Functional importance of Ca2+-deficient N-terminal lobe of molluscan troponin C in troponin regulation

Ca(2+)-binding sites I and II in the N-terminal lobe of molluscan troponin C (TnC) have lost the ability to bind Ca(2+) due to substitutions of the amino acid residues responsible for Ca(2+) liganding. To evaluate the functional importance of the Ca(2+)-deficient N-terminal lobe in the Ca(2+)-regulatory function of molluscan troponin, we constructed chimeric TnCs comprising the N-terminal lobes from rabbit fast muscle and squid mantle muscle TnCs and the C-terminal lobe from akazara scallop TnC, TnC(RA), and TnC(SA), respectively. We characterized their biochemical properties as compared with those of akazara scallop wild-type TnC (TnC(AA)). According to equilibrium dialysis using (45)Ca(2+), TnC(RA), and TnC(SA) bound stoichiometrically 3 mol Ca(2+)/mol and 1 mol Ca(2+)/mol, respectively, as expected from their primary structures. All the chimeric TnCs exhibited difference-UV-absorption spectra at around 280-290 nm upon Ca(2+) binding and formed stable complexes with akazara scallop troponin I, even in the presence of 6M urea, if Ca(2+) was present. However, when the troponin complexes were constructed from chimeric TnCs and akazara scallop troponin T and troponin I, they showed different Ca(2+)-regulation abilities from each other depending on the TnC species. Thus, the troponin containing TnC(SA) conferred as high a Ca(2+) sensitivity to Mg-ATPase activity of rabbit actomyosin-akazara scallop tropomyosin as did the troponin containing TnC(AA), whereas the troponin containing TnC(RA) conferred virtually no Ca(2+) sensitivity. Our findings indicate that the N-terminal lobe of molluscan TnC plays important roles in molluscan troponin regulation, despite its inability to bind Ca(2+).     

Enhancement by Mg2+ of domain specificity in Ca2+-dependent interactions of calmodulin with target sequences

Mg2+ binds to calmodulin without inducing the changes in secondary structure that are characteristic of Ca2+ binding, or the exposure of hydrophobic surfaces that are involved in typical Ca2+-dependent target interactions. The binding of Mg2+ does, however, produce significant spectroscopic changes in residues located in the Ca2+-binding loops, and the Mg-calmodulin complex is significantly different from apo-calmodulin in loop conformation. Direct measurement of Mg2+ binding constants, and the effects of Mg2+ on Ca2+ binding to calmodulin, are consistent with specific binding of Mg2+, in competition with Ca2+. Mg2+ increases the thermodynamic stability of calmodulin, and we conclude that under resting, nonstimulated conditions, cellular Mg2+ has a direct role in conferring stability on both domains of apo-calmodulin. Apo-calmodulin binds typical target sequences from skeletal muscle myosin light chain kinase and neuromodulin with Kd approximately 70-90 nM (at low ionic strength). These affinities are virtually unchanged by 5 mM Mg2+, in marked contrast to the strong enhancement of peptide affinity induced by Ca2+. Under conditions of stimulation and increased [Ca2+], Mg2+ has a role in directing the mode of initial target binding preferentially to the C-domain of calmodulin, due to the opposite relative affinities for binding of Ca2+ and Mg2+ to the two domains. Mg2+ thus amplifies the intrinsic differences of the domains, in a target specific manner. It also contributes to setting the Ca2+ threshold for enzyme activation and increases the importance of a partially Ca2+-saturated calmodulin-target complex that can act as a regulatory kinetic and equilibrium intermediate in Ca2+-dependent target interactions.     

