Metallothionein-3 is a component of a multiprotein complex in the mouse brain.

Metallothionein (MT)-3, originally called growth inhibitory factor (GIF), was initially identified through its ability to inhibit the growth of neuronal cells in the presence of brain extract. MT-3 is the brain specific isoform of the MT family whose specific biological activity associates it with neurological disorders. Indeed, studies report that MT-3 is decreased by ~30% in brains of patients with Alzheimer disease (AD). Furthermore, many lines of evidence suggest that MT-3 engages in specific protein interactions. To address this, we conducted immunoaffinity chromatography experiments using an immobilized anti-mouse MT-3 antibody. We identified five associated proteins from the pool of sixteen recovered using mass spectrometry and tandem mass spectrometry after in-gel trypsin digestion of bands from the affinity chromatography. The proteins identified were: heat shock protein 84 (HSP84), heat shock protein 70 (HSP70), dihydropyrimidinase-like protein-2 (DRP-2), creatine kinase (CK) and beta-actin. Coimmunoprecipitation experiments, also conducted on whole mouse brain extract using the anti-mouse MT-3 antibody along with commercially available antibodies against HSP84 and CK, confirmed that these three proteins were in a single protein complex. Immunohistochemical experiments were then conducted on the perfused mouse brain that confirmed the in situ colocalization of CK and MT-3 in the hippocampus region. These data provide new insights into the involvement of MT-3 in a multiprotein complex, which will be used to understand the biological activity of MT-3 and its role in neurological disease.

Reversible trifluoroacetic acid-induced conformational changes in glycophorin A as detected by proton nuclear magnetic resonance spectroscopy.

High-field (270 MHz) 1H-NMR has been employed to study the solution conformation of glycophorin A, a sialoglycoprotein which spans the human erythrocyte membrane. Glycophorin A is one of the most fully characterized integral membrane proteins known, making it an excellent model for the study of membrane-bound proteins. This protein consists of three distinct domains: a glycosylated extracellular N-terminus, a hydrophobic intramembranous segment, and a polar cytoplasmic C-terminus. These domains contain aromatic residues which serve as convenient 1H-NMR conformational probes. The aromatic region of the NMR spectrum of glycophorin A in 2H2O shows single, well-resolved His and Tyr resonances. No resonances are observed, however, for the Phe residues which are located in or near the hydrophobic domain. These observations suggest that considerable heterogeneity with respect to segmental motions exists within the protein. This is consistent with circular dichroism data showing the intramembranous segment to be completely helical with the extremities of the protein being predominantly random coils. The helix of the hydrophrobic domain is remarkably resistant to conventional denaturing conditions including variations in pH, and temperature, and treatment with guanidine hydrochloride. However, in trifluoroacetic acid, which strongly solvates peptide backbones, there is extensive reversible unfolding of the helical structure as evidenced by the appearance of Phe resonances. Solvent titration experiments indicate that approximately a 1 : 1 volume ratio of trifluoroacetic acid to 2H2O is required to initiate unfolding of the helix.

Three-dimensional solution structure of mouse [Cd7]-metallothionein-1 by homonuclear and heteronuclear NMR spectroscopy.

Sequential 1H-NMR assignments of mouse [Cd7]-metallothionein-1 (MT1) have been carried out by standard homonuclear NMR methods and the use of an accordion-heteronuclear multiple quantum correlation (HMQC) experiment for establishing the metal, 113Cd2+, to cysteine connectivities. The three-dimensional structure was then calculated using the distance constraints from two-dimensional nuclear Overhauser effect (NOE) spectroscopy spectra and the Cys-Cd connectivities as input for a distance geometry-dynamical simulated annealing protocol in X-PLOR 3.851. Similar to the mammalian MT2 isoforms, the homologous primary structure of MT1 suggested two separate domains, each containing one metal cluster. Because there were no interdomain constraints, the structure calculation for the N-terminal beta- and the C-terminal alpha-domain were carried out separately. The structures are based on 409 NMR constraints, consisting of 381 NOEs and 28 cysteine-metal connectivities. The only elements of regular secondary structure found were two short stretches of 3(10) helices along with some half-turns in the alpha-domain. Structural comparison with rat liver MT2 showed high similarity, with the beta-domain structure in mouse MT1 showing evidence of increased flexibility compared to the same domain in MT2. The latter was reflected by the presence of fewer interresidue NOEs, no slowly exchanging backbone amide protons, and enhanced cadmium-cadmium exchange rates found in the beta-domain of MT1.

3D solution structure of copper and silver-substituted yeast metallothioneins.

