Interaction of calcium channel antagonists with calcium: spectroscopic and modeling studies on diltiazem and its Ca2+ complex

Using spectral techniques, the solution conformation of diltiazem was studied in acetonitrile with special reference to the effect of Ca2+ on the drug structure. Complete assignment of the proton resonances in the 1H-NMR spectrum of the drug was made using one-and two-dimensional spectral analyses. A two-dimensional 1H-NOESY spectrum (in the phase-sensitive mode) was obtained to identify the interproton connectivities in the drug molecule. A molecular modeling program involving Monte Carlo simulation and energy minimization was employed to arrive at the structure of the drug. The program was run with and without the input of the interproton distances derived from the NOESY cross peaks. Both the protocols led to a structure of the drug which was generally similar to that reported from X-ray diffraction data on crystalline diltiazem hydrochloride (Kojic-Prodic, et al. Helv. Chim. Acta 1984, 67, 916-926). However, significant differences between the two structures were seen in the orientations of the substituent groups attached to the benzothiazepine ring. Substantial changes in the circular dichroic (CD) and 1H-NMR spectra of diltiazem were observed on addition of Ca2+ up to a mole ratio of 0.5 Ca2+ per drug. Relatively large changes were seen in 1H resonances of the N-methyl protons and the methylene protons attached to the heterocyclic nitrogen. Analysis of the binding isotherms from CD data at 22 +/- 1 degrees C indicated a 2:1 drug:Ca2+ "sandwich" complex with an estimated dissociation constant of 140 microM. One-dimensional difference NOE and two-dimensional NOESY spectra revealed interproton connectivities between two drug molecules that were compatible with the sandwich complex formation. The interproton distances derived from the volume integrals of the NOESY cross peaks were used as geometrical constraints in modeling the Ca(2+)-bound conformation of diltiazem. The minimum-energy conformation corresponded to the sandwich complex where Ca2+ was coordinated to three oxygens in each of the two drug molecules. Combined with our earlier data on the ability of diltiazem to translocate Ca2+ across the lipid bilayer in synthetic liposomes (Ananthanarayanan, V.S.; Taylor, L.; Pirritano, S.Biochem. Cell Biol. 1992, 70, 608-612), the structural data presented here point to a role for Ca2+ in the interaction of diltiazem with its membrane-bound receptor.     

Interaction of calcium channel antagonists with calcium: structural studies on verapamil and its Ca2+ complex

The conformation of the calcium channel antagonist verapamil has been determined in acetonitrile, in the absence and presence of Ca2+, using two-dimensional 1H-NMR and molecular modeling techniques. Interproton connectivities in the drug molecule were identified from the observed NOESY cross peaks and interproton distances were estimated from the magnitudes of the volume integrals of the cross peaks. The molecular modeling program utilized the Monte Carlo simulation to generate a random ensemble of conformers complying with the NOESY-derived distance constraints. The energies of these conformers were subsequently computed. The minimum-energy structure of the free drug obtained in this manner exhibited some significant differences from the structure of verapamil determined by X-ray crystallography. In particular, the torsional angles in the middle region of the molecule containing the aliphatic "backbone" were such that the two aromatic rings at either end of the drug molecules were moved farther apart from each other in solution than in the crystal structure. The nearly perpendicular orientation of the aromatic rings seen in the crystal was, however, maintained in the solution structure as well. The addition of Ca2+ to a solution of verapamil in acetonitrile caused marked changes in the difference absorbance of the drug in the 200-300-nm region and in many of its 1H-NMR resonances. The changes were most significant up to a mole ratio of about 0.5 Ca2+:drug. Analysis of the binding data at 25 degrees C showed the presence of both 2:1 and 1:1 drug:Ca2+ complexes in equilibrium, the former "sandwich" complex being dominant at the lower cation concentrations with an estimated dissociation constant of about 300 microM. All of the NOESY cross peaks of the free drug remained on addition of 0.5 mol ratio of Ca2+ to verapamil in deuterated acetonitrile and only two new connectivities were observed. Using the interproton distances calculated from these NOESY data, molecular modeling of the 2:1 drug:Ca2+ complex was carried out to yield the minimum-energy conformer. In this conformer, Ca2+ was coordinated to two methoxy oxygens from each of the two drug molecules. The implications of the verapamil-Ca2+ interaction are discussed in terms of available experimental data on the binding of verapamil to the dihydropyridine-sensitive channel and in terms of a hypothesis on the formation of a drug-Ca(2+)-receptor complex in the lipid bilayer environment.     

