indicated that the desensitized myosin preparations were largely monomeric and therefore the change in pi appears to reflect an intramolecular event. Addition of EDTA to spin-labelled scallop heavy meromyosin caused an immediate 2.5 to 4-fold increase in pi and a partial desensitization of the ATPase activity. Comparable experiments with subfragment 1 yielded a barely detectable increase in pi (1.5-fold) in the first ten minutes. The restricted rotational motion observed in desensitized myosin and heavy meromyosin could arise by a conformational change in the subfragment 1-subfragment 2 hinge region or by an association of one head with its partner. The latter mechanism, involving the exposed light chain binding site, would also explain the preferential release of one regulatory light chain from scallop myosin, and might account for some other co-operative effects observed in this molecule (Bagshaw, 1980).     

Inhibitory effect of MgATP on the release of regulatory light chain from scallop myosin and light chain composition of scallop myosin hybridized with abalone light chain 2 at 30 degrees C

The experimental conditions for release of the regulatory light chain (RLC) of scallop myosin at 30 degrees C were studied. Substantially all RLC was released from myosin by incubation for 5 min in medium containing buffer and KCl. This release of RLC was inhibited strongly by Ca2+, while the effect of Mg2+ was about 10,000 times weaker than that of Ca2+. Even in the absence of Ca2+, MgATP and MgADP inhibited the release of RLC, while the protective effect of AMPPNP was negligible. Other Mg nucleotides also showed some protective effect, though appreciably less than MgATP. The incubation of scallop myosin with abalone regulatory light chain (LC2) at 30 degrees C for 5 min produced a hybrid myosin. In the presence of 5 mM MgCl2, 1 of the 2 mol of RLC per mol of scallop myosin was exchanged with 1 mol of LC2. In the presence of Ca2+ or MgATP, myosin bound 1 extra mole of LC2 besides the 2 mol each of SH-LC and RLC.     

The On-Off Switch in Regulated Myosins: Different Triggers but Related Mechanisms

In regulated myosin, motor and enzymatic activities are toggled between the on-state and off-state by a switch located on its lever arm domain, here called the regulatory domain (RD). This region consists of a long α-helical “heavy chain” stabilized by a “regulatory” light chain (RLC) and an “essential” light chain (ELC). The on-state is activated by phosphorylation of the RLC of vertebrate smooth muscle RD or by direct binding of Ca²⁺ to the ELC of molluscan RD. Crystal structures are available only for the molluscan RD. To understand in more detail the pathway between the on-state and the off-state, we have now also determined the crystal structure of a molluscan (scallop) RD in the absence of Ca²⁺. Our results indicate that loss of Ca²⁺ abolishes most of the interactions between the light chains and may increase the flexibility of the RD heavy chain. We propose that disruption of critical links with the C-lobe of the RLC is the key event initiating the off-state in both smooth muscle myosins and molluscan myosins.     

Hybrid myosin light chains containing a calcium-specific site from troponin C.

Recombinant DNA approaches have allowed us to probe the mechanisms by which the regulatory light chains (RLCs) regulate myosin function by identifying the functional importance of specific regions of the RLC molecule. For example, we have demonstrated that the presence of high-affinity Ca2+/Mg(2+)-binding site in the N-terminal domain of the RLC is essential for the regulation of myosin-actin interaction [Reinach, F. C., Nagai, K. & Kendrick-Jones, J. (1986) Nature 322, 80-83]. To explore further the role of this metal-binding site in the RLC and generate an RLC with a Ca(2+)-specific site, we constructed four chicken skeletal muscle myosin regulatory light chain hybrid 'genes'. In these, the first domain containing the high-affinity Ca2+/Mg(2+)-binding site in the RLC was replaced with that containing the lower-affinity, Ca(2+)-specific, regulatory site from troponin C (TnC). In two of these hybrids, we replaced only the Ca(2+)-binding EF hand, while in the other two the EF hand and the N-terminal helix of TnC were transplanted. These hybrids were expressed in Escherichia coli in high yields and the purified proteins were used in calcium-binding experiments to assay the affinity and specificity of the sites and incorporated into scallop myosin to assay their regulatory behaviour. The results obtained show that the calcium-binding site from TnC, when transplanted into the RLC backbone, had a low affinity although most of its specificity appeared to be retained. As a result, although the TnC/RLC hybrids bound to scallop myosin and were able to activate the MgATPase activity of scallop acto-myosin, they were unable to regulate it. These results are in agreement with our previous findings that occupancy of the Ca2+/Mg2+ site in the RLC is essential for regulation. Our results suggest that the specificity and affinity of the calcium-binding site in troponin C is dependent on both intra- and inter-domain interactions within troponin C and that these latter interactions appear to be missing when this binding site is transplanted into the light chain backbone.     