3D solution structural calculations for yeast silver(I)-substituted metallothionein (MT) and native copper(I) MT were completed using experimentally determined NOE and dihedral angle constraints, in conjunction with experimentally derived metal-to-Cys connectivities for AgMT which were assumed identical for CuMT. For the first 40 residues in both structures, the polypeptide backbone wraps around the metal cluster in two large parallel loops separated by a deep cleft containing the metal cluster. Minor differences between the two structures include differences in hydrogen bonds and the orientation of the N-terminus with the overall protein volume conserved to within 6.5%.

Characterization of the calcium-binding sites of calcineurin B.

Calcineurin (CaN) is a calcium- and calmodulin-dependent serine/threonine phosphatase whose inhibition by the immunosuppressant-immunophilin complexes (cyclosporin-cyclophilin and FK506-FKBP) is considered key to the mechanism of immunosuppression. CaN is a heterodimer, consisting of a 59 kDa catalytic subunit (A) and a 19 kDa calcium-binding regulatory subunit (B). The latter is postulated to harbor four calcium binding domains of the EF hand type. The titration of the CaN B apoprotein with the isomorphic Cd2+ was followed by 113Cd NMR and these data support one high-affinity metal binding site and three lower-affinity ones. Flow dialysis data with Ca2+ indicate one high affinity calcium binding site with Kd approximately 2.4 x 10(-8) M and three other sites with Kd approximately 1.5 x 10(-5) M. The chemical shifts of all four 113Cd resonances (-75, -93, -106 and -119 ppm) are in the same range as found in other 113Cd substituted calcium-binding proteins, and are indicative of all-oxygen coordination of pentagonal bipyramidal geometry.

HIV protease substrate conformation: modulation by cyclophilin A.

Cyclophilin A (CyPA), a cytosolic peptidyl-prolyl trans-cis isomerase can accelerate the trans-cis isomerization of Xxx-Pro peptide bonds. One- and two-dimensional 1H-NMR spectroscopy were used to determine that the heptapeptide Ser-Gln-Asn-Tyr-Pro-Ile-Val, a model peptide of an HIV-1 protease cleavage site in the gag polyprotein of HIV-1, is a substrate for CyPA. Experiments revealed a slow exchange about the Tyr-Pro peptide bond with 30 +/- 5% in the cis conformation (pH 1-9). While the interconversion rate is too slow to measure by kinetic NMR methods in the absence of CyPA, these methods, saturation transfer and NOE experiments, established that CyPA enhanced the rate of trans-cis interconversion, a process inhibited by cyclosporin A (CsA). With a substrate:CyPA ratio of 40:1, an interconversion rate of 2.5 s(-1) at 25 degrees C was observed.

A binding site peptide fragment of the nicotinic acetylcholine receptor. Sequence-specific assignment of 1H-NMR resonances in the dodecamer alpha 185-196.

The total sequence-specific 1H assignment for the alpha 185-peptide was accomplished by analysis of COSY spectra along with spin-decoupling and confirmatory NOE difference experiments. Some ambiguities in the assignments were successfully addressed utilizing additional peptides with selective amino acid substitutions. The chemical shifts of several of the C alpha H resonances, along with evidence for a slowly exchanging amide at Thr-191 suggest that the alpha 185-peptide may contain a certain amount of non-random coil structure. The role of any such ordered structure in the mechanism of binding to alpha-bungarotoxin remains to be determined. The assignment of the peptide 1H resonances will facilitate the analysis and identification of chemical shift perturbations observed upon formation of the complex between alpha-bungarotoxin and the alpha 185-peptide [7].

Structural elements pertinent to the interaction of cyclosporin A with its specific receptor protein, cyclophilin.

Cyclophilin (163 amino acids; 17,737 daltons) is a ubiquitous cytosolic protein that specifically binds the potent immunosuppressive drug cyclosporin A (CsA). To characterize the structural details of this interaction, extensive use has been made of two-dimensional (2D) NMR methods. For studies on CsA, these methods are being used to assign the conformational space accessible to CsA by analysis of the spectra from the multiple CsA conformers present in slow exchange in mixed solvent systems. These same 2D NMR methods also have been used for extensive studies of the major bovine thymus cyclophilin (CyP) isoform and its complex with stoichiometric amounts of CsA. In the former case, these studies have revealed 81% of the 156 expected HN-H alpha crosspeaks. The complete spin-coupled spin systems for one-third of these amide resonances have been assigned according to amino acid type. After exhaustive D2O exchange, there remain 44 amide protons which exhibit 2D NMR features indicative of a hydrophobic domain with beta-sheet secondary structure. The CsA-complexed form of CyP exhibits a discrete structure and set of resonances in slow exchange with the drug-free CyP. The amino acids that have been specifically identified to be affected by the interaction are limited in number and include three Phe residues, the unique Trp at position 120, and two Ala residues.