NMR investigations of recombinant 15N/13C/2H-labeled bradykinin bound to a Fab mimic of the B2 receptor

NMR spectroscopy has been used to obtain structural information on the bioactive conformation of the nonapeptide hormone bradykinin (Arg-Pro-Pro-Gly-Ser-Pro-Phe-Arg, BK) bound to the Fab-fragment of an antibody that mimics the hormone binding site of the natural bradykinin B2-receptor. Using 15N or 15N,13C, 60% 2H isotope labelled bradykinin, complete 1H, 13C and 15N assignments for bradykinin bound to the Fab-fragment have been obtained. Preliminary interpretation of 15N edited NOE spectra indicates that the conformation of bradykinin bound to the model receptor differs substantially from previous computer models of the bioactive conformation of bradykinin.     

NMR studies of Ca2+ binding to the regulatory domains of cardiac and E41A skeletal muscle troponin C reveal the importance of site I to energetics of the induced structural changes

Ca2+ binding to the N-domain of skeletal muscle troponin C (sNTnC) induces an "opening" of the structure [Gagné, S. M., et al. (1995) Nat. Struct. Biol. 2, 784-789], which is typical of Ca2+-regulatory proteins. However, the recent structures of the E41A mutant of skeletal troponin C (E41A sNTnC) [Gagné, S. M., et al. (1997) Biochemistry 36, 4386-4392] and of cardiac muscle troponin C (cNTnC) [Sia, S. K., et al. (1997) J. Biol. Chem. 272, 18216-18221] reveal that both of these proteins remain essentially in the "closed" conformation in their Ca2+-saturated states. Both of these proteins are modified in Ca2+-binding site I, albeit differently, suggesting a critical role for this region in the coupling of Ca2+ binding to the induced structural change. To understand the mechanism and the energetics involved in the Ca2+-induced structural transition, Ca2+ binding to E41A sNTnC and to cNTnC have been investigated by using one-dimensional 1H and two-dimensional {1H,15N}-HSQC NMR spectroscopy. Monitoring the chemical shift changes during Ca2+ titration of E41A sNTnC permits us to assign the order of stepwise binding as site II followed by site I and reveals that the mutation reduced the Ca2+ binding affinity of the site I by approximately 100-fold [from KD2 = 16 microM [sNTnC; Li, M. X., et al. (1995) Biochemistry 34, 8330-8340] to 1.3 mM (E41A sNTnC)] and of the site II by approximately 10-fold [from KD1 = 1.7 microM (sNTnC) to 15 microM (E41A sNTnC)]. Ca2+ titration of cNTnC confirms that cNTnC binds only one Ca2+ with a determined dissociation constant KD of 2.6 microM. The Ca2+-induced chemical shift changes occur over the entire sequence in cNTnC, suggesting that the defunct site I is perturbed when site II binds Ca2+. These measurements allow us to dissect the mechanism and energetics of the Ca2+-induced structural changes.     

Structural characterization of an N-acetyl-2-aminofluorene (AAF) modified DNA oligomer by NMR, energy minimization, and molecular dynamics

