Solution structure of the DNA-binding domain of human telomeric protein, hTRF1.

BACKGROUND: Mammalian telomeres consist of long tandem arrays of the double-stranded TTAGGG sequence motif packaged by a telomere repeat binding factor, TRF1. The DNA-binding domain of TRF1 shows sequence homology to each of three tandem repeats of the DNA-binding domain of the transcriptional activator c-Myb. The isolated c-Myb-like domain of human TRF1 (hTRF1) binds specifically to telomeric DNA as a monomer, in a similar manner to that of homeodomains. So far, the only three-dimensional structure of a telomeric protein to be determined is that of a yeast telomeric protein, Rap 1p. The DNA-binding domain of Rap 1p contains two subdomains that are structurally closely related to c-Myb repeats. We set out to determine the solution structure of the DNA-binding domain of hTRF1 in order to establish its mode of DNA binding. RESULTS: The solution structure of the DNA-binding domain of hTRF1 has been determined and shown to comprise three helices. The architecture of the three helices is very similar to that of each Rap 1p subdomain and also to that of each c-Myb repeat. The second and third helix form a helix-turn-helix (HTH) variant. The length of the third helix of hTRF1 is similar to that of the second subdomain of Rap 1p. CONCLUSIONS: The hTRF1 DNA-binding domain is likely to bind to DNA in a similar manner to that of the second subdomain of Rap 1p. On the basis of the Rap 1p-DNA complex, a model of the hTRF1 DNA-binding domain in complex with human telomeric DNA was constructed. In addition to DNA recognition by the HTH variant, a flexible N-terminal arm of hTRF1 is likely to interact with DNA.

Cell death induced by a caspase-cleaved transmembrane fragment of the Alzheimer amyloid precursor protein.

The Alzheimer amyloid precursor protein (APP) is a transmembrane protein whose abnormal processing is associated with the pathogenesis of Alzheimer's disease. Activated caspases cleave APP and generate its carboxyl-terminally truncated fragment (APPdeltaC31). We have previously reported that overexpression of wild-type APP induces caspase-3 activation and apoptosis in postmitotic neurons. We now report that APPdeltaC31 potentially plays pathophysiological roles in neuronal death. Adenovirus-mediated overexpression of wild-type APP695 induced activation of caspase-3 and accumulation of APPdeltaC31 in postmitotic neurons derived from human NT2 embryonal carcinoma cells, whereas an APP mutant lacking the Abeta(1-20) region induced neither caspase-3 activation nor APPdeltaC31 generation. Inhibition of caspase-3 suppressed the generation of APPdeltaC31 in APP-overexpressing neurons. Forced expression of APPdeltaC31 induced apoptotic changes of neurons and non-neuronal cells, but failed to activate caspase-3. The cytotoxicity of APPdeltaC31 was also dependent on the Abeta(1-20) region. These results suggest that accumulation of wild-type APP activates neuronal caspase-3 to generate APPdeltaC31 that mediates caspase-3-independent cell death.

Anion and pH-dependent conformational transition of an amphiphilic polypeptide.

While several proteins, including beta-lactamase, cytochrome c and apomyoglobin, are maximally unfolded at pH 2 by HCl in the absence of salt, the addition of anions, either from salt or acid, co-operatively induces the unfolded proteins to refold to a molten globule state, because anions bind preferentially to the compact molten globule state compared to the extended unfolded state. To study the role of the anion-dependent conformational transition at neutral pH, we synthesized a model polypeptide of 51 amino acid residues, consisting of tandem repeats of a Lys-Lys-Leu-Leu sequence and containing a turn sequence, Asn-Pro-Gly, at the center of the molecule. The model polypeptide showed no significant conformation by circular dichroism under conditions of low salt at neutral pH. However, addition of anions, either from salt or acid, induced the folding transition to an alpha-helical conformational state. The order of effectiveness of various anions in inducing the folding transition was consistent with the series of anions in inducing the molten globule of the acid-denatured protein. This suggests that the helical state of the model polypeptide is equivalent to the molten globule state. At pH values above 9, the model polypeptide also took an alpha-helical conformation, which was very similar to that induced by anions. On the basis of the chloride and pH-dependent conformational transitions, a phase diagram for the conformational states was constructed. The phase diagram was explained simply by assuming that the conformational transition is linked to the proton and the anion bindings to a limited number of amino groups and that anions bind only to the protonated groups.

Guanidine hydrochloride-induced folding of proteins.

Guanidine hydrochloride (Gdn-HCl) is the most commonly used denaturant for proteins. Contrary to expectation, we found that Gdn-HCl at low concentrations refolds acid-unfolded apomyoglobin and cytochrome c, stabilizing the molten globule state, i.e. a compact denatured state with a significant amount of secondary structure, but substantially disordered tertiary structure. A further increase in Gdn-HCl concentration, above 1 M, caused co-operative unfolding of the molten globule state. Similar sequential folding and unfolding transitions were also observed at neutral pH with a synthetic amphiphilic peptide consisting of Lys and Leu residues, indicating the generality of the phenomenon. Although the Gdn-HCl-induced refolding and unfolding transitions were puzzling at first glance, we show that they are readily interpreted in terms of the differential action of Gdn-HCl. We also show that the comparison of the unfolding curves for the molten globule and native states provides a measure of the buried surface area upon formation of the molten globule state.