NMR analysis of the structure and metal sequestering properties of metallothioneins.

113Cd-NMR studies have been used to elucidate the structure of the metal-binding sites in mammalian and invertebrate ( Scylla serrata) metallothioneins (MTs). Chemical shift data have shown that all Cd ions are tetrahedrally coordinated to four cysteine thiolate ligands with single cysteinyl sulfurs bridging adjacent metals. Homonuclear decoupling experiments have shown that the 7 g-atoms of metal bound per mole of mammalian protein are located in a three- and a four-metal cluster while the 6 g-atoms of metal in the invertebrate MT are located in two three-metal clusters. The different metal binding affinities of the two mammalian clusters have been determined by 113Cd-NMR. The three-metal cluster prefers Cu greater than Zn greater than Cd whereas exactly the reverse order applies in the four-metal cluster. Proteolytic cleavage of the protein produced a 32-residue fragment which contained the four-metal cluster and demonstrated the presence of two separate domains in the protein. 500 MHz 1H-NMR has been employed to elucidate the arrangement of these metal clusters in the tertiary structure of the protein. The 1H resonances were assigned from their scalar and dipolar connectivities obtained from extensive one and two-dimensional NMR experiments. A specific application of 2D correlation spectroscopy ( COSY ) to the assignment of the 1H resonances in crab MT-1 is discussed. A molecular model, representing the three-dimensional solution structure of this protein, has been constructed based on an analysis of all these data. Detailed structural features of this model are discussed, with particular emphasis on their relationship to the function and evolution of the protein.

Magnetic resonance, pathology and physiology of the BA1112 rhabdomyosarcoma in vivo.

We studied histology, findings on H-1 magnetic resonance (MR) imaging, and correlations of P-31 MR spectroscopy with microelectrode pH and pO2 measurements in the BA1112 rhabdomyosarcoma in WAG/Rij/Y rats. Intratumoral hemorrhage was a prominent feature on MR images and pathologic specimens. Eosinophilic necrosis could be seen microscopically but was not discernible on images. The peaks seen on P-31 MR spectra were similar to those reported in other tumors. The intratumoral pH was neutral despite low pO2 values and P-31 MR evidence for impaired metabolic status.

Augmentation of experimental skin flap survival via postoperative phosphocreatine replacement.

Despite improvements in our understanding of cutaneous vascular territories, clinical skin flap necrosis resulting from ischemic compromise is still a reality. Therapy with vasodilators has been generally unsuccessful, but replacement of high-energy phosphometabolites through the use of ATP-MgCl2 and fructose 1,6-diphosphate has been effective. Recently we reported that phosphocreatine is the major high-energy phosphometabolite in mammalian skin and that ATP levels and cellular well-being in skin flaps are dependent on adequate supply of this phosphometabolite. We report herein the successful augmentation of survival of ischemically compromised skin flaps through postoperative phosphocreatine infusion. This metabolite effectively circumvents the nonfunctioning mitochondrial creatine-phosphocreatine energy shuttle without disturbing the delicate [ATP]/[ADP] balance in the cytosol. In addition, phosphocreatine may favorably redistribute blood flow from muscle to the ischemically compromised skin.

Sensitivity-enhanced detection of fast exchanging protons by an exchange-edited gradient HEHAHA-HSQC experiment.

An experiment is presented which allows for the sensitivity-enhanced measurement of proton exchange rates in a HSQC type experiment. Instead of using INEPT type transfer of magnetization from protons to heteronuclei and vice versa, we have used heteronuclear Hartmann-Hahn transfer, which is known to have higher sensitivity in the presence of chemical exchange. Direct NOE's between NH protons and alpha-protons are suppressed by an exchange-editing step unless the alpha-resonances are degenerate with the water resonance. A comparison between the exchange-edited HEHAHA-HSQC and a standard exchange-edited HSQC experiment performed on the uniformly 15N-labeled staphylococcal nuclease H124L shows an enhancement of approximately 100% with the former experiment. A set of one-dimensional exchange-edited spectra of urea was used for evaluating the ability to extract exchange rates using the presented experiment.

113Cd nmr study of the metal cluster structure of human liver metallothionein.