An N-acetyl-2-aminofluorene (AAF) modified deoxyoligonucleotide duplex, d(C1-C2-A3-C4-[AAF-G5]-C6-A7-C8-C9).d(G10-G11-T12-G13-C14-++ +G15-T16-G17-G18), was studied by one- and two-dimensional NMR spectroscopy. Eight of the nine complementary nucleotides form Watson-Crick base pairs, as shown by NOEs between the guanine imino proton and cytosine amino protons for G.C base pairs or by an NOE between the thymine imino proton and adenine H2 proton for A.T base pairs. The AAF-G5 and C14 bases show no evidence of complementary hydrogen bond formation to each other. The AAF-G5 base adopts a syn conformation, as indicated by NOEs between the G5 imino proton and the A3-H3' and A3-H2'/H2" protons and by NOEs between the fluorene-H1 proton of AAF and the G5-H1' or C6-H1' proton. The NOEs from the C4-H6 proton to C4 sugar protons are weak, and thus the glycosidic torsion angle in this nucleotide is not well defined by these NMR data. The remaining bases are in the anti conformation, as depicted by the relative magnitude of the H8/H6 to H2' NOEs when compared to the H8/H6 to H1' NOEs. The three base pairs on each end of the duplex exhibit NOEs characteristic of right-handed B-form DNA. Distance restraints obtained from NOESY data recorded at 32 degrees C using a 100-ms mixing time were used in conformational searches by molecular mechanics energy minimization studies. The final, unrestrained, minimum-energy conformation was then used as input for an unrestrained molecular dynamics simulation. Chemical exchange cross peaks are observed, and thus the AAF-9-mer exists in more than a single conformation on the NMR time scale. The NMR data, however, indicate the presence of a predominant conformation (> or = 70%). The structure of the predominant conformation of the AAF-9-mer shows stacking of the fluorene moiety on an adjacent base pair, exhibiting features of the base-displacement [Grunberger, D., Nelson, J. H., et al. (1970) Proc. Natl. Acad. Sci. U.S.A. 66, 488-494] and insertion-denaturation models [Fuchs, R.P.P., & Daune, M. (1971) FEBS Lett. 14, 206-208], while the distal ring of the fluorene moiety protrudes into the minor groove.     

1H, 13C, 15N nuclear magnetic resonance backbone assignments and secondary structure of human calcineurin B

The calmodulin- and calcium-stimulated protein phosphatase calcineurin, PP2B, consists of two subunits: calcineurin B, which binds Ca2+, and calcineurin A, which contains the catalytic site and a calmodulin binding site. Heteronuclear 3D and 4D NMR experiments were carried out on a recombinant human calcineurin B which is a 170-residue protein of molecular mass 19.3 kDa, uniformly labeled with 15N and 13C. The nondenaturing detergent CHAPS was used to obtain a monomeric form of calcineurin B. Three-dimensional triple resonance experiments yielded complete sequential assignment of the backbone nuclei (1H, 13C, and 15N). This assignment was verified by a 4D HN(COCA)NH experiment carried out with 50% randomly deuteriated and uniformly 15N- and 13C-enriched calcineurin B. The secondary structure of calcineurin B has been determined on the basis of the 13C alpha and 13C beta secondary chemical shifts, J(HNH alpha) couplings, and NOE connectivities obtained from 3D 15N-separated and 4D 13C/15N-separated NOESY spectra. Calcineurin B has eight helices distributed in four EF-hand, helix-loop-helix [Kretsinger, R. H. (1980) CRC Crit. Rev. Biochem. 8, 119-174] calcium binding domains. The secondary structure of calcineurin B is highly homologous to that of calmodulin. In comparison to calmodulin, helices B and C are shorter while helix G is considerably longer. As was observed for calmodulin in solution, calcineurin B does not have a single long central helix; rather, helices D and E are separated by a six-residue sequence in a flexible nonhelical conformation.     

Alterations in calcium antagonist receptors and calcium content in senescent brain and attenuation by nimodipine and nicardipine

Characteristics of L- and N-type calcium (Ca++) channel antagonist receptors in brains of senescence-accelerated prone mouse (SAMP8) showing age-related deterioration of learning and memory were examined by using (+)-[3H]PN 200-110 and [125I]omega-conotoxin GVIA as radioligands. There was a tendency toward consistent decrease in Bmax for both radioligands in seven brain regions of SAMP8 compared with the control mouse. The reduction in (+)-[3H]Pn 200-110 binding sites was statistically significant in the hippocampus, midbrain and pons/medulla oblongata, and that in [125I]omega-conotoxin binding sites was significant in the cerebral cortex, corpus striatum and pons/medulla oblongata. On the other hand, there was a marked elevation in Ca++ content in the brain of SAMP8. Chronic p.o. administration (0.3, 1 and 3 mg/kg/day for 3 weeks) of nimodipine and nicardipine to SAMP8 caused a significant increase in the Bmax values of (+)-[3H]PN 200-110 binding in the cerebral cortex and hippocampus. This may reflect up-regulation of brain Ca++ channel antagonist receptors as a result of the prolonged blockade by nimodipine and nicardipine. On the other hand, similar administration of amlodipine and nilvadipine failed to produce an enhancement of Bmax values of (+)-[3H]PN 200-110 binding, whereas both drugs at high doses evoked a significant increase in the apparent dissociation constant. Furthermore, the brain Ca++ content in SAMP8 was markedly reduced by chronic p.o. administration of Ca++ channel antagonists, and the decrease was equivalently observed for all of four 1,4-dihydropyridine antagonists in spite of the difference in the effect on brain receptors. In conclusion, the present study suggests that there is an altered Ca++ homeostasis in the SAMP8 brain that is effectively attenuated by chronic administration of nimodipine and nicardipine. Hence SAMP8 may be a suitable animal model for evaluating the therapeutic effects of Ca++ channel antagonists on neurological disorders associated with the aging brain.     