Helical structure of phospholamban in membrane bilayers.

The regulation of calcium levels across the membrane of the sarcoplasmic reticulum involves the complex interplay of several membrane proteins. Phospholamban is a 52 residue integral membrane protein that is involved in reversibly inhibiting the Ca(2+) pump and regulating the flow of Ca ions across the sarcoplasmic reticulum membrane during muscle contraction and relaxation. The structure of phospholamban is central to its regulatory role. Using homonuclear rotational resonance NMR methods, we show that the internuclear distances between [1-(13)C]Leu7 and [3-(13)C]Ala11 in the cytoplasmic region, between [1-(13)C]Pro21 and [3-(13)C]Ala24 in the juxtamembrane region and between [1-(13)C]Leu42 and [3-(13)C]Cys46 in the transmembrane domain of phospholamban are consistent with alpha-helical secondary structure. Additional heteronuclear rotational-echo double-resonance NMR measurements confirm that the secondary structure is helical in the region of Pro21 and that there are no large conformational changes upon phosphorylation. These results support the model of the phospholamban pentamer as a bundle of five long alpha-helices. The long extended helices provide a mechanism by which the cytoplasmic region of phospholamban interacts with residues in the cytoplasmic domain of the Ca(2+) pump.

Role of heme axial ligands in the conformational stability of the native and molten globule states of horse cytochrome c.

One unique aspect of cytochrome c folding concerns the involvement of the covalently attached heme group and its axial ligands. To elucidate the role of the ligands in stabilizing the native and molten globule states, we studied the conformational and thermodynamic features of the iron-free derivative of horse cyctochrome c (porphyrin-cytochrome c). At neutral pH, far-UV circular dichroism suggested that porphyrin-cytochrome c has native-like alpha-helices, whereas near-UV CD suggested that the side-chains are flexible. Its stability against heat or denaturants was much less than that of the intact protein, and similar to that of the acidic molten globule state of the holoprotein. These results indicate that, at neutral pH, the ligation of His18 of the iron is important for the maintenance of the native structure whereas the Met80 ligation is not essential, and that porphyrin-cytochrome c assumes a molten globule-like state. Porphyrin-cytochrome c was largely unfolded at pH 2.0 in the absence of salt, but assumed another molten globule-like structure in the presence of anions. The salt-induced stabilization of the molten globule-like state was the same as that of apocytochrome c, requiring a much higher salt concentration than holocytochrome c. These results indicate that, at acidic pH, the His18 ligation is important, although not essential, for stabilizing the molten globule state. Taken together, both specific (i.e. the His18 axial ligand, as observed at acidic pH) and nonspecific interactions (the hydrophobic effects of the heme, as observed at neutral pH) contribute to stabilizing the molten globule state.

NMR structure of the hRap1 Myb motif reveals a canonical three-helix bundle lacking the positive surface charge typical of Myb DNA-binding domains.

Mammalian telomeres are composed of long tandem arrays of double-stranded telomeric TTAGGG repeats associated with the telomeric DNA-binding proteins, TRF1 and TRF2. TRF1 and TRF2 contain a similar C-terminal Myb domain that mediates sequence-specific binding to telomeric DNA. In the budding yeast, telomeric DNA is associated with scRap1p, which has a central DNA-binding domain that contains two structurally related Myb domains connected by a long linker, an N-terminal BRCT domain, and a C-terminal RCT domain. Recently, the human ortholog of scRap1p (hRap1) was identified and shown to contain a BRCT domain and an RCT domain similar to scRap1p. However, hRap1 contained only one recognizable Myb motif in the center of the protein. Furthermore, while scRap1p binds telomeric DNA directly, hRap1 has no DNA-binding ability. Instead, hRap1 is tethered to telomeres by TRF2. Here, we have determined the solution structure of the Myb domain of hRap1 by NMR. It contains three helices maintained by a hydrophobic core. The architecture of the hRap1 Myb domain is very close to that of each of the Myb domains from TRF1, scRap1p and c-Myb. However, the electrostatic potential surface of the hRap1 Myb domain is distinguished from that of the other Myb domains. Each of the minimal DNA-binding domains, containing one Myb domain in TRF1 and two Myb domains in scRap1p and c-Myb, exhibits a positively charged broad surface that contacts closely the negatively charged backbone of DNA. By contrast, the hRap1 Myb domain shows no distinct positive surface, explaining its lack of DNA-binding activity. The hRap1 Myb domain may be a member of a second class of Myb motifs that lacks DNA-binding activity but may interact instead with other proteins. Other possible members of this class are the c-Myb R1 Myb domain and the Myb domains of ADA2 and Adf1. Thus, while the folds of all Myb domains resemble each other closely, the function of each Myb domain depends on the amino acid residues that are located on the surface of each protein.

