Functional delineation of the Ca(2+)-deficient EF-hand in cardiac muscle, with genetically engineered cardiac-skeletal chimeric troponin C.

Cardiac and fast skeletal isoforms of TnC each comprise four putative EF-hand (helix-loop-helix) motifs as potential Ca(2+)-binding sites (sites 1-4), except that site 1 in cardiac TnC is deficient in Ca2+ coordination. In skeletal TnC, the N-terminal sites 1 and 2 are both essential for the trigger mechanism of the contraction switch. However, the mechanism in cardiac muscle is unsettled; it is obscure whether the cardiac site 1 is functionally inert due to calcium deficiency and consequently site 2 is the lone trigger site, or whether sites 1 and 2 perform interactively despite the impairment. These possibilities were addressed by mutagenizing site 1 in skeletal TnC to mimic the cardiac response. In one mutant (STnC-1), two selected Ca(2+)-ligands were abolished. In another (C1/S chimera), 41 N-terminal residues from cardiac TnC were spliced to STnC. The Ca(2+)-binding capacities as well as skinned fiber responses were measured. The STnC-1 derivative failed to switch on contraction. In contrast, the chimeric construct expressed close to full contractile potential in myocardium (74 +/- 3% Po; Po = maximal tension) and also the manifest cardiac phenotype. By devising supplemental chimeric constructs, cardiac-type N-terminal overhang together with cardiac-type EF-hand for site 1 both were found essential for the phenotype. We conclude that cardiac TnC site 1 is actively engaged in the trigger mechanism and in fact dominates the phenotype despite the inability to chelate Ca2+. The N-terminal overhang also participates in this mechanism, which is a novel finding. The conclusion that a non-chelating site functions interactively with a proximal site in cardiac TnC may have wider significance, inasmuch as similar pairings of disparate EF-hands are of common occurrence.     

Parallel measurement of Ca2+ binding and fluorescence emission upon Ca2+ titration of recombinant skeletal muscle troponin C. Measurement of sequential calcium binding to the regulatory sites

Calcium binding to chicken recombinant skeletal muscle TnC (TnC) and its mutants containing tryptophan (F29W), 5-hydroxytryptophan (F29HW), or 7-azatryptophan (F29ZW) at position 29 was measured by flow dialysis and by fluorescence. Comparative analysis of the results allowed us to determine the influence of each amino acid on the calcium binding properties of the N-terminal regulatory domain of the protein. Compared with TnC, the Ca(2+) affinity of N-terminal sites was: 1) increased 6-fold in F29W, 2) increased 3-fold in F29ZW, and 3) decreased slightly in F29HW. The Ca(2+) titration of F29ZW monitored by fluorescence displayed a bimodal curve related to sequential Ca(2+) binding to the two N-terminal Ca(2+) binding sites. Single and double mutants of TnC, F29W, F29HW, and F29ZW were constructed by replacing aspartate by alanine at position 30 (site I) or 66 (site II) or both. Ca(2+) binding data showed that the Asp --> Ala mutation at position 30 impairs calcium binding to site I only, whereas the Asp --> Ala mutation at position 66 impairs calcium binding to both sites I and II. Furthermore, the Asp --> Ala mutation at position 30 eliminates the differences in Ca(2+) affinity observed for replacement of Phe at position 29 by Trp, 5-hydroxytryptophan, or 7-azatryptophan. We conclude that position 29 influences the affinity of site I and that Ca(2+) binding to site I is dependent on the previous binding of metal to site II.     