Lipid binding ridge on loops 2 and 3 of the C2A domain of synaptotagmin I as revealed by NMR spectroscopy

The C2A domain of synaptotagmin I, which binds Ca2+ and anionic phospholipids, serves as a Ca2+ sensor during excitation-secretion coupling. We have used multidimensional NMR to locate the region of C2A from rat synaptotagmin I that interacts, in the presence of Ca2+, with phosphatidylserine. Untagged, recombinant C2A was double-labeled with 13C and 15N, and triple-resonance NMR data were collected from C2A samples containing either Ca2+ alone or Ca2+ plus 6:0 phosphatidylserine. Phospholipid binding led to changes in chemical shifts of backbone atoms in residues Arg233 and Phe234 of loop 3 (a loop that also binds Ca2+) and His198, Val205, and Phe206 of loop 2. These residues lie along a straight line on a surface ridge of the C2A domain. The only other residue that exhibited appreciable chemical shift changes upon adding lipid was His254; however, because His254 is located on the other side of the molecule from the phospholipid docking site defined by the other residues, its shifts may result from nonspecific interactions. The results show that the "docking ridge" responsible for Ca2+-dependent membrane association is localized on the opposite side of the C2A domain from the transmembrane and C2B domains of synaptotagmin.     

Structural changes of sarcoplasmic reticulum Ca(2+)-ATPase upon Ca2+ binding studied by simultaneous measurement of infrared absorbance changes and changes of intrinsic protein fluorescence

Ca2+ binding to sarcoplasmic reticulum Ca(2+)-ATPase was investigated by Fourier transform infrared (FTIR) spectroscopy using the photolytic release of Ca2+ from the photolabile Ca2+ chelator 1-(2-nitro-4,5-dimethoxy)-N,N,N',N',- tetrakis[(oxycarbonyl)]methyl-1,2-ethandiamine (DM-nitrophen). IR absorbance changes in 1H2O and 2H2O were detected in the spectral region from 1800 cm-1 to 1200 cm-1, reflecting photolysis of DM-nitrophen and Ca2+ binding to the Ca(2+)-ATPase. As an independent probe for protein conformational changes, intrinsic fluorescence changes upon Ca2+ release were monitored simultaneously to the FTIR measurements. Both the IR absorbance changes and the fluorescence intensity changes correlated well with the Ca2+ binding activity of the ATPase in this specific step. Ca2+ binding caused IR difference bands mainly in the region of amide I absorption of the polypeptide backbone, reflecting conformational changes of the protein. The small amplitude of the signals indicates that only a few residues perform local structural changes such as changes of bond angles or hydrogen bonding. Other absorbance changes appearing above 1700 cm-1 can be assigned to Ca2+ binding to Glu or Asp side chain carboxyl groups and concomitant deprotonation of these residues. This assignment is strengthened by downshifts of these bands by 4 cm-1 to 6 cm-1 upon 1H2O/2H2O exchange. This is in line with results of mutagenesis studies where such residues containing carboxyl groups were associated with the high affinity Ca2+ binding site (Clarke, D.M., Loo, T.W. and MacLennan, D.H. (1990) J. Biol. Chem. 265, 6262-6267).     