Ca2(+)-dependent protein phosphatase which dephosphorylates regulatory light chain-a in scallop smooth muscle myosin

Ca2(+)-dependent protein phosphatase was purified from scallop adductor smooth muscle by a combination of DEAE-Toyoperal 650S ion exchange chromatographies and gel filtration on Sephacryl S-300. The phosphatase consisted of two subunits having molecular weights of 60 and 19 kDa. Phosphorylated regulatory light chain-a (RLC-a) was dephosphorylated by this phosphatase both in free and bound states in myosin prepared from the opaque portion of scallop smooth muscle (opaque myosin). The dephosphorylation was activated by Ca2+. The half maximal activation was a 1 microM free Ca2+ in the presence of calmodulin and 7 microM free Ca2+ in the absence of calmodulin. Opaque myosin phosphorylated at the heavy chain was not dephosphorylated with this phosphatase. p-Nitrophenyl phosphate was dephosphorylated. In addition to Ca2+, the phosphatase activity for RLC-a was activated by Mn2+, while p-nitrophenylphosphatase activity was activated by Mg2+ more strongly than by Mn2+. The pH-activity curves showed a maximum at pH 7 in the presence of Mn2+, but at around pH 8 in the presence of Mg2+. This phosphatase is similar to phosphatase 2B or calcineurin. The possible regulatory function of this phosphatase in scallop catch muscle is discussed.     

Calcium binding and conformation of regulatory light chains of smooth muscle myosin of scallop

Calcium binding was studied with two regulatory light chains (RLC-a and RLC-b) of smooth muscle myosin of scallop. With the equilibrium dialysis method, the binding of 0.98 mol Ca2+ per mol of RLC-b was observed with a dissociation constant of 2.3 X 10(-5) M. Similar values for RLC-b, 1.9 X 10(-5) M, and RLC-a, 1.5 X 10(-5) M, were obtained by measuring the difference absorption spectrum induced by Ca2+. The difference molar absorption coefficient at 288 nm was 159 and 209 M-1 X cm-1 for RLC-a and RLC-b, respectively, while it was -34 M-1 X cm-1 for the regulatory light chain of striated muscle myosin of scallop (RLC-st). Proton NMR spectra of the three light chains were very similar to each other and were broader than those of other Ca2+ binding proteins, parvalbumin and calmodulin. The regulatory light chains may be more rigid than in these Ca2+ binding proteins. CD spectra were measured for the three light chains, and the estimated helix contents were 27, 29, and 24%, respectively, for RLC-a, RLC-b, and RLC-st. All these results in comparison with the primary structures led us to suppose that the polypeptide of regulatory light chains is folded in such a way that domain 4 becomes near to the calcium binding site of domain 1. The decrease in intact light chains on trypsin digestion was determined for the gel electrophoretic patterns. RLC-a was 6 times more susceptible to the tryptic digestion than RLC-b.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early events during folding of wild-type staphylococcal nuclease and a single-tryptophan variant studied by ultrarapid mixing

A continuous-flow mixing device with a dead time of 100 micros coupled with intrinsic tryptophan and 1-anilinonaphthalene-8-sulfonate (ANS) fluorescence was used to monitor structure formation during early stages of the folding of staphylococcal nuclease (SNase). A variant with a unique tryptophan fluorophore in the N-terminal beta-barrel domain (Trp76 SNase) was obtained by replacing the single Trp140 in wild-type SNase with His in combination with Trp substitution of Phe76. A common background of P47G, P117G and H124L mutations was chosen in order to stabilize the protein and prevent accumulation of cis proline isomers under native conditions. In contrast to WT(*) SNase, which shows no changes in tryptophan fluorescence prior to the rate-limiting folding step ( approximately 100 ms), the F76W/W140H variant shows additional changes (enhancement) during an early folding phase with a time constant of 75 micros. Both proteins exhibit a major increase in ANS fluorescence and identical rates for this early folding event. These findings are consistent with the rapid accumulation of an ensemble of states containing a loosely packed hydrophobic core involving primarily the beta-barrel domain while the specific interactions in the alpha-helical domain involving Trp140 are formed only during the final stages of folding. The fact that both variants exhibit the same number of kinetic phases with very similar rates confirms that the folding mechanism is not perturbed by the F76W/W140H mutations. However, the Trp at position 76 reports on the rapid formation of a hydrophobic cluster in the N-terminal beta-sheet region while the wild-type Trp140 is silent during this early stage of folding. Quantitative modeling of the (un)folding kinetics and thermodynamics of these two proteins versus urea concentration revealed that the F76W/W140H mutation selectively destabilizes the native state relative to WT(*) SNase while the stability of transient intermediates remains unchanged, leading to accumulation of intermediates under equilibrium conditions at moderate denaturant concentrations.     

