Effect of troponin C on the cooperativity in Ca2+ activation of cardiac muscle

The presence of protein kinase C (PKC), a key enzyme in signal transduction, has not been investigated in fungal cells. The phorbol ester TPA, an activator of PKC, may be used as an indicator of the presence and role of PKC in Phycomyces blakesleeanus spores. Activation of spore germination by acetate was prevented by 6 nM TPA. The TPA analog 4 alpha PDD, an ineffective activator of PKC, did not affect spore germination. 3 mM dbcAMP, on the other hand, reversed the inhibition of germination caused by TPA. TPA-stimulated protein kinase activity was detected in spores. The possible relationship between PKC and the increased levels of cAMP that accompany the induction of spore germination is discussed.     

Apparent cooperativity of Ca2+ binding associated with crystallization of Ca2+-binding protein from sarcoplasmic reticulum

Needle-shaped crystals of the Ca2+-binding protein (CBP) isolated from rabbit skeletal muscle sarcoplasmic reticulum were studied with regard to the influence of Ca2+, K+, and H+ on its solubility and cation binding. The solubility of CBP is sharply decreased with concentration of Ca2+, whereas K+ increased it. Aggregation of the CBP and crystal formation is correlated with the binding of Ca2+. The Ca2+ bound to the crystalline CBP is two to three times higher than that of the soluble form. A strong apparent positive cooperative behavior of Ca2+ binding by CBP was observed concomitant with the shift in equilibrium from the soluble to the crystalline form. From the steepest Hill slope we obtained Hill coefficients of 3.3 for soluble CBP and 14 for the transition between soluble and crystalline forms of CBP. A detailed treatment is presented to validate the applicability of Hill plots for the combined binding and crystallization process. Two-thirds of the Ca2+-binding sites were K+ sensitive and one-third were K+ insensitive. An increase in H+ concentration decreased the Ca2+ binding by crystalline CBP without affecting its solubility, with a pK value of 6.2 determined for this process. These results indicate that the equilibrium between the soluble and crystalline forms of CBP is determined by the amount and nature of the bound cations, Ca2+, K+, and H+. They suggest the possibility that a cycle of aggregation and solubilization of CBP attends the uptake and release of Ca2+ in the sarcoplasmic reticulum, respectively.     

The cPLA2 C2alpha domain in solution: structure and dynamics of its Ca2+-activated and cation-free states

Cytosolic phospholipase A2 is involved in several signal transduction pathways where it catalyses release of arachidonic acid from intracellular lipid membranes. Its membrane insertion is facilitated by its independently folding C2alpha domain, which is activated by the binding of two intracellular Ca2+ ions. However, the details of its membrane-insertion mechanism, including its Ca2+-activation mechanism, are not understood. There are several unresolved issues, including the following. There are two experimentally resolved structures of the Ca2+-activated state of its isolated C2alpha domain, one determined using x-ray crystallography and the other determined using NMR spectroscopy, which differ from each other significantly in the spatial region that inserts into the membrane. This by itself adds to ambiguities associated with investigations targeting its mechanism of membrane insertion. Furthermore, there is no experimentally determined structure of its cation-free state, which hinders investigations associated with its cation-activation mechanism. In this work, we generate several unrestrained molecular dynamics trajectories of its isolated C2alpha domain in solution (equivalent to approximately 60 ns) and investigate these issues. Our main results are as follows: a), the Ca2+ coordination scheme of the domain is consistent with the x-ray structure and with previous mutagenesis studies; b), the helical segment of the Ca2+-binding loop, CBL-I, undergoes nanosecond timescale flexing (but not an unwinding), as can be inferred from physiological temperature NMR data and in contrast to low temperature x-ray data; and c), removal of the two activating Ca2+ ions from their binding pockets does not alter the backbone structure of the domain, a result consistent with electron paramagnetic resonance data.     

Common structural framework of the two Ca2+/Mg2+ binding loops of troponin C and other Ca2+ binding proteins

The refinement of the crystal structure of turkey skeletal muscle troponin C at 2.2-A resolution reveals that the two calcium binding loops that are occupied by Ca2+ ions adopt conformations very similar to those of the two homologous loops of parvalbumin and to that of loop III-IV of the intestinal calcium binding protein. This specific fold assures suitable spatial positioning of the Ca2+ ligands. It consists of two reverse turns, one located at each end of the loop, and four Asx turns (a cyclic hydrogen-bonded structure involving an oxygen of the side chain of residue n and the main-chain amide nitrogen of residue n + 2) whenever such a side chain coordinates to the metal ion. The fifth Ca2+ coordination position in both loops of troponin C is occupied by a water molecule that is within hydrogen-bonding distance of an aspartic acid, thus mediating indirect interaction between the cation and the negatively charged carboxylate. The same loop framework is conserved in the two Ca2+ binding loops of parvalbumin and loop III-IV of the intestinal Ca2+ binding protein in spite of the variability in the nature of the side chains at equivalent positions. The disposition of the Ca2+ and of its coordinating water molecule relative to the protein main chain is conserved in all these cases.     