Cadmium-113 nuclear magnetic resonance (113Cd nmr) was used to elucidate the structural properties of the cadmium binding sites in human liver metallothionein. The isotopically labeled 113Cd-metallothionein was prepared by the in vitro exchange of the native metals (greater than 94% zinc) for 113CdCl2 during isolation. The two isoproteins, MT-1 and MT-2, showed 113Cd nmr resonances in the chemical shift range 610-670 ppm. The multiplet structure of the resonances is due to two bond scalar interactions between adjacent 113Cd ions linked by cysteine thiolate ligands. Homonuclear 113Cd decoupling experiments allowed the determination of the metal cluster structure, which, similar to the rabbit liver metallothionein, consists of a four- and a three-metal cluster designated cluster A and cluster B, respectively. Chemical shift similarities in the 113Cd nmr spectra of the human, rabbit and calf liver MT-1 and MT-2 are observed, especially for cluster A. Small variations in chemical shifts are explained in terms of differences in the primary structure between the two human isoproteins.

Nuclear magnetic resonance spectroscopy of skin: predictive correlates for clinical application.

The first application of phosphorous 31 (31P) and proton (1H) nuclear magnetic resonance (NMR) spectroscopy to the analysis of the metabolic profiles of skin flaps in a rat model and of human skin grafts is presented. Resonances of adenosine triphosphate (ATP), phosphocreatine (PCr), and inorganic phosphate (Pi) were identified in 31P nuclear magnetic resonance spectra. Resonances of phosphocreatine, creatine (Cr), and lactate (Lac) were identified in 1H nuclear magnetic resonance spectra. The most significant finding was the substantial presence of phosphocreatine as the major high-energy phosphometabolite in mammalian skin, a finding which heretofore has not been widely recognized. An energy shuttle between phosphocreatine and ATP is operative in skin to buffer the fall in ATP during ischemic (anaerobic) insult. Inability to replenish exhausted phosphocreatine reserves predictively correlates with eventual flap necrosis. We have defined and analyzed temporal fluxes in the phosphocreatine-creatine and phosphocreatine plus creatine-lactate ratios by proton nuclear magnetic resonance. Both are sensitive, accurate, and unambiguous early prognostic indices of eventual flap outcome. These findings support the concept that the fate of a flap may be established as early as 3 hours after elevation and have laid the groundwork for development and application of noninvasive in vivo nuclear magnetic resonance spectroscopy to the study of skin flaps in animals and humans.

Phosphorus-31 NMR studies of E. coli ribosomes.

Phosphorus-31 nuclear magnetic resonance spectra, relaxation times and nuclear Overhauser (NOE) enhancement have been measured for E. coli ribosomes, subunits and rRNA. NOE and T1 experiments reveal that the phosphorus relaxation in this organelle is largely dipolar in origin. Moreover these results imply the presence of internal motion within the RNA chain with a correlation time of about 3-5 x 10(-9) sec. In all cases the predominant resonance is centered at about -1.5 ppm (relative to 85% H3PO4) as expected for a phosphodiester linkage where there is a large degree of double helix. The linewidth narrows by about a factor of four when the ribosomal proteins are removed indicating a substantial immobilization of the RNA when it is assembled into the ribosome. In addition to the phosphodiester resonance, ribosomes also reveal one or two narrower resonances shifted to low field by 1-4 ppm. Based on the observation that these resonances show a pH dependent chemical shift, we assign them to phosphate monoesters i.e. terminal 3' or 5' phosphate groups. These terminal phosphates are due to short oligomers of RNA derived from the terminus of the chain.

Characterization of the properties of the multiple metal binding sites in alkaline phosphatase by carbon-13 nuclear magnetic resonance.

Carbon-13 nuclear magnetic resonance (13C NMR) of Escherichia coli alkaline phosphatase labeled biosynthetically with beta,beta-[gamma-13C]dideuteriohistidine has been used to determine the number and identity of the histidine residues that participate in metal ion coordination at the three classes of binding sites in this dimeric Zn2+ metalloenzyme. Detailed 13C NMR titrations of the apoenzyme with 113Cd2+ and Mg2+, in conjunction with parallel 13 Cd NMR measurements [Otvos, J.D., & Armitage, I.M. (1980) Biochemistry (third of three papers in this issue)], permitted the assignment of four histidine residues as ligands to the "catalytic", or A site, metal ions, two coordinated via their N pi imidazole nitrogens and two via N pi. In addition, a fifth histidyl ligand, coordinated through N pi, was shown to be located at the "structural", or B, sites on the dimer. The "regulatory", or C, sites do not contain histidyl metal ligands. Unambiguous identification of the three histidines coordinated to metal ion via N pi was provided by the observation of resolved 113Cd-13C spin-spin coupling (3J = 12-19 Hz) in their gamma-carbon resonances. Once assigned, the 13C resonances of the five histidyl metal ligands were used to monitor the relative affinities of the A, B, and C sites for Cd2+ and Zn2+. At pH 6.3, Cd2+ was found to bind to the A sites at least 10 times tighter than to the B or C sites, which have roughly equal affinities. In marked contrast, Zn2+ was found to have similar affinities for the A and B sites at both pH 6.3 and 8.0. The affinity of the C sites for Zn2+ and Mg2+ was shown to be at least an order of magnitude lower. The binding constants of all three sites for Cd2+ and Zn2+ are greater than 10(5) M-1. Evidence is also presented that suggests that the A, B, and C sites may be located in close proximity to one another in the monomers.