A new hypothesis for Ca2+ flows in skeletal muscle and its implications for other cell types

We offer a new hypothesis to explain calcium flows in skeletal muscle cells. Our model accounts for the uptake of Ca²⁺ from the extracellular fluid, and the release of Ca²⁺ from the sarcoplasmic reticulum (SR/ER) (the endoplasmic reticulum in muscle is named sarcoplasmic reticulum); this has engendered difficulty in reviews encompassing both muscle and nonmuscle cells. Here we will typically refer to the organelle as ER, except when specifically discussing muscle cells. The broad consideration of two major, still unexplained properties of skeletal muscle function, namely excitation contraction coupling and capacitative calcium entry are accounted for in a unitary hypothesis. This model allows a reinterpretation of existing data, and points to areas where new investigation may be fruitful. While primarily aimed at explaining Ca²⁺ flows in skeletal muscle, we consider findings of other systems to explore the implications of this hypothesis for other cell types.     

A model for the calmodulin-peptide complex based on the troponin C crystal packing and its similarity to the NMR structure of the calmodulin-myosin light chain kinase peptide complex.

In the crystal structure of troponin C, the holo C-domain is bound in a head-to-tail fashion to the A-helix of the apo N-domain of a symmetry-related molecule. Using this interaction, we have proposed a model for the calmodulin-peptide complex. We find that the interaction of the C-domain with the A-helix is similar to that observed in the NMR structure of the calmodulin-myosin light chain kinase (MLCK) peptide complex. This similarity in binding has enabled us to make a precise sequence alignment of the target peptides in the calmodulin-binding cleft and to rationalize the amino acid sequence-dependent binding strengths of various peptides. Our model differs from that proposed by Strynadka and James (Proteins Struct. Funct. Genet. 7, 234-248, 1990) in that the peptides are rotated by 100 degrees in the calmodulin binding cleft.     

Cellular and molecular aspects of familial hypertrophic cardiomyopathy caused by mutations in the cardiac troponin I gene

Mutations in the cardiac troponin I (CTnI) gene occur in 5% of families with familial hypertrophic cardiomyopathy (FHC) and 20 mutations in this gene that cause FHC have now been described. The clinical manifestations of CTnI mutations that cause FHC are diverse, ranging from asymptomatic with high life expectancy to severe heart failure and sudden cardiac death. Most of these FHC mutations in CTnI result in cardiac hypertrophy unlike cardiac troponin T FHC mutations. All CTnI FHC mutations investigated in vitro affect the physiological function of CTnI, but other factors such as environmental or genetic factors (other genes that may affect the CTnI gene) are likely to be involved in influencing the severity of the phenotype produced by these mutations, since the distribution of hypertrophy among affected individuals varies within and between families. CTnI mutations mainly alter myocardial performance via changes in the Ca²⁺-sensitivity of force development and in some cases alter the muscle relaxation kinetics due to haemodynamic or physical obstructions of blood flow from the left ventricle. (Mol Cell Biochem 263: 99–114, 2004)     

Long-term modulation of mitochondrial Ca2+ signals by protein kinase C isozymes

The modulation of Ca2+ signaling patterns during repetitive stimulations represents an important mechanism for integrating through time the inputs received by a cell. By either overexpressing the isoforms of protein kinase C (PKC) or inhibiting them with specific blockers, we investigated the role of this family of proteins in regulating the dynamic interplay of the intracellular Ca2+ pools. The effects of the different isoforms spanned from the reduction of ER Ca2+ release (PKCalpha) to the increase or reduction of mitochondrial Ca2+ uptake (PKCzeta and PKCbeta/PKCdelta, respectively). This PKC-dependent regulatory mechanism underlies the process of mitochondrial Ca2+ desensitization, which in turn modulates cellular responses (e.g., insulin secretion). These results demonstrate that organelle Ca2+ homeostasis (and in particular mitochondrial processing of Ca2+ signals) is tuned through the wide molecular repertoire of intracellular Ca2+ transducers.     