Calcium-binding properties of troponin C in detergent-skinned heart muscle fibers

In order to obtain information with regard to behavior of the Ca2+ receptor, troponin C (TnC), in intact myofilament lattice of cardiac muscle, we investigated Ca2+-binding properties of canine ventricular muscle fibers skinned with Triton X-100. Analysis of equilibrium Ca2+-binding data of the skinned fibers in ATP-free solutions suggested that there were two distinct classes of binding sites which were saturated over the physiological range of negative logarithm of free calcium concentration (pCa): class I (KCa = 7.4 X 10(7) M-1, KMg = 0.9 X 10(3) M-1) and class II (KCa = 1.2 X 10(6) M-1, KMg = 1.1 X 10(2) M-1). The class I and II were considered equivalent, respectively, to the Ca2+-Mg2+ and Ca2+-specific sites of TnC. The assignments were supported by TnC content of the skinned fibers determined by electrophoresis and 45Ca autoradiograph of electroblotted fiber proteins. Dissociation of rigor complexes by ATP caused a downward shift of the binding curve between pCa 7 and 5, an effect which could be largely accounted for by lowering of KCa of the class II sites. When Ca2+ binding and isometric force were measured simultaneously, it was found that the threshold pCa for activation corresponds to the range of pCa where class II sites started to bind Ca2+ significantly. We concluded that the low affinity site of cardiac TnC plays a key role in Ca2+ regulation of contraction under physiological conditions, just as it does in the regulation of actomyosin ATPase. Study of kinetics of 45Ca washout from skinned fibers and myofibrils revealed that cardiac TnC in myofibrils contains Ca2+-binding sites whose off-rate constant for Ca2+ is significantly lower than the Ca2+ off-rate constant hitherto documented for the divalent ion-binding sites of either cardiac/slow muscle TnC or fast skeletal TnC.     

Binary interactions of troponin subunits

The association constants for the formation of the binary complexes of rabbit fast skeletal muscle troponin subunits have been determined for three solution conditions: (a) 1 mM CaCl2, (b) 3 mM MgCl2 and 1 mM EGTA, and (c) 2 mM EDTA. The subunits were labeled with extrinsic fluorescence probes, either 5-(iodoacetamido)eosin (IAE) or dansylaziridine (DANZ), and the binding was detected by enhancement or quenching of the probe fluorescence. The association constant for the TnI X TnT (where TnI and TnT are the inhibitory subunit and the tropomyosin-binding subunit, respectively, of troponin) complex was measured with two different probes, IAE-TnI and IAE-TnT. The measured values were not affected by the presence of Ca2+ or Mg2+, and the mean values for the three buffer conditions are, respectively, 8.0 X 10(6) and 9.0 X 10(6) M-1 for the two probes. The association constant for TnC-TnI (where TnC is the Ca2+-binding subunit of troponin) interaction was measured with three probes, IAE-TnC, DANZ-TnC, and IAE-TnI. Values of 1.7 X 10(9), 1.2 X 10(8), and 1.0 X 10(6) M-1 were obtained, respectively, in the presence of calcium ion, in the presence of magnesium ion (no calcium), and in the absence of divalent metal ions. A mean value of 4.0 X 10(7) M-1 was obtained for the association constant of TnC X TnT using DANZ-TnC and IAE-TnC as probes in the presence of calcium or magnesium ions. A value of 4.5 X 10(6) M-1 was obtained in the absence of divalent metal ions. The results show that the presence of magnesium ion in the Ca2+-Mg2+ sites strengthens the TnC-TnI and the TnC-TnT interactions and suggest that the troponin structure would be stabilized. This likely results from the effect of magnesium ion on the Ca2+-Mg2+ domains of TnC. The presence of calcium ion in the Ca2+-specific sites provides an additional binding free energy for the TnC-TnI interaction which presumably reflects the changes in the subunit interactions required for the calcium regulatory switch.     

Infrared spectroscopic study of the binding of divalent cations to Akazara scallop troponin C: the effect of the methylene side chain of glutamate residue