Effect of pH and calcium concentration on calcium diffusion in streptococcal model-plaque biofilms

Demineralization during a cariogenic episode is affected by storage and transport in dental plaque of ions released from enamel, and by the effect on both of plaque fluid pH and ion concentrations. To investigate this, 45Ca effusion from a condensed film of streptococci was measured at pH 7, 6 and 5, and 0-20 mmol/l calcium. Cells were loaded into effusion chambers and the appearance of 45Ca and [3H]-inulin in carrier-containing but initially tracer-free buffer was measured. Ratios of 45Ca and [3H]-inulin activity in the initial suspending solution and at equilibrium in the clearance solution, permitted calculation of extracellular volume and bound calcium. The rate of Ca appearance was proportional to the retarded diffusion coefficient (rDe), which was related to the effective diffusion coefficient (De) by: rDe = De/(1 + R) in which R is the ratio of bound to free Ca2+. The rate of Ca2+ effusion increased with calcium concentration, converging on a value of 2.8 x 10(-10) m2/sec. At low pH it reached convergence at a lower [Ca]. This demonstrates that calcium effusion is dependent on binding, so a high proportion of binding sites in plaque will reduce mineral loss in vivo. Loss of binding sites at low pH will increase mineral loss.     

Ca2+ differentially regulates conventional protein kinase Cs' membrane interaction and activation

The regulation of conventional protein kinase Cs by Ca2+ was examined by determining how this cation affects the enzyme's 1) membrane binding and catalytic function and 2) conformation. In the first part, we show that significantly lower concentrations of Ca2+ are required to effect half-maximal membrane binding than to half-maximally activate the enzyme. The disparity between binding and activation kinetics is most striking for protein kinase C betaII, where the concentration of Ca2+ promoting half-maximal membrane binding is approximately 40-fold higher than the apparent Km for Ca2+ for activation. In addition, the Ca2+ requirement for activation of protein kinase C betaII is an order of magnitude greater than that for the alternatively spliced protein kinase C betaI; these isozymes differ only in 50 amino acids at the carboxyl terminus, revealing that residues in the carboxyl terminus influence the enzyme's Ca2+ regulation. In the second part, we use proteases as conformational probes to show that Ca2+dependent membrane binding and Ca2+-dependent activation involve two distinct sets of structural changes in protein kinase C betaII. Three separate domains spanning the entire protein participate in these conformational changes, suggesting significant interdomain interactions. A highly localized hinge motion between the regulatory and catalytic halves of the protein accompanies membrane binding; release of the carboxyl terminus accompanies the low affinity membrane binding mediated by concentrations of Ca2+ too low to promote catalysis; and exposure of the amino-terminal pseudosubstrate and masking of the carboxyl terminus accompany catalysis. In summary, these data reveal that structural determinants unique to each isozyme of protein kinase C dictate the enzyme's Ca2+-dependent affinity for acidic membranes and show that, surprisingly, some of these determinants are in the carboxyl terminus of the enzyme, distal from the Ca2+-binding site in the amino-terminal regulatory domain.     

Interaction of a novel GDP exchange inhibitor with the Ras protein

Mutated, tumorigenic Ras is present in a variety of human tumors. Compounds that inhibit tumorigenic Ras function may be useful in the treatment of Ras-related tumors. The interaction of a novel GDP exchange inhibitor (SCH-54292) with the Ras-GDP protein was studied by NMR spectroscopy. The binding of the inhibitor to the Ras protein was enhanced at low Mg2+ concentrations, which enabled the preparation of a stable complex for NMR study. To understand the enhanced inhibitor binding and the increased GDP dissociation rates of the Ras protein, the conformational changes of the Ras protein at low Mg2+ concentrations was investigated using two-dimensional 1H-15N HSQC experiments. The Ras protein existed in two conformations in slow exchange on the NMR time scale under such conditions. The conformational changes mainly occurred in the GDP binding pocket, in the switch I and the switch II regions, and were reversible. The Ras protein resumed its regular conformation after an excess amount of Mg2+ was added. A model of the inhibitor in complex with the Ras-GDP protein was derived from intra- and intermolecular NOE distance constraints, and revealed that the inhibitor bound to the critical switch II region of the Ras protein.     