Calpain activation by cooperative Ca2+ binding at two non-EF-hand sites

The active site residues in calpain are mis-aligned in the apo, Ca(2+)-free form. Alignment for catalysis requires binding of Ca2+ to two non-EF-hand sites, one in each of the core domains I and II. Using domain swap constructs between the protease cores of the mu and m isoforms (which have different Ca2+ requirements) and structural and biochemical characterization of site-directed mutants, we have deduced the order of Ca2+ binding and the basis of the cooperativity between the two sites. Ca2+ binds first to the partially preformed site in domain I. Knockout of this site through D106A substitution eliminates binding to this domain as shown by the crystal structure of D106A muI-II. However, at elevated Ca2+ concentrations this mutant still forms the double salt bridge that links the two Ca2+ sites and becomes nearly as active as muI-II. Elimination of the bridge in E333A muI-II has a more drastic effect on enzyme action, especially at low Ca2+ concentrations. Domain II Ca2+ binding appears essential, because Ca(2+)-coordinating side-chain mutants E302R and D333A have severely impaired muI-II activation and activity. The introduction of mutations into the whole heterodimeric enzyme that eliminate the salt bridge or Ca2+ binding to domain II produce similar phenotypes, suggesting that the protease core Ca2+ switch is crucial and cannot be overridden by Ca2+ binding to other domains.     

Ca2+ binding effects on the C2 domain conformation of human cytosolic phospholipase A2

It has been reported that the cooperative binding of calcium ions indicated a local conformational change of the human cytosolic phospholipase A2 (cPLA2) C2 domain (Nalefski et al., (1997) Biochemistry 36, 12011-12018). However its structural evidence is less known (Malmberg et al., (2003) Biochemistry 42, 13227-13240). In this letter, life-time decay and fluorescence quenching techniques were employed to compare the calcium-induced conformational changes. The life-time decay parameters and fluorescence quenching constant changes were small between the apo- and holo-C2 domains when tryptophan residue was excited at 295 nm. In contrast, the quenching constant change was large, from 0.52 M(-1) for the apo-C2 to 8.8 M(-1) for the holo-C2 domain, when tyrosine residues were excited at 284 nm. Our results provide new information on amino acid side chain orientation change at calcium binding loop 3, which is necessary for Ca2+ binding regulated membrane targeting of human cytosolic phospholipase A2.     

Intra- and intersubunit cooperativity in activation of BK channels by Ca2+

The activation of BK channels by Ca(2+) is highly cooperative, with small changes in intracellular Ca(2+) concentration having large effects on open probability (Po). Here we examine the mechanism of cooperative activation of BK channels by Ca(2+). Each of the four subunits of BK channels has a large intracellular COOH terminus with two different high-affinity Ca(2+) sensors: an RCK1 sensor (D362/D367) located on the RCK1 (regulator of conductance of K(+)) domain and a Ca-bowl sensor located on or after the RCK2 domain. To determine interactions among these Ca(2+) sensors, we examine channels with eight different configurations of functional high-affinity Ca(2+) sensors on the four subunits. We find that the RCK1 sensor and Ca bowl contribute about equally to Ca(2+) activation of the channel when there is only one high-affinity Ca(2+) sensor per subunit. We also find that an RCK1 sensor and a Ca bowl on the same subunit are much more effective in increasing Po than when they are on different subunits, indicating positive intrasubunit cooperativity. If it is assumed that BK channels have a gating ring similar to MthK channels with alternating RCK1 and RCK2 domains and that the Ca(2+) sensors act at the flexible (rather than fixed) interfaces between RCK domains, then a comparison of the distribution of Ca(2+) sensors with the observed responses suggest that the interface between RCK1 and RCK2 domains on the same subunit is flexible. On this basis, intrasubunit cooperativity arises because two high-affinity Ca(2+) sensors acting across a flexible interface are more effective in opening the channel than when acting at separate interfaces. An allosteric model incorporating intrasubunit cooperativity nested within intersubunit cooperativity could approximate the Po vs. Ca(2+) response for eight possible subunit configurations of the high-affinity Ca(2+) sensors as well as for three additional configurations from a previous study.     

Conformational transitions in the Ca2+ + Mg2+-activated ATPase and the binding of Ca2+ ions.