Akazara scallop striated adductor muscle troponin C (TnC) binds only one Ca2+ because the three EF-hand motifs are short of critical residues for the coordination of Ca2+. Fourier-transform infrared spectroscopy was applied to study coordination structures of M2+ (= Mg2+, Ca2+, Sr2+, and Ba2+) bound in an Akazara scallop TnC mutant (E142D) and the wild-type TnC C-lobe in D2O solution. The region of the COO- antisymmetric stretch provides information regarding the coordination modes of a COO- group to a metal ion. The side chain COO- group of Asp142 did not bind to Ca2+ in the bidentate coordination mode, suggesting that the absence of a methylene group is critical for the Ca2+ coordination structure of Akazara scallop TnC (Nara et al., Vib Spect 2006, 42, 188-191). The present study has shown that the absence of a methylene group is not compensated for by a larger metal ion such as Sr2+ or Ba2+. CD spectra showed that the secondary structures are conserved between M2+-free (apo), Mg2+-loaded, Ca2+-loaded, Sr2+-loaded, and Ba2+-loaded states, which was consistent with the results estimated from their amide I band patterns. The metal-ligand interaction at position 12 of site IV is discussed in comparison with the coordination mode of the side chain COO- group of the wild-type TnC C-lobe.     

Structure of a trapped intermediate of calmodulin: calcium regulation of EF-hand proteins from a new perspective

Calmodulin (CaM) is a multifunctional Ca2+-binding protein that regulates the activity of many enzymes in response to changes in the intracellular Ca2+ concentration. There are two globular domains in CaM, each containing a pair of helix-loop-helix Ca2+-binding motifs called EF-hands. Ca2+-binding induces the opening of both domains thereby exposing hydrophobic pockets that provide binding sites for the target enzymes. Here, I present a 2.4 A resolution structure of a calmodulin mutant (CaM41/75) in which the N-terminal domain is locked in the closed conformation by a disulfide bond. CaM41/75 crystallized in a tetragonal lattice with the Ca2+ bound in all four EF-hands. In the closed N-terminal domain Ca ions are coordinated by the four protein ligands in positions 1, 3, 5 and 7 of the loop, and by two solvent ligands. The glutamate side-chain in the 12th position of the loop (Glu31 in site I and Glu67 in site II), which in the wild-type protein provides a bidentate Ca2+ ligand, remains in a distal position. Based on a comparison of CaM41/75 with other CaM and troponin C structures a detailed two-step mechanism of the Ca2+-binding process is proposed. Initially, the Ca2+ binds to the N-terminal part of the loop, thus generating a rigid link between the incoming helix (helix A, or helix C) and the central beta structure involving the residues in the sixth, seventh and eighth position of the loop. Then, the exiting helix (helix B or helix D) rotates causing the glutamate ligand in the 12th position to move into the vicinity of the immobilized Ca2+. An adjustment of the phi, psi backbone dihedral angles of the Ile residue in the eighth position is necessary and sufficient for the helix rotation and functions as a hinge. The model allows for a significant independence of the Ca2+-binding sites in a two-EF-hand domain.     

A comparative study of the interactions of synthetic peptides of the skeletal and cardiac troponin I inhibitory region with skeletal and cardiac troponin C

The cardiac and skeletal TnI inhibitory regions have identical sequences except at position 110 which contains Pro in the skeletal sequence and Thr in the cardiac sequence. The effect of the synthetic TnI inhibitory peptides [skeletal TnI peptide (104-115), cardiac TnI peptide (137-148), and a single Gly-substituted analogue at position 110] on the secondary structure of skeletal and cardiac TnC was investigated. The biphasic increases in ellipticity and tyrosine fluorescence were analyzed to determine the Ca2+ binding constants for the high- and low-affinity Ca2+ binding sites of TnC. Importantly, the skeletal and cardiac TnI peptides altered Ca2+ binding at the low-affinity sites of TnC, but the magnitude and direction of the pCa shifts depended on whether the peptides were bound to skeletal or cardiac TnC. For example, binding of skeletal TnI peptide to skeletal TnC (monitored by CD) caused a pCa shift of +0.30 unit such that a lower Ca2+ concentration was required to fill sites I and II, while binding of this peptide to cardiac TnC caused a pCa shift of -0.35 unit such that a higher Ca2+ concentration was required to fill site II. This is the first report of the alteration at the low-affinity regulatory sites (located in the N-terminal domain) by the skeletal TnI inhibitory peptide, even though the primary peptide binding site is located in the C-terminal domain of TnC, a finding which strongly indicates that there is communication between the two halves of the TnC molecule.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitating and engineering the ion specificity of an EF-hand-like Ca2+ binding.