The kinetics of bivalent metal ion dissociation from myosin subfragments.

Bivalent metal ions have multiple roles in subunit association and ATPase regulation in scallop adductor-muscle myosin. To help elucidate these functions, the rates of Ca2+ and Mg2+ dissociation from the non-specific high-affinity sites on the regulatory light chains were measured and compared with those of rabbit skeletal-muscle myosin subfragments. Ca2+ dissociation had a rate constant of about 0.7 s-1 in both species, as measured by the time course of the pH change on EDTA addition. Mg2+ dissociation had a rate constant of 0.05 s-1, as monitored by its displacement with the paramagnetic Mn2+ ion. It is concluded that the exchange between Ca2+ and Mg2+ at the non-specific site, on excitation of both skeletal and adductor muscles, is too slow to contribute to the activation itself. The release of bivalent metal ions from the non-specific site is, however, the first step in release of the scallop regulatory light chain (Bennett & Bagshaw (1986) Biochem. J. 233, 179-186). In scallop myosin additional specific sites are present, which can bind Ca2+ rapidly, to effect activation of the ATPase. In the course of this work, Ca2+ dissociation from EGTA was studied as a model system. This gave rates of 1 s-1 and 0.3 s-1 at pH 7.0 and pH 8.0 respectively.     

Complete primary structure of a scallop striated muscle myosin heavy chain. Sequence comparison with other heavy chains reveals regions that might be critical for regulation

We have determined the primary structure of the myosin heavy chain (MHC) of the striated adductor muscle of the scallop Aequipecten irradians by cloning and sequencing its cDNA. It is the first heavy chain sequence obtained in a directly Ca(2+)-regulated myosin. The 1938-amino acid sequence has an overall structure similar to other MHCs. The subfragment-1 region of the scallop MHC has a 59-62% sequence identity with sarcomeric and a 52-53% identity with nonsarcomeric (smooth and metazoan nonmuscle) MHCs. The heavy chain component of the regulatory domain (Kwon, H., Goodwin, E. B., Nyitray, L., Berliner, E., O'Neall-Hennessey, E., Melandri, F. D., and Szent-Gy?rgyi, A. G. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4771-4775) starts at either Leu-755 or Val-760. Ca(2+)-sensitive Trp residues (Wells, C., Warriner, K. E., and Bagshaw, C. R. (1985) Biochem. J. 231, 31-38) are located near the C-terminal end of this segment (residues 818-827). More detailed sequence comparison with other MHCs reveals that the 50-kDa domain and the N-terminal two-thirds of the 20-kDa domain differ substantially between sarcomeric and nonsarcomeric myosins. In contrast, in the light chain binding region of the regulatory domain (residues 784-844) the scallop sequence shows greater homology with regulated myosins (smooth muscle, nonmuscle, and invertebrate striated muscles) than with unregulated ones (vertebrate skeletal and heart muscles). The N-terminal 25-kDa domain also contains several residues which are preserved only in regulated myosins. These results indicate that certain heavy chain sites might be critical for regulation. The rod has features typical of sarcomeric myosins. It is 52-60% and 30-33% homologous with sarcomeric and nonsarcomeric MHCs, respectively. A Ser-rich tailpiece (residues 1918-1938) is apparently nonhelical.     

Effects of removal and reconstitution of myosin regulatory light chain and troponin C on the Ca(2+)-sensitive ATPase activity of myofibrils from scallop striated muscle

In order to examine the involvement of troponin-linked Ca(2+)-regulation, in addition to well-known myosin-linked Ca(2+)-regulation, in the contraction of molluscan striated muscle, myofibrils from Ezo-giant scallop striated muscle were desensitized to Ca(2+) by removing both myosin regulatory light chain and troponin C by treatment with a strong divalent cation chelator, CDTA. The ATPase level in the desensitized myofibrils was about half the maximum level in intact myofibrils regardless of the Ca(2+)-concentration at 25 and 15 degrees C. In the absence of Ca(2+), the ATPase of the desensitized myofibrils was suppressed by myosin regulatory light chain but not affected by troponin C at either temperature. The ATPase was activated at higher Ca(2+)-concentrations by both myosin regulatory light chain and troponin C, but the activating effects of these two proteins were affected differently by temperature. The activation of ATPase by myosin regulatory light chain was much greater than that by troponin C at 25 degrees C, whereas the activation by troponin C was much greater than that by myosin regulatory light chain at 15 degrees C. The maximum activation was only obtained in the presence of both myosin regulatory light chain and troponin C at these temperatures. These findings strongly suggest that the contraction of scallop striated muscle is regulated through both myosin-linked and troponin-linked Ca(2+)-regulation, and that the troponin-linked Ca(2+)-regulation is more significant at lower temperature.     