Binding of Zn2+ to a Ca2+ loop allosterically attenuates the activity of factor VIIa and reduces its affinity for tissue factor

The protease domain of coagulation factor VIIa (FVIIa) is homologous to trypsin with a similar active site architecture. The catalytic function of FVIIa is regulated by allosteric modulations induced by binding of divalent metal ions and the cofactor tissue factor (TF). To further elucidate the mechanisms behind these transformations, the effects of Zn2+ binding to FVIIa in the free form and in complex with TF were investigated. Equilibrium dialysis suggested that two Zn2+ bind with high affinity to FVIIa outside the N-terminal gamma-carboxyglutamic acid (Gla) domain. Binding of Zn2+ to FVIIa, which was influenced by the presence of Ca2+, resulted in decreased amidolytic activity and slightly reduced affinity for TF. After binding to TF, FVIIa was less susceptible to zinc inhibition. Alanine substitutions for either of two histidine residues unique for FVIIa, His216, and His257, produced FVIIa variants with decreased sensitivity to Zn2+ inhibition. A search for putative Zn2+ binding sites in the crystal structure of the FVIIa protease domain was performed by Grid calculations. We identified a pair of Zn2+ binding sites in the Glu210-Glu220 Ca2+ binding loop adjacent to the so-called activation domain canonical to serine proteases. Based on our results, we propose a model that describes the conformational changes underlying the Zn2+-mediated allosteric down-regulation of FVIIa's activity.     

Phosphorylation of myosin light chain kinase: a cellular mechanism for Ca2+ desensitization

Phosphorylation of the regulatory light chain of myosin by the Ca²⁺/calmodulin-dependent myosin light chain kinase plays an important role in smooth muscle contraction, nonmuscle cell shape changes, platelet contraction, secretion, and other cellular processes. Smooth muscle myosin light chain kinase is also phosphorylated, and recent results from experiments designed to satisfy the criteria of Krebs and Beavo for establishing the physiological significance of enzyme phosphorylation have provided insights into the cellular regulation and function of this phosphorylation in smooth muscle. The multifunctional Ca²⁺/calmodulin-dependent protein kinase II phosphorylates myosin light chain kinase at a regulatory site near the calmodulin-binding domain. This phosphorylation increases the concentration of Ca²⁺/calmodulin required for activation and hence increases the Ca²⁺ concentrations required for myosin light chain kinase activity in cells. However, the concentration of cytosolic Ca²⁺ required to effect myosin light chain kinase phosphorylation is greater than that required for myosin light chain phosphorylation. Phosphorylation of myosin light chain kinase is only one of a number of mechanisms used by the cell to down regulate the Ca²⁺ signal in smooth muscle. Since both smooth and nonmuscle cells express the same form of myosin light chain kinase, this phosphorylation may play a regulatory role in cellular processes that are dependent on myosin light chain phosphorylation.     

Prostaglandin E2 stimulates insulin-like growth factor binding protein-4 expression and synthesis in cultured human articular chondrocytes: Possible mediation by Ca++ -calmodulin regulated processes