The Escherichia coli D-galactose and D-glucose receptor, an aqueous periplasmic receptor that triggers sugar sensing and transport, possesses a single Ca2+ binding site similar in structure and specificity to the EF-hand class of sites found in eukaryotic Ca2+ signaling proteins including calmodulin and its homologues. A universal feature of these sites is the use of a pentagonal bipyramidal array of seven oxygens to coordinate bound Ca2+. Here we investigate the mechanisms used by this coordinating array to control ion specificity. To vary the cavity size and charge of the array, we have replaced axial glutamine 142 in the prokaryotic site with asparagine, glutamate, and aspartate. The ion selectivities of the resulting engineered sites have been quantitated by measuring dissociation constants for a series of spherical metal ions, differing in increments of radius and charge, from groups Ia, IIa, and IIIa and the lanthanides. Dramatic specificity changes are observed: sites containing an engineered smaller side chain (Asn or Asp) bind the largest cations up to 50-fold more tightly than the native site; and sites containing an engineered negative side chain (Glu or Asp) exhibit preferences for trivalent over divalent cations up to 1900-fold higher than the native site. The results indicate that the cavity size and negative charge of the coordination array play key roles in selective Ca2+ binding and that the array can be engineered to preferentially bind other cations.     

Coordination structures of Ca2+ and Mg2+ in Akazara scallop troponin C in solution. FTIR spectroscopy of side-chain COO- groups.

FTIR spectroscopy has been applied to study the coordination structures of Mg2+ and Ca2+ ions bound in Akazara scallop troponin C (TnC), which contains only a single Ca2+ binding site. The region of the COO- antisymmetric stretch provides information about the coordination modes of COO- groups to the metal ions: bidentate, unidentate, or pseudo-bridging. Two bands were observed at 1584 and 1567 cm-1 in the apo state, whereas additional bands were observed at 1543 and 1601 cm-1 in the Ca2+-bound and Mg2+-bound states, respectively. The intensity of the band at 1567 cm-1 in the Mg2+-bound state was identical to that in the apo state. Therefore, the side-chain COO- group of Glu142 at the 12th position in the Ca2+-binding site coordinates to Ca2+ in the bidentate mode but does not interact with Mg2+ directly. A slight upshift of COO- antisymmetric stretch due to Asp side-chains was also observed upon Mg2+ and Ca2+ binding. This indicates that the COO- groups of Asp131 and Asp133 interact with both Ca2+ and Mg2+ in the pseudo-bridging mode. Therefore, the present study directly demonstrated that the coordination structure of Mg2+ was different from that of Ca2+ in the Ca2+-binding site. In contrast to vertebrate TnC, most of the secondary structures remained unchanged among apo, Mg2+-bound and Ca2+-bound states of Akazara scallop TnC, as spectral changes upon either Ca2+ or Mg2+ binding were very small in the infrared amide-I' region as well as in the CD spectra. Fluorescence spectroscopy indicated that the spectral changes upon Ca2+ binding were larger than that upon Mg2+ binding. Moreover, gel-filtration experiments indicated that the molecular sizes of TnC had the order apo TnC > Mg2+-bound TnC > Ca2+-bound TnC. These results suggest that the tertiary structures are different in the Ca2+- and Mg2+-bound states. The present study may provide direct evidence that the side-chain COO- groups in the Ca2+-binding site are directly involved in the functional on/off mechanism of the activation of Akazara scallop TnC.     