Comparison of the complexation of fluoroquinolone antimicrobials with metal ions by nuclear magnetic resonance spectroscopy

The complexation of fluoroquinolone antimicrobials with various metal ions have been studied in aqueous solution (pD 2.5, 37 degrees C) by 1H and 13C-NMR spectroscopy. The compounds examined are levofloxacin, ciprofloxacin and lomefloxacin. In each drug, new signals have appeared by the addition of Al3+, suggesting that the complexes are formed between the drug and Al3+ and that the ligand exchange is slow on the NMR time scale. Solution structure of the major species in the presence of 2.0 mol equiv of Al3+ has been proposed based on the large downfield shifts of some specific protons. Signals of both the coordinated and free drugs have shown slight broadening at 90 degrees C due to the enhanced rate in ligand dissociation process, though the coalescence phenomena are not observed even at this temperature. Thus, the complexes are supposed to be stable at the physiological condition. Titration experiments have revealed that the binding ability of levofloxacin toward Al3+ is much stronger than that of ciprofloxacin and lomefloxacin at pD 2.5. In contrast to the complexation with Al3+, the binding of these drugs with other metal ions such as Ca2+ and Mg2+ is much weaker; NMR signals have shown no appreciable downfield shift by the addition of Ca2+ and Mg2+. Based on these results, it is concluded that the fluoroquinolone antimicrobials examined in the present study at pD 2.5 exist as stable complexes in the presence of Al3+ and the absorptivity of the drugs on oral administration could be affected by Al3+.     

The paternal effect gene ms(3)sneaky is required for sperm activation and the initiation of embryogenesis in Drosophila melanogaster

Calcium sensor proteins translate transient increases in intracellular calcium levels into metabolic or mechanical responses, by undergoing dramatic conformational changes upon Ca2+ binding. A detailed analysis of the calcium binding-induced conformational changes in the representative calcium sensors calmodulin (CaM) and troponin C was performed to obtain insights into the underlying molecular basis for their response to the binding of calcium. Distance difference matrices, analysis of interresidue contacts, comparisons of interhelical angles, and inspection of structures using molecular graphics were used to make unbiased comparisons of the various structures. The calcium-induced conformational changes in these proteins are dominated by reorganization of the packing of the four helices within each domain. Comparison of the closed and open conformations confirms that calcium binding causes opening within each of the EF-hands. A secondary analysis of the conformation of the C-terminal domain of CaM (CaM-C) clearly shows that CaM-C occupies a closed conformation in the absence of calcium that is distinct from the semi-open conformation observed in the C-terminal EF-hand domains of myosin light chains. These studies provide insight into the structural basis for these changes and into the differential response to calcium binding of various members of the EF-hand calcium-binding protein family. Factors contributing to the stability of the Ca2+-loaded open conformation are discussed, including a new hypothesis that critical hydrophobic interactions stabilize the open conformation in Ca2+ sensors, but are absent in "non-sensor" proteins that remain closed upon Ca2+ binding. A role for methionine residues in stabilizing the open conformation is also proposed.     

Comparative conformational studies on cyclic hexapeptides corresponding to message sequence His-Phe-Arg-Trp of alpha-melanotropin by NMR

Solution conformation of cyclo(Gly1-His2-Phe3-Arg4-Trp5-Gly6) and its D-Phe analog corresponding to the message sequence [Gly-alpha-MSH5-10] of alpha-MSH has been studied by 1D and 2D proton magnetic resonance spectroscopy in dimethyl sulfoxide (DMSO)-d6 solution and in a DMSO-d6/H2O cryoprotective mixture. The NMR data for both the analogs in solution at 300 K cannot be interpreted based on a single ordered conformation, as evidenced by the broadening of only -NH resonances as well as the temperature coefficients of the amide protons. An analysis of the nuclear Overhauser effect (NOE) cross-peaks in conjunction with temperature coefficient data indicates an equilibrium of multiple conformers with a substantial population of particular conformational states at least in the D-analog. The molecular dynamics simulations without and with NOE constraints also reveal numerous low-energy conformers with two gamma-turns, a gamma-turn and a beta-turn, two beta-turns, etc. for both the analogs. The observed NMR spectra can be rationalized by a dynamic equilibrium of conformers characterized by a gamma-bend at Gly6, two gamma-bends at Phe3 and Gly6 and a conformer with a single beta-turn and a gamma-bend for the L-Phe analog. On the other hand, a conformation with two fused beta-turns around the two tetrads His2-D-Phe3-Arg4-Trp5 and Trp5-Gly6-Gly1-His2 dominates the equilibrium mixture for the D-Phe analog. For the D-Phe analog, the experimentally observed average conformation is corroborated by molecular dynamics simulations as well as by studies in cryoprotective solvent.     