We have studied the fluorescence of the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum labelled with fluorescein isothiocyanate. The change in intensity of fluorescein fluorescence caused by addition of Ca2+ to the labelled ATPase can be interpreted in terms of a two-conformation model for the ATPase, one conformation (E1) having a high affinity for Ca2+, the other (E2) a low affinity. Effects of Ca2+ as a function of pH allow an estimate of the effect of pH on the E1/E2 ratio, consistent with kinetic studies. A model is presented for binding of Ca2+ to the ATPase as a function of pH that is consistent both with the data on the E1/E2 equilibrium and with literature data on Ca2+ binding.     

The battle of two ions: Ca2+ signalling against Na+ stress

Soil salinity adversely affects plant growth, crop yield and the composition of ecosystems. Salinity stress impacts plants by combined effects of Na⁺ toxicity and osmotic perturbation. Plants have evolved elaborate mechanisms to counteract the detrimental consequences of salinity. Here we reflect on recent advances in our understanding of plant salt tolerance mechanisms. We discuss the embedding of the salt tolerance‐mediating SOS pathway in plant hormonal and developmental adaptation. Moreover, we review newly accumulating evidence indicating a crucial role of a transpiration‐dependent salinity tolerance pathway, that is centred around the function of the NADPH oxidase RBOHF and its role in endodermal and Casparian strip differentiation. Together, these data suggest a unifying and coordinating role for Ca²⁺ signalling in combating salinity stress at the cellular and organismal level.     

Sr2+ binding to the Ca2+ binding site of the synaptotagmin 1 C2B domain triggers fast exocytosis without stimulating SNARE interactions

Sr(2+) triggers neurotransmitter release similar to Ca(2+), but less efficiently. We now show that in synaptotagmin 1 knockout mice, the fast component of both Ca(2+)- and Sr(2+)-induced release is selectively impaired, suggesting that both cations partly act by binding to synaptotagmin 1. Both the C(2)A and the C(2)B domain of synaptotagmin 1 bind Ca(2+) in phospholipid complexes, but only the C(2)B domain forms Sr(2+)/phospholipid complexes; therefore, Sr(2+) binding to the C(2)B domain is sufficient to trigger fast release, although with decreased efficacy. Ca(2+) induces binding of the synaptotagmin C(2) domains to SNARE proteins, whereas Sr(2+) even at high concentrations does not. Thus, triggering of the fast component of release by Sr(2+) as a Ca(2+) agonist involves the formation of synaptotagmin/phospholipid complexes, but does not require stimulated SNARE binding.     

Co-operative binding of Ca2+ ions to the regulatory binding sites of gelsolin.

The rate of association of actin with gelsolin was measured at various Ca2+ and ATP concentrations. The fraction of Ca2+-activated gelsolin was determined by quantitative evaluation of the association rates thereby assuming that Ca2+-binding gelsolin associates with actin and Ca2+-free gelsolin does not. A plot of the fraction of Ca2+-activated gelsolin vs. the free Ca2+ concentration revealed a sigmoidal shape suggesting that co-operative binding of Ca2+ ions is required for activation of gelsolin. A good fit of the experimental data by calculated binding curves was obtained if two Ca2+ ions were assumed to bind to actin in a highly co-operative manner. ATP decreased the rate of association of gelsolin with actin and bound to gelsolin at a low affinity (Kd = 32 microm for Ca2+-free and Kd = 400 microm for Ca2+-activated gelsolin). In contrast, a 1 : 1 gelsolin-actin complex was found to be activated for association with actin by a single Ca2+ ion in a non-co-operative manner.     

The conformation of the activation peptide of protein C is influenced by Ca2+ and Na+ binding

Previous studies have suggested that the conformation of the activation peptide of protein C is influenced by the binding of Ca(2+). To provide direct evidence for the linkage between Ca(2+) binding and the conformation of the activation peptide, we have constructed a protein C mutant in the gamma-carboxyglutamic acid-domainless form in which the P1 Arg(169) of the activation peptide is replaced with the fluorescence reporter Trp. Upon binding of Ca(2+), the intrinsic fluorescence of the mutant decreases approximately 30%, as opposed to only 5% for the wild-type, indicating that Trp(169) is directly influenced by the divalent cation. The K(d) of Ca(2+) binding for the mutant protein C was impaired approximately 4-fold compared with wild-type. Interestingly, the conformation of the activation peptide was also found to be sensitive to the binding of Na(+), and the affinity for Na(+) binding increased approximately 5-fold in the presence of Ca(2+). These findings suggest that Ca(2+) changes the conformation of the activation peptide of protein C and that protein C is also capable of binding Na(+), although with a weaker affinity compared with the mature protease. The mutant protein C can no longer be activated by thrombin but remarkably it can be activated efficiently by chymotrypsin and by the thrombin mutant D189S. Activation of the mutant protein C by chymotrypsin proceeds at a rate comparable to the activation of wild-type protein C by the thrombin-thrombomodulin complex.     