Evidence that both Ca(2+)-specific sites of skeletal muscle TnC are required for full activity

To investigate the role of the Ca2(+)-specific (I and II) sites of fast skeletal muscle troponin C (TnC) in the regulation of contraction, we have produced two TnC mutants which have lost the ability to bind Ca2+ at either site I (VG1) or at site II (VG2). Both mutants were able to partially restore force to TnC-depleted skinned muscle fibers (approximately 25% for VG1 and approximately 50% for VG2). In contrast, bovine cardiac TnC (BCTnC), which like VG1 binds Ca2+ only at site II, could fully reactivate the contraction of TnC-depleted fibers. Higher concentrations of both mutants were required to restore force to the TnC-depleted fibers than with wild type TnC (WTnC) or BCTnC. VG1 and VG2 substituted fibers could not bind additional WTnC, indicating that all of the TnC-binding sites were saturated with the mutant TnC's. The Ca2+ concentration required for force activation was much higher for VG1 and VG2 substituted fibers than for WTnC or BCTnC substituted fibers. Also, the steepness of force activation was much less in VG1 and VG2 versus WTnC and BCTnC substituted fibers. These results suggest cooperative interactions between sites I and II in WTnC. In contrast, BCTnC has essentially the same apparent Ca2+ affinity and steepness of force activation as does WTnC. Thus, cardiac TnC must have structural differences from WTnC which compensate for the lack of site I, while in WTnC, both Ca2(+)-specific sites are probably crucial for full functional activity.     

1H-NMR studies of synthetic peptide analogues of calcium-binding site III of rabbit skeletal troponin C: effect on the lanthanum affinity of the interchange of aspartic acid and asparagine residues at the metal ion coordinating positions

The present work determines the contribution of liganding aspartic acid (Asp) residues, at the +X, +Y, and +Z metal ion coordinating positions, to the lanthanum(3+) (La3+) ion binding affinity of synthetic analogues of calcium-binding site III of rabbit skeletal troponin C. Eight 13-residue synthetic analogues were prepared by solid-phase synthesis; the primary sequences of these analogues represent all possible combinations having aspartic acid and asparagine at the +X, +Y, and +Z positions. High-field proton nuclear magnetic resonance (NMR) spectroscopy was used to monitor the binding of the La3+ ion to each of the analogues. Comparison of the chemical shift changes showed large variations in the magnitude of the shift; these were reflected in the La3+ ion association constants determined for each analogue. The association constants ranged from 9.1 x 10(2) M-1 to 2.5 x 10(5) M-1. It was observed that those analogues with the larger number of acidic residues to coordinate the La3+ ion yielded the higher association constants. The La3+ ion binding results demonstrate that the Asp residues at the positions of study contribute equally and in an additive manner to the association constant and that the presence of neighboring Asp residues at either the +X and +Y, the +Y and +Z, or the +X and +Y and +Z metal ion coordinating positions introduced dentate-dentate repulsion, which, acts as to detract from the La3+ ion association constant of the analogues.     

The role of GyrB in the DNA cleavage-religation reaction of DNA gyrase: a proposed two metal-ion mechanism

We have examined the role of the DNA gyrase B protein in cleavage and religation of DNA using site-directed mutagenesis. Three aspartate residues and a glutamate residue: E424, D498, D500 and D502, thought to co-ordinate a magnesium ion, were mutated to alanine; in addition, the glutamate residue and one aspartate residue were mutated to glutamine and asparagine, respectively. We have shown that these residues are important for the cleavage-religation reaction and are likely to be involved in magnesium ion co-ordination. On separate mutation of two of these aspartate residues to cysteine or histidine, the metal ion preference for the DNA relaxation activity of gyrase changed from magnesium to manganese (II). We present evidence to support the idea that cleavage of each DNA strand involves two or more metal ions, and suggest a scheme for the DNA cleavage chemistry of DNA gyrase involving two metal ions.     

Effects of cardiac thin filament Ca2+: statistical mechanical analysis of a troponin C site II mutant.