Insulin-like growth factor-1, IGF-1, is believed to be an important anabolic modulator of cartilage metabolism whose action is mediated by high affinity cell surface receptors and bioactivity and bioavailability regulated, in part, by IGF-1 binding proteins (IGFBPs). Prostaglandin E₂ (PGE₂) stimulates collagen and proteoglycan synthesis in cartilage via an autocrine feedback loop involving IGF-1. We determined whether the eicosanoid could regulate IGFBP-4, a major form expressed by chondrocytes and, as such, act as a modifier of IGF-1 action at another level. Using human articular chondrocytes in high-density primary culture, Western and Western ligand blotting to measure secreted IGFBP-4 protein, and Northern analysis to monitor IGFBP-4 mRNA levels, we demonstrated that PGE₂ provoked a 2.7 ± 0.3- and 3.8 ± 0.5- (n = 3) fold increase in IGFBP-4 mRNA and protein, respectively. This effect was reversed by the Ca⁺⁺ channel blocker, verapamil, and the Ca⁺⁺/calmodulin inhibitor, W-7. The Ca⁺⁺ ionophore, ionomycin, mimicked the effects of PGE₂. The phorbol ester, PMA, which activated phospholipid-dependent protein kinase C (PKC) in chondrocytes, had no effect on IGFBP-4 production. Cyclic AMP mimetics and PKA activators, IBMX, and Sp-cAMP, inhibited the expression of the binding protein as did the PGE₂ secretagogue, interleukin-1β (IL-β). The inhibitory effect of the latter cytokine was mediated by a erbstatin/genistein (tyrosine) sensitive kinase. Dexamethasone, an inhibitor of cyclooxygenase (COX-2) expression and PGE₂ synthesis, down-regulated control, constitute levels of IGFBP-4 mRNA and protein, eliminating the previously demonstrated possibility of cross-talk between glucocorticoid receptor (GR) and PGE₂-receptor signalling pathways. The results suggest that extracellular signals control IGFBP-4 production by a number of different transducing networks with changes in Ca⁺⁺ and calmodulin activity exerting a strong positive influence, possibly maintaining the constitutivity of IGFBP-4 synthesis under basal conditions. PGE₂ activation of the IGF-1/IGFBP axis may play a pivotal role in the metabolism of cartilage and possibly connective tissues in general. Eicosanoid biosynthesis may be a rate-limiting step in cartilage repair processes. J. Cell. Biochem. 65:408–419. © 1997 Wiley-Liss, Inc.     

Investigating dual Ca2+ modulation of the ryanodine receptor 1 by molecular dynamics simulation

The ryanodine receptors (RyR) are essential to calcium signaling in striated muscles. A deep understanding of the complex Ca²⁺-activation/inhibition mechanism of RyRs requires detailed structural and dynamic information for RyRs in different functional states (eg, with Ca²⁺ bound to activating or inhibitory sites). Recently, high-resolution structures of the RyR isoform 1 (RyR1) were solved by cryo-electron microscopy, revealing the location of a Ca²⁺ binding site for activation. Toward elucidating the Ca²⁺-modulation mechanism of RyR1, we performed extensive molecular dynamics simulation of the core RyR1 structure in the presence and absence of activating and solvent Ca²⁺ (total simulation time is >5 μs). In the presence of solvent Ca²⁺, Ca²⁺ binding to the activating site enhanced dynamics of RyR1 with higher inter-subunit flexibility, asymmetric inter-subunit motions, outward domain motions and partial pore dilation, which may prime RyR1 for subsequent channel opening. In contrast, the solvent Ca²⁺ alone reduced dynamics of RyR1 and led to inward domain motions and pore contraction, which may cause inhibition. Combining our simulation with the map of disease mutation sites in RyR1, we constructed a wiring diagram of key domains coupled via specific hydrogen bonds involving the mutation sites, some of which were modulated by Ca²⁺ binding. The structural and dynamic information gained from this study will inform future mutational and functional studies of RyR1 activation and inhibition by Ca²⁺.     

Crystallographic identification of Ca2+ and Sr2+ coordination sites in synaptotagmin I C2B domain

Synaptotagmin I has two tandem Ca(2+)-binding C(2) domains, which are essential for fast synchronous synaptic transmission in the central nervous system. We have solved four crystal structures of the C(2)B domain, one of them in the cation-free form at 1.50 A resolution, two in the Ca(2+)-bound form at 1.04 A (two bound Ca(2+) ions) and 1.65 A (three bound Ca(2+) ions) resolution and one in the Sr(2+)-bound form at 1.18 A (one bound Sr(2+) ion) resolution. The side chains of four highly conserved aspartic acids (D303, D309, D363, and D365) and two main chain oxygens (M302:O and Y364:O), together with water molecules, are in direct contact with two bound Ca(2+) ions (sites 1 and 2). At higher Ca(2+) concentrations, the side chain of N333 rotates and cooperates with D309 to generate a third Ca(2+) coordination site (site 3). Divalent cation binding sites 1 and 2 in the C(2)B domain were previously identified from NMR NOE patterns and titration studies, supplemented by site-directed mutation analysis. One difference between the crystal and NMR studies involves D371, which is not involved in coordination with any of the identified Ca(2+) sites in the crystal structures, while it is coordinated to Ca(2+) in site 2 in the NMR structure. In the presence of Sr(2+), which is also capable of triggering exocytosis, but with lower efficiency, only one cation binding site (site 1) was occupied in the crystallographic structure.     