Spectroscopic characterization of the EF-hand domain of phospholipase C delta1: identification of a lipid interacting domain

The interaction of the isolated EF-hand domain of phospholipase C delta1 with arachidonic acid (AA) was characterized using circular dichroism (CD) and fluorescence spectroscopy. The far-UV CD spectral changes indicate that AA binds to the EF domain. The near-UV CD spectra suggest that the orientations of aromatic residues in the peptide are affected when AA binds to the protein. The fluorescence of the single intrinsic tryptophan located in EF1 was enhanced by the addition of dodecylmaltoside (DDM) and AA suggesting that this region of the protein is involved in hydrophobic interactions. In the presence of a low concentration of DDM it was found that AA induced a change in fluorescence resonance energy transfer, which is indicative of a conformational change. The lipid induced conformational change may play a role in calcium binding because the isolated EF-hand domain did not bind Ca2+ in the absence of lipids, but Ca2+-dependent changes in the intrinsic tryptophan emission were observed when free fatty acids were present. These studies identify specific EF-hand domains as allosteric regulatory domains that require hydrophobic ligands such as lipids.     

Ca(2+) induces an extended conformation of the inhibitory region of troponin I in cardiac muscle troponin.

The inhibitory region of troponin I (TnI) plays a central regulatory role in the contraction and relaxation cycle of skeletal and cardiac muscle through its Ca(2+)-dependent interaction with actin. Detailed structural information on the interface between TnC and this region of TnI has been long in dispute. We have used fluorescence resonance energy transfer (FRET) to investigate the global conformation of the inhibitory region of a full-length TnI mutant from cardiac muscle (cTnI) in the unbound state and in reconstituted complexes with the other cardiac troponin subunits. The mutant contained a single tryptophan residue at the position 129 which was used as an energy transfer donor, and a single cysteine residue at the position 152 labeled with IAEDANS as energy acceptor. The sequence between Trp129 and Cys152 in cTnI brackets the inhibitory region (residues 130-149), and the distance between the two sites was found to be 19.4 A in free cTnI. This distance was insensitive to reconstitution of cTnI with cardiac troponin T (cTnT), cTnC, or cTnC and cTnT in the absence of bound regulatory Ca(2+) in cTnC. An increase of 9 A in the Trp129-Cys152 separation was observed upon saturation of the Ca(2+) regulatory site of cTnC in the complexes. This large increase suggests an extended conformation of the inhibitory region in the interface between cTnC and cTnI in holo cardiac troponin. This extended conformation is different from a recent model of the Ca(2+)-saturated skeletal TnI-TnC complex in which the inhibitory region is modeled as a beta-turn. The observed Ca(2+)-induced conformational change may be a switch mechanism by which movement of the regulatory region of cTnI to the exposed hydrophobic patch of the open regulatory N-domain of cTnC pulls the inhibitory region away from actin upon Ca(2+) activation in cardiac muscle.     

Anisodamine causes the changes of structure and function in the transmembrane domain of the Ca(2+)-ATPase from sarcoplasmic reticulum

The effects of anisodamine on the Ca(2+)-ATPsae of sarcoplasmic reticulum (SR) were investigated by using differential scanning calorimetry to measure the ability of anisodamine to denature the transmembrane domain and the cytoplasmic domain. Anisodamine significantly altered the thermotropic phase behaviors of the transmembrane domain of purified Ca(2+)-ATPase. Specifically, the melting temperature of the transmembrane domain moved toward lower temperatures with the concentrations of anisodamine increasing and the thermotropic phase peak was abolished at 10 mM, indicating that the stabilized structure of the transmembrane domain in the presence of Ca2+ could be destabilized by anisodamine. Decreases of the intrinsic fluorescence and increases of the extrinsic fluorescence of ANS, a fluorescent probe, showed the exposure of tryptophan and hydrophobic region, respectively, suggesting again that anisodamine caused a less compact conformation in the transmembrane domain. A marked inhibition of the Ca2+ uptake activity of SR Ca(2+)-ATPase was observed when the addition of anisodamine. The drug did not affect the cytoplasmic domain of the enzyme and only slightly decreased the ATPase activity of the enzyme at concentrations up to 10 mM. This was likely due to the destabilized protein transmembrane domain. To sum up, our results revealed that anisodamine interacted specifically with the transmembrane domain of SR Ca(2+)-ATPase and inhibited the Ca2+ uptake activity of the enzyme.