Cardiac thin filaments contain many troponin C (TnC) molecules, each with one regulatory Ca2+ binding site. A statistical mechanical model for the effects of these sites is presented and investigated. The ternary troponin complex was reconstituted with either TnC or the TnC mutant CBMII, in which the regulatory site in cardiac TnC (site II) is inactivated. Regardless of whether Ca2+ was present, CBMII-troponin was inhibitory in a thin filament-myosin subfragment 1 MgATPase assay. The competitive binding of [3H]troponin and [14C]CBMII-troponin to actin.tropomyosin was measured. In the presence of Mg2+ and low free Ca2+ they had equal affinities for the thin filament. When Ca274+ was added, however, troponin's affinity for the thin filament was 2.2-fold larger for the mutant than for the wild type troponin. This quantitatively describes the effect of regulatory site Ca2+ on troponin's affinity for actin.tropomyosin; the decrease in troponin-thin filament binding energy is small. Application of the theoretical model to the competitive binding data indicated that troponin molecules bind to interdependent rather than independent sites on the thin filament. Ca2+ binding to the regulatory site of TnC has a long-range rather than a merely local effect. However, these indirect TnC-TnC interactions are weak, indicating that the cooperativity of muscle activation by Ca2+ requires other sources of cooperativity.     

The calcium and magnesium binding sites on cardiac troponin and their role in the regulation of myofibrillar adenosine triphosphatase

The cardiac troponin (Tn) complex, consisting of a Ca2+-binding subunit (TnC), an inhibitory subunit (TnI), and a tropomyosin-binding subunit (TnT), has been reconstituted from purified troponin subunits isolated from bovine heart muscle. The Ca2+-binding properties of cardiac Tn were determined by equilibrium dialysis using either EGTA or EDTA to regulate the free Ca2+ concentration. Cardiac Tn binds 3 mol Ca2+/mol and contains two Ca2+-binding sites with a binding constant of 3 X 10(8) M-1 and one binding site with a binding constant of 2 X 10(6) M-1. In the presence of 4 mM MgC12, the binding constant of the sites of higher affinity is reduced to 3 X 10(7) M-1, while Ca2+ binding to the site at the lower affinity is unaffected. The two high affinity Ca2+-binding sites of cardiac Tn are analogous to the two Ca2+-Mg2+ sites of skeletal Tn, while the single low affinity site is similar to the two Ca2+-specific sites of skeletal Tn (Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4625-5633). The Ca2+-binding properties of the complex of TnC and TnI (1:1 molar ratio) were similar to those of Tn. Cardiac TnC also binds 3 mol of Ca2+/mol and contains two sites with a binding constant of 1 X 10(7) M-1 and a single site with a binding constant of 2 X 10(5) M-1. Assuming competition between Mg2+ and Ca2+ for the high affinity sites of TnC and Tn, the binding constants for Mg2+ were 0.7 and 3.0 X 10(3) M-1, respectively. The Ca2+ dependence of cardiac myofibrillar ATPase activity was similar to that of an actomyosin preparation regulated by the reconstituted troponin complex. Comparison by the Ca2+-binding properties of cardiac Tn and the cardiac myofibrillar ATPase activity as a function of [Ca2+] and at millimolar [Mg2+] suggests that activation of the ATPase occurs over the same range of [Ca2+] where the Ca2+-specific site of cardiac Tn binds Ca2+.     

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.     

Cross-linking between the regulatory regions of troponin-I and troponin-C abolishes the inhibitory function of troponin

We reported previously that both residues 48 and 82 on opposite sides of troponin-C's (TnC's) N-terminal regulatory hydrophobic cleft photo-cross-linked to Met121 of troponin-I (TnI) [Luo, Y., Leszyk, J., Qian, Y., Gergely, J., and Tao, T. (1999) Biochemistry 38, 6678-6688]. Here we report that the Ca2+-absent inhibitory activity of troponin (Tn) was progressively lost as the extent of photo-cross-linking increased. To extend these studies, we constructed a mutant TnI with a single cysteine at residue 121 (TnI121). In Tn complexes containing TnI121 and mutant TnCs with a single cysteine at positions 12, 48, 82, 98, or 125 (TnC12, TnC48 etc.), TnI121 formed disulfide cross-links primarily with TnC48 and TnC82 when Ca2+ was present, and with only TnC48 when Ca2+ was absent. These results indicate that TnI Met121 is situated within the N-domain hydrophobic cleft of TnC in the presence of Ca2+, and that it moves out of the cleft upon Ca2+ removal but remains within the vicinity of TnC. Activity assays revealed that the Met121 to Cys mutation in TnI121 reduced the Ca2+-present activation of Tn, indicating that Met121 is important in hydrophobic interactions between this TnI region and TnC's N-domain cleft. The formation of a disulfide cross-link between TnI121 and TnC48 or TnC82 abolished the Ca2+-absent inhibitory activity of Tn, indicating that the movement of the Met121 region of TnI out of TnC's N-domain cleft is essential for the occurrence of further events in the inhibitory process of skeletal muscle contraction. On the basis of these and other results, a simple mechanism for Ca2+ regulation of skeletal muscle contraction is presented and discussed.     