Determination by cadmium-113 nuclear magnetic resonance of the structural basis for metal ion dependent anticooperativity in alkaline phosphatase.

Cadmium-113 nuclear magnetic resonance (113Cd NMR) has been used to probe the binding characteristics of 113Cd2+ to the three classes of metal binding sites in Escherichia coli alkaline phosphatase to help elucidate the molecular origin of the metal ion dependent "half-sites" reactivity exhibited by this dimeric Zn2+ metalloenzyme [Otvos, J.D., Armitage, I.M., Chlebowski, J.F., & Coleman, J.E. (1979) J. Biol. Chem. 254, 4707-4713]. In the absence of phosphate, the first two 113Cd2+ ions added to the apodimer give rise to a single 113Cd resonance (169 ppm), indicating selective binding to the pair of symmetrically disposed A sites. Resonances arising from additional 113Cd2+ bound to the B and C sites cannot be observed; B- and/or C-site occupation also renders the A-site 113Cd resonance undetectable. Both these observations have been attributed to severe chemical exchange broadening in the A-, B-, and C-site 113Cd signals induced by an unknown modulation process(es). Interestingly, covalent phosphorylation of the active-site serine residues abolishes this exchange modulation, allowing three separate resonances to be detected and assigned to 113Cd2+ located at each of the three classes of metal binding sites in the enzyme. By varying the metal composition of the phosphorylated enzyme, we have characterized the correlations that exist between the chemical shifts ana intensities of these 113Cd resonances and the metal occupancies of the A, B, and C sites in the individual subunits. This information has allowed us to conclude that the half-sites phosphorylation of the Cd2 2+ enzyme is accompanied by a slow migration of half the Cd2+ originally located at the A sites to the B sites on the phosphorylated subunits. The driving force for this metal redistribution, which at equilibrium leaves half the subnits devoid of metal ion and thereby incapable of binding phosphate, is apparently the dramatic stabilization of the complex of Cd2+ with the B sites, which was demonstrated to occur in those subunits that become phosphorylated. From the kinetics of both phosphorylation and metal redistribution in Cd2 2+ enzyme, we suggest that population of the A and B sites in a subunit, rather than the A site alone, constitutes the minimum requirement for induction of catalytic function in alkaline phosphatase. The spin relaxation properties of the enzyme-bound 113Cd2+ ions are also briefly discussed.

Evidence for site-selective metal binding in calf liver metallothionein.

Two isoproteins of calf liver metallothionein (MT) have been isolated, purified, and characterized by atomic absorption, ultraviolet absorption, electron spin resonance, and 113Cd nuclear magnetic resonance spectroscopy. Native calf liver MT was found to contain both Cu+ and Zn2+ in a mole ratio of approximately 0.75. Selective replacement of the native Zn2+ with 113Cd2+ can be accomplished in vitro by adding 113CdCl2 to the homogenate before chromatography. Both isoproteins of metallothionein thus prepared contain approximately 3.9 g atoms of Cd2+ and 2.6 g atoms of Cu+/mol of protein. No ESR signal was found, indicating that either Cu+ or antiferromagnetically coupled Cu2+ is the form of copper present. Arguments in support of the former state are presented. Unlike the native 113Cd,Zn MT from rabbit liver, calf liver 113Cd,Cu MT exhibits a remarkably simple 113Cd NMR spectrum. Four major resonances were found for each isoprotein, in the same positions as the resonances assigned to the metals in the four-metal cluster A of rabbit liver metallothionein. This conclusion was confirmed by homonuclear decoupling experiments. This result in conjunction with the stoichiometry of bound metal ions found in the native protein suggests that Cu+ is bound selectively to the three-metal cluster B sites, and that one homogeneous protein fraction predominates. Three minor resonances to higher field are observed in the 113Cd NMR spectrum of calf liver MT-1 and one in calf liver MT-2, which may be attributed to a small fraction of cluster B with one Cu+ replaced by 113Cd2+. The possible biological significance of the different metal ion specificities of cluster A versus cluster B is discussed.