Integrating cytosolic calcium signals into mitochondrial metabolic responses

Stimulation of hepatocytes with vasopressin evokes increases in cytosolic free Ca2+ ([Ca2+]c) that are relayed into the mitochondria, where the resulting mitochondrial Ca2+ ([Ca2+]m) increase regulates intramitochondrial Ca2+-sensitive targets. To understand how mitochondria integrate the [Ca2+]c signals into a final metabolic response, we stimulated hepatocytes with high vasopressin doses that generate a sustained increase in [Ca2+]c. This elicited a synchronous, single spike of [Ca2+]m and consequent NAD(P)H formation, which could be related to changes in the activity state of pyruvate dehydrogenase (PDH) measured in parallel. The vasopressin-induced [Ca2+]m spike evoked a transient increase in NAD(P)H that persisted longer than the [Ca2+]m increase. In contrast, PDH activity increased biphasically, with an initial rapid phase accompanying the rise in [Ca2+]m, followed by a sustained secondary activation phase associated with a decline in cellular ATP. The decline of NAD(P)H in the face of elevated PDH activity occurred as a result of respiratory chain activation, which was also manifest in a calcium-dependent increase in the membrane potential and pH gradient components of the proton motive force (PMF). This is the first direct demonstration that Ca2+-mobilizing hormones increase the PMF in intact cells. Thus, Ca2+ plays an important role in signal transduction from cytosol to mitochondria, with a single [Ca2+]m spike evoking a complex series of changes to activate mitochondrial oxidative metabolism.     

Solution NMR structure and backbone dynamics of the major cold-shock protein (CspA) from Escherichia coli: evidence for conformational dynamics in the single-stranded RNA-binding site

The major cold-shock protein (CspA) from Escherichia coli is a single-stranded nucleic acid-binding protein that is produced in response to cold stress. We have previously reported its overall chain fold as determined by NMR spectroscopy [Newkirk, K., Feng, W., Jiang, W., Tejero, R., Emerson, S. D., Inouye, M., and Montelione, G. T. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 5114-5118]. Here we describe the complete analysis of 1H, 13C, and 15N resonance assignments for CspA, together with a refined solution NMR structure based on 699 conformational constraints and an analysis of backbone dynamics based on 15N relaxation rate measurements. An extensive set of triple-resonance NMR experiments for obtaining the backbone and side chain resonance assignments were carried out on uniformly 13C- and 15N-enriched CspA. Using a subset of these triple-resonance experiments, the computer program AUTOASSIGN provided automatic analysis of sequence-specific backbone N, Calpha, C', HN, Halpha, and side chain Cbeta resonance assignments. The remaining 1H, 13C, and 15N resonance assignments for CspA were then obtained by manual analysis of additional NMR spectra. Dihedral angle constraints and stereospecific methylene Hbeta resonance assignments were determined using a new conformational grid search program, HYPER, and used together with longer-range constraints as input for three-dimensional structure calculations. The resulting solution NMR structure of CspA is a well-defined five-stranded beta-barrel with surface-exposed aromatic groups that form a single-stranded nucleic acid-binding site. Backbone dynamics of CspA have also been characterized by 15N T1, T2, and heteronuclear 15N-1H NOE measurements and analyzed using the extended Lipari-Szabo formalism. These dynamic measurements indicate a molecular rotational correlation time taum of 4.88 +/- 0.04 ns and provide evidence for fast time scale (taue < 500 ps) dynamics in surface loops and motions on the microsecond to millisecond time scale within the proposed nucleic acid-binding epitope.     