M42 aminopeptidase catalytic site: the structural and functional role of a strictly conserved aspartate residue

The M42 aminopeptidases are a family of dinuclear aminopeptidases widely distributed in Prokaryotes. They are potentially associated to the proteasome, achieving complete peptide destruction. Their most peculiar characteristic is their quaternary structure, a tetrahedron-shaped particle made of twelve subunits. The catalytic site of M42 aminopeptidases is defined by seven conserved residues. Five of them are involved in metal ion binding which is important to maintain both the activity and the oligomeric state. The sixth conserved residue, a glutamate, is the catalytic base deprotonating the water molecule during peptide bond hydrolysis. The seventh residue is an aspartate whose function remains poorly understood. This aspartate residue, however, must have a critical role as it is strictly conserved in all MH clan enzymes. It forms some kind of catalytic triad with the histidine residue and the metal ion of the M2 binding site. We assess its role in TmPep1050, an M42 aminopeptidase of Thermotoga maritima, through a mutational approach. Asp-62 was substituted with alanine, asparagine, or glutamate residue. The Asp-62 substitutions completely abolished TmPep1050 activity and impeded dodecamer formation. They also interfered with metal ion binding as only one cobalt ion is bound per subunit instead of two. The structure of Asp62Ala variant was solved at 1.5 Å showing how the substitution has an impact on the active site fold. We propose a structural role for Asp-62, helping to stabilize a crucial loop in the active site and to position correctly the catalytic base and a metal ion ligand of the M1 site.     

Effect of Ca2+ binding on troponin C. Changes in spin label mobility, extrinsic fluorescence, and sulfhydryl reactivity.

The Ca2+ binding component (TnC) of troponin has been selectively labeled with either a spin label, N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) iodoacetamide, or with a fluorescent probe, S-mercuric-N-dansyl cysteine, presumably at its single cysteine residue (Cys-98) in order to probe the interactions of TnC with divalent metals and with other subunits of troponin. The modified protein has the same Ca2+ binding properties as native TnC (Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4628), viz. two Ca2+ binding sites at which Mg2+ appears to compete (Ca2+-Mg2+ sites, KCa = 2 X 10(7) M-1) and two sites at which Mg2+ does not compete (Ca2+-specific sites, KCa = 2 X 10(5) M-1). Either Ca2+ or Mg2+ alters the ESR spectrum of spin-labeled TnC in a manner that indicates a decrease in the mobility of the label, Ca2+ having a slightly greater effect. In systems containing both Ca2+ and Mg2+ the mobility of the spin label is identical with that in systems containing Ca2+ alone. The binding constants for Ca2+ and Mg2+ deduced from ESR spectral changes are 10(7) and 10(3) M-1, respectively, and the apparent affinity for Ca2+ decreases by about an order of magnitude on adding 2 mM Mg2+. Thus, the ESR spectral change is associated with binding of Ca2+ to one or both of the Ca2+-Mg2+ sites. Addition of Ca2+ to the binary complexes of spin-labeled TnC with either troponin T (TnT) or troponin I (TnI) produces greater reduction in the mobility of the spin label than in the case of spin-labeled TnC alone, and in the case of the complex with TnI the affinity for Ca2+ is increased by an order of magnitude. The fluorescence of dansyl (5-dimethylaminonaphthalene-1-sulfonyl)-labeled TnC is enhanced by Ca2+ binding to both high and low affinity sites with apparent binding constants of 2.6 X 10(7) M-1 and 2.9 X 10(5) M-1, respectively, calculated from the transition midpoints. The presence of 2 mM Mg2+, which produces no effect on dansyl fluorescence itself, in contrast to its effect on the spin label, shifts the high affinity constant to 2 X 10(6) M-1. Spectral changes produced by Ca2+ binding to the TnC-TnI complex furnish evidence that the affinity of TnC for Ca2+ is increased in the complex. The reactivity of Cys-98 to the labels and to 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) is decreased by Ca2+ or Mg2+ both with native TnC and in 6 M urea. The reaction rate between Cys-98 and Nbs2 decreases to one-half the maximal value at a Ca2+ concentration that suggests binding to the Ca2+-Mg2+ sites. Formation of a binary complex between TnI and TnC reduces the rate of reaction, and there is a further reduction by Ca2+. The effect of Ca2+ takes place at concentrations that are 1 order of magnitude lower than in the case of TnC alone. These results suggest that the Ca2+ binding site adjacent to Cys-98 is one of the Ca2+-Mg2+ binding sites.     