Intermolecular tuning of calmodulin by target peptides and proteins: differential effects on Ca2+ binding and implications for kinase activation.

Ca(2+)-activated calmodulin (CaM) regulates many target enzymes by docking to an amphiphilic target helix of variable sequence. This study compares the equilibrium Ca2+ binding and Ca2+ dissociation kinetics of CaM complexed to target peptides derived from five different CaM-regulated proteins: phosphorylase kinase. CaM-dependent protein kinase II, skeletal and smooth myosin light chain kinases, and the plasma membrane Ca(2+)-ATPase. The results reveal that different target peptides can tune the Ca2+ binding affinities and kinetics of the two CaM domains over a wide range of Ca2+ concentrations and time scales. The five peptides increase the Ca2+ affinity of the N-terminal regulatory domain from 14- to 350-fold and slow its Ca2+ dissociation kinetics from 60- to 140-fold. Smaller effects are observed for the C-terminal domain, where peptides increase the apparent Ca2+ affinity 8- to 100-fold and slow dissociation kinetics 13- to 132-fold. In full-length skeletal myosin light chain kinase the inter-molecular tuning provided by the isolated target peptide is further modulated by other tuning interactions, resulting in a CaM-protein complex that has a 10-fold lower Ca2+ affinity than the analogous CaM-peptide complex. Unlike the CaM-peptide complexes, Ca2+ dissociation from the protein complex follows monoexponential kinetics in which all four Ca2+ ions dissociate at a rate comparable to the slow rate observed in the peptide complex. The two Ca2+ ions bound to the CaM N-terminal domain are substantially occluded in the CaM-protein complex. Overall, the results indicate that the cellular activation of myosin light chain kinase is likely to be triggered by the binding of free Ca2(2+)-CaM or Ca4(2+)-CaM after a Ca2+ signal has begun and that inactivation of the complex is initiated by a single rate-limiting event, which is proposed to be either the direct dissociation of Ca2+ ions from the bound C-terminal domain or the dissociation of Ca2+ loaded C-terminal domain from skMLCK. The observed target-induced variations in Ca2+ affinities and dissociation rates could serve to tune CaM activation and inactivation for different cellular pathways, and also must counterbalance the variable energetic costs of driving the activating conformational change in different target enzymes.     

A model for the Ca2+-induced conformational transition of troponin C. A trigger for muscle contraction

The initial contractile event in muscle is the binding of Ca2+ ions to troponin C of the troponin complex, leading to a series of conformational changes in the members of the thin and thick filaments. Knowledge of the crystal structure of turkey skeletal muscle troponin C has provided a structural basis for the modeling of the first stage of this process in atomic detail. This crystal structure probably represents the molecule in the relaxed state of muscle, with two of the maximum of 4 Ca2+ ions bound. The basis for the model presented here is that upon binding of the additional two Ca2+ ions, the regulatory domain of the molecule undergoes a conformational transition to become closely similar in structure to the domain which always binds Ca2+ or Mg2+ under physiological conditions. The root mean square discrepancy in atomic coordinates between the apo and the modeled Ca2+-bound states of the regulatory domain is 4.8 A, with some shifts as large as 10-15 A in the region near the linker between the two Ca2+ binding sites. It is demonstrated that this Ca2+-bound conformation of the regulatory domain conforms to accepted protein structure rules and that the change in conformation can be accomplished without encountering any barriers too high to be surmounted on the physiological time scale.     

Role of interchain alpha-helical hydrophobic interactions in Ca2+ affinity, formation, and stability of a two-site domain in troponin C.