Determination of calcium binding sites in gas-phase small peptides by tandem mass spectrometry

Low-energy (LE) and high-energy (HE) collisionally activated decompositions (CAD) of calcium/peptide complexes of the form [M - H + Ca]+ and [M + Ca]2+ reflect the site of calcium binding in various gas-phase peptides that are models of the calcium binding site III of rabbit skeletal troponin C. The Ca2+ binding sites involve an aspartic acid, glutamic acid, and asparagine, which are in the metal-binding loops of calcium-binding proteins. Both fast atom bombardment (FAB) and electrospray ionization (ESI) were used to generate the metal/peptide complexes. When submitted to LE CAD, ESI-produced Ca2+/peptide complexes undergo fragmentations that are controlled by Ca2+ binding and provide information on the Ca2+ binding site. The LE CAD spectra are simple, indicating that Ca2+ binding involves specific oxygen ligands including acidic side chains and that only a few low-energy fragmentation channels exist. The HE CAD spectra of FAB-produced Ca2+/peptide complexes are more complex, owing to the introduction of high internal energy into the precursor ion. Interactions of the other alkaline-earth metal ions Mg2+ and Ba2+ with these peptides reveal that the ligand preferences of these metal ions are slightly different than those of Ca2+.     

An NMR study on the DNA-binding SPKK motif and a model for its interaction with DNA

The solution structure of one and two repeats of the 'SPKK' DNA-binding motif is reported on the basis of NMR measurements. In dimethylsulphoxide (DMSO) the major population (approximately 90%) of peptides, SPRKSPRK(S2) and GSPKKSPRK(S2b), adopts a conformation, which has two trans prolines. The two 'SP(R/K)K' units in these peptides are equivalent and each adopts a turn structure exchanging with an extended structure. This is suggested by an NOE connectivity of the beta-turn type, between the backbone amide protons of residues (i+2) and (i+3) and NOE connectivities of the Asx(sigma)-turn type, between protons of the ith Ser and the backbone amide proton on residue (i+2). This suggests that each SP(R/K)K unit has a structural intermediate between (or a combination of) a beta-turn and an Asx(sigma)-turn. In 90-10% DMSO/H2O at 4 degrees C the two units of S2 are connected more tightly by folding into a short 3(10) helix, broken at the second proline. For another peptide, Thr-Pro-Arg-Lys(T1), the major population (75%) in 100% DMSO comprises a beta-turn in rapid exchange with an extended structure. We did not observe an NOE connectivity of the Asx(sigma) type with the T1 peptide. A possible structure of the SPKK motif in the complex with DNA is discussed.     

Fatty acid-mediated calcium sequestration within intracellular calcium pools

Intracellular Ca2+ pools play an essential role in generating Ca2+ signals. The heterogeneity of intracellular Ca2+ pools reflects the complex and dynamic character of the endoplasmic reticulum within which they reside. Translocation of Ca2+ between distinct subcompartments of the endoplasmic reticulum is mediated by a sensitive and specific GTP-activated process involving formation of reversible communicating junctions (Rys-Sikora, K. E., Ghosh, T. K., and Gill, D. L. (1994) J. Biol. Chem. 269, 31607-31613). In the presence of palmitate at 10 microM or above, this GTP-activated mechanism mediates substantial Ca2+ accumulation within a specific Ca2+-pumping pool. The fatty acid- and GTP-dependent accumulation of Ca2+ was highly chain length-specific; pentadecanoate (C15) and palmitate (C16) were equally effective, whereas fatty acids of shorter or longer chain length were either marginally effective or devoid of effect. Fatty acids with one or more unsaturated carbons were without effect, regardless of chain length. Palmitate-induced Ca2+ accumulation was immediately terminated with 2 microM palmitoyl-CoA, a blocker of the GTP-activated Ca2+-translocating mechanism. The anion transport inhibitor 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid completely prevented both palmitate- and oxalate-mediated GTP-dependent Ca2+ accumulation, with EC50 approximately 30 microM. Ca2+ sequestered in the presence of palmitate and GTP could be immediately and completely released by A23187, whereas the sequestered Ca2+ was remarkably resistant to release induced by inositol 1,4,5-trisphosphate (InsP3). In contrast, oxalate-sequestered Ca2+ within the same pool could be effectively released by either ionophore or InsP3. The results indicate that fatty acids are specifically transported into the lumen of a subset of Ca2+ pools, wherein they mediate substantial sequestration of Ca2+ in a distinct membrane-associated substate that is not readily releasable by opened InsP3-sensitive Ca2+ channels.