Glutamine metabolism of isolated rat hepatocytes. Evidence for catecholamine activation of alpha-ketoglutarate dehydrogenase

Effects of norepinephrine on gluconeogenesis and ureogenesis from glutamine by hepatocytes from fasted rats were assessed. Comparisons were made to asparagine metabolism and to the effects of NH4Cl and dibutyryl cyclic AMP. With asparagine as substrate, aspartate content was very high but norepinephrine, dibutyryl cyclic AMP, or NH4Cl had little effect on gluconeogenesis or ureogenesis. Metabolism of asparagine could be greatly enhanced by the combination of oleate, ornithine, and NH4Cl. However, even under these conditions, asparatate content remained high, and norepinephrine and dibutyryl cyclic AMP had little influence on glucose or urea synthesis. With glutamine as substrate, aspartate content was much lower, but was greatly elevated by norepinephrine, dibutyryl cyclic AMP, or NH4Cl. Each of these effectors strongly stimulated glucose and urea formation from glutamine. NH4Cl stimulation was accompanied by an increased glutamate and decreased alpha-ketoglutarate content. This suggests the mechanism for NH4Cl stimulation is a near-equilibrium adjustment to ammonia by glutamate dehydrogenase and aspartate aminotransferase rather than a principal involvement of glutaminase. Although both norepinephrine and dibutyryl cyclic AMP lowered alpha-ketoglutarate to the same extent, norepinephrine more rapidly increased aspartate content and led to a smaller accumulation of glutamate than did dibutyryl cyclic AMP. Moreover, only norepinephrine led to a rapid increase in succinyl-CoA concentration. The catecholamine effect could not be explained by specific changes in cytosolic or mitochondrial redox states. The results suggest that alpha-ketoglutarate dehydrogenase is a site of catecholamine action in rat liver. Since purified alpha-ketoglutarate dehydrogenase is known to be Ca2+ stimulated and Ca2+ flux is involved in catecholamine action, these findings also suggest that mitochondrial Ca2+ is elevated by catecholamines.     

Aluminum fluoride interactions with troponin C.

The increasing interest in the metal ion aluminum fluoride and beryllium fluoride complexes as phosphate analogs in the myosin ATPase reaction and in muscle fiber studies prompted the examination of their interactions with the regulatory system of troponin and tropomyosin. In this work, the effects of these metal ion analogs on the spectral properties of the Ca(2+)-binding subunit of troponin, troponin C (TnC), were examined. In contrast to beryllium fluoride which did not change the spectral properties of TnC, aluminum fluoride binding induced an increase in both the alpha-helicity and the tyrosine fluorescence of TnC and exposed a hydrophobic region on this protein for fluorescent probe binding. Aluminum fluoride also reduced the Ca2+ and/or Mg(2+)-induced changes on TnC. These results indicate a direct interaction of aluminum fluoride with TnC and merit consideration in designing muscle fiber experiments with this phosphate analog.