We have previously shown that a 34-residue synthetic peptide representing the calcium-binding site III of troponin C formed a symmetric two-site dimer consisting of two helix-loop-helix motifs arranged in a head-to-tail fashion (Shaw, G.S., Hodges, R.S., & Sykes, B.D., 1990, Science 249, 280-283). In this study the hydrophobicities of the alpha-helices were altered by replacing L-98 and F-102 in the N-terminal region and/or I-121 and L-122 in the C-terminal region with alanine residues. Our results showed that substitution of hydrophobic residues either in the N- or C-terminal region have little effect on alpha-helix formation but resulted in a 100- and 300-fold decrease in Ca2+ affinity, respectively. Simultaneous substitution of both hydrophobes in the N- and C-terminal region resulted in a 1,000-fold decrease in Ca2+ affinity. Data from guanidine hydrochloride denaturation studies suggested that intermolecular interactions occur and that the less hydrophobic analogs had a lower overall conformational stability. These data support the contention that the hydrophobic residues are important in the formation of the two-site domain in troponin C, and this hydrophobic association stabilizes Ca2+ affinity.     

An atomic model of the thin filament in the relaxed and Ca2+-activated states

Contraction of striated muscles is regulated by tropomyosin strands that run continuously along actin-containing thin filaments. Tropomyosin blocks myosin-binding sites on actin in resting muscle and unblocks them during Ca2+-activation. This steric effect controls myosin-crossbridge cycling on actin that drives contraction. Troponin, bound to the thin filaments, couples Ca2+-concentration changes to the movement of tropomyosin. Ca2+-free troponin is thought to trap tropomyosin in the myosin-blocking position, while this constraint is released after Ca2+-binding. Although the location and movements of tropomyosin are well known, the structural organization of troponin on thin filaments is not. Its mechanism of action therefore remains uncertain. To determine the organization of troponin on the thin filament, we have constructed atomic models of low and high-Ca2+ states based on crystal structures of actin, tropomyosin and the "core domain" of troponin, and constrained by distances between filament components and by their location in electron microscopy (EM) reconstructions. Alternative models were also built where troponin was systematically repositioned or reoriented on actin. The accuracy of the different models was evaluated by determining how well they corresponded to EM images. While the initial low and high-Ca2+ models fitted the data precisely, the alternatives did not, suggesting that the starting models best represented the correct structures. Thin filament reconstructions were generated from the EM data using these starting models as references. In addition to showing the core domain of troponin, the reconstructions showed additional detail not present in the starting models. We attribute this to an extension of TnI linking the troponin core domain to actin at low (but not at high) Ca2+, thereby trapping tropomyosin in the OFF-state. The bulk of the core domain of troponin appears not to move significantly on actin, regardless of Ca2+ level. Our observations suggest a simple model for muscle regulation in which troponin affects the charge balance on actin and hence tropomyosin position.     

High‐affinity cooperative Ca2+ binding by MICU1–MICU2 serves as an on–off switch for the uniporter

The mitochondrial calcium uniporter is a Ca²⁺‐activated Ca²⁺ channel that is essential for dynamic modulation of mitochondrial function in response to cellular Ca²⁺ signals. It is regulated by two paralogous EF‐hand proteins—MICU1 and MICU2, but the mechanism is unknown. Here, we demonstrate that both MICU1 and MICU2 are stabilized by Ca²⁺. We reconstitute the MICU1–MICU2 heterodimer and demonstrate that it binds Ca²⁺ cooperatively with high affinity. We discover that both MICU1 and MICU2 exhibit affinity for the mitochondria‐specific lipid cardiolipin. We determine the minimum Ca²⁺ concentration required for disinhibition of the uniporter in permeabilized cells and report a close match with the Ca²⁺‐binding affinity of MICU1–MICU2. We conclude that cooperative, high‐affinity interaction of the MICU1–MICU2 complex with Ca²⁺ serves as an on–off switch, leading to a tightly controlled channel, capable of responding directly to cytosolic Ca²⁺ signals.