Ca2+-induced structural change in the Ca2+Mg2+ domain of troponin C detected by crosslinking

Rabbit muscle troponin C was selectively modified at Cys-98 by 1,3-difluoro-4,6-dinitrobenzene. The second function of the bifunctional reagent was triggered at alkaline pH in the presence and absence of Ca²⁺. The crosslinked troponin C was hydrolyzed by trypsin and the peptides containing a dinitrobenzene moiety were isolated. When troponin C was crosslinked in the presence of Ca²⁺, the single dinitrobenzene-containing peptide was Gly-89-Arg-100, in which Cys-98 was crosslinked with Lys-90. When crosslinking was performed in the absence of Ca²⁺, beside the above peptide two additional peptides containing dinitrobenzene were found. One of these peptides is made up of two fragments, Ser-91-Arg-100 and Asn-105-Arg-120, crosslinked between Cys-98 and Tyr-109. The second peptide, Ala-121-Lys-140, contains modified Lys-136, presumably crosslinked with His-135. The data indicate that the distances between the α-carbon of Cys-98 and those of Lys-90, Tyr-109, Lys-136 and probably the α-carbon distance His-125-Lys-136, do not exceed 14 Å. Comparison with the X-ray structure of troponin C (Herzberg, O, and James, M.N.G. (1985) Nature 313, 653–659) indicates that some of the above distances increase on Ca²⁺-binding.     

A calmodulin binding domain of RyR increases activation of spontaneous Ca2+ sparks in frog skeletal muscle

The calmodulin C lobe binding region (residues 3614-3643) on the sarcoplasmic reticulum Ca2+ release channel (RyR1) is thought to be a region of contact between subunits within RyR1 homotetramer Ca2+ release channels. To determine whether the 3614-3643 region is a regulatory site/interaction domain within RyR in muscle fibers, we have investigated the effect of a synthetic peptide corresponding to this region (R3614-3643) on Ca2+ sparks in frog skeletal muscle fibers. R3614-3643 (0.2-3.0 microM) promoted the occurrence of Ca2+ sparks in a highly cooperative dose-dependent manner, with a half-maximal activation at 0.47 microM and a maximal increase in frequency of approximately 5-fold. A peptide with a single amino acid substitution within R3614-3643 (L3624D) retained the ability to bind Ca(2+)-free calmodulin but did not increase Ca2+ spark frequency, suggesting that R3614-3643 does not modulate Ca2+ sparks by removal of endogenous calmodulin. Our data support a model in which the calmodulin binding domain of RyR1 modulates channel activity by at least two mechanisms: direct binding of calmodulin as well as interactions with other regions of RyR.     

Stationary Ca2+ nanodomains in the presence of buffers with two binding sites

We examine closed-form approximations for the equilibrium Ca²⁺ and buffer concentrations near a point Ca²⁺ source representing a Ca²⁺ channel, in the presence of a mobile buffer with two Ca²⁺ binding sites activated sequentially and possessing distinct binding affinities and kinetics. This allows us to model the impact on Ca²⁺ nanodomains of realistic endogenous Ca²⁺ buffers characterized by cooperative Ca²⁺ binding, such as calretinin. The approximations we present involve a combination or rational and exponential functions, whose parameters are constrained using the series interpolation method that we recently introduced for the case of simpler Ca²⁺ buffers with a single Ca²⁺ binding site. We conduct extensive parameter sensitivity analysis and show that the obtained closed-form approximations achieve reasonable qualitative accuracy for a wide range of buffer’s Ca²⁺ binding properties and other relevant model parameters. In particular, the accuracy of the derived approximants exceeds that of the rapid buffering approximation in large portions of the relevant parameter space.     

Change in the ultraviolet spectrum of solubilized Ca2+-dependent ATPase from sarcoplasmic reticulum due to binding with Ca2+ ions.

Solubilized sarcoplasmic reticulum (SSR) was prepared by solubilizing fragmented sarcoplasmic reticulum (FSR) with a nonionic detergent (C12E8) then displacing the detergent with Tween 80, using a DEAE-cellulose column. The UV absorption of SSR decreased reversibly at about 286 and 292 nm on removal of free Ca2+ ions, while no change in the fluorescence spectrum was detectable. On the other hand, the fluorescence intensity of FSR decreased 3-4% on removal of free Ca2+ ions, as previously reported by Dupont [(1976) Biochem. Biophys. Res. Commun. 71, 544-550]. The UV absorption of FSR increased reversibly at about 270-280 nm on removal of free Ca2+ ions, but the rate of the change was very slow (k = about 0.1 min-1).