Structural model of the phospholamban ion channel complex in phospholipid membranes.

Phospholamban is a 52 amino acid residue membrane protein involved with the regulation of calcium levels across sarcoplasmic reticulum membranes in cardiac muscle cells. The N-terminal 30 amino acid residues of the protein are largely hydrophilic and include two sites whose phosphorylation is thought to dissociate an inhibitory complex between phospholamban and Ca2+ ATPase. The C-terminal 22 amino acid residues are largely hydrophobic, anchor the protein in the membrane and are responsible for Ca2+ selective ion conductance. Specific interactions between the transmembrane domains stabilize a pentameric protein complex. We have obtained circular dichroism (CD), transmission Fourier transform infrared (FTIR) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectra of the full-length protein and have compared these results to those from a 28 residue peptide that includes the transmembrane domain. Both proteins reconstituted into phospholipid membranes are largely alpha-helical by CD and FTIR. Polarized ATR-FTIR measurements show that both the cytosolic and transmembrane helices are oriented perpendicular to the membrane plane with a tilt of 28 (+/- 6) degrees with respect to the membrane normal. This tilt angle is in close agreement to that calculated from a model for the transmembrane domain of phospholamban suggested by mutagenesis and molecular modeling. Phosphorylation does not significantly change the secondary structure or orientation of the protein. The pentameric complex is modeled as a left-handed coiled-coil of five long helices (40 (+/- 3) residues) that extend across the membrane from the lumenal carboxy terminus to the phosphorylation site in the cytoplasm. The helix bundle forms a perpendicular ion pore that may begin at a distance (17 to 29 A) from the membrane surface. Based on the above, we propose a mechanism by which phospholamban regulates Ca2+ levels across membranes that takes into account both its selective ion conductance and inhibitory association with the Ca2+ pump.

Conformational changes in melittin upon complexation with an anionic melittin analog.

Melittin and its Glu-(7,21,22,23,24) analog upon mixing in equimolar concentrations form a hybrid oligomer with significant helical structure, in conditions in which each peptide separately adopts a largely disordered structure. The hybrid exhibits both cold- and heat-induced denaturations similar to the phenomena exhibited by proteins. The hybrid also retains significant residual structure at higher temperature, similar to the 'molten globular state' that has been suggested for protein. Melittin, at concentrations in which it forms helical tetramers, also exhibits these phenomena and may be used as a model for protein-denaturation studies.

Mast cell degranulating (MCD) peptide and its optical isomer activate GTP binding protein in rat mast cells.

The MCD peptide in bee venom induces degranulation in mast cells. The internal calcium concentration of mast cells increased and remained high following MCD stimulation. This calcium increase was blocked by pertussis toxin (Ptx) treatment, suggesting that MCD peptide activates Ptx-sensitive G-protein. Even in the absence of external calcium in the incubation medium, the calcium concentration increased by MCD treatment, but soon returned to the original level. D-MCD, the optical isomer of the MCD peptide, also increased the internal calcium concentration through a Ptx-sensitive pathway. We suggest that cationic clusters at one side of the surface are more important in activating the G-protein than the alpha-helix conformation.

Presence of non-selective type of endothelin receptor on vascular endothelium and its linkage to vasodilation.

We studied the role of non-selective type (ETB) of endothelin (ET) receptor in the vasculature, using a ligand specific to the ETB receptor, [Glu9]-sarafotoxin S6b ([Glu9]SRTb). Endothelium-containing rat thoracic aorta possessed specific binding sites for 125I-[Glu9]SRTb, which were almost eliminated by removal of the endothelium, while ET-3-specific binding sites were not detected in the endothelium-intact rat aorta. Only ETB receptor was detected in the membranes from the endothelium of porcine thoracic aorta. [Glu9]SRTb exerted only vasodilation in rat aortic ring. These findings indicate that ETB receptors are located on vascular endothelium and linked to vasodilation.

Mode of disulfide bond formation of a heat-stable enterotoxin (STh) produced by a human strain of enterotoxigenic Escherichia coli.

To determine the modes of three disulfide linkages in the heat-stable enterotoxin (STh) produced by a human strain of enterotoxigenic Escherichia coli, we synthesized STh(6-18), which consists of 13 amino acid residues and has the same intramolecular disulfide linkages as native STh [(1985) FEBS Lett. 181, 138-142], by stepwise and selective formation of disulfide bonds using different types of removable protecting groups for the Cys residues. Synthesis of the peptide with different modes of disulfide bond formation provided three peptides consistent with standard STh(6-18) in their physicochemical and biological properties, thereby indicating that the disulfide bonds in STh(6-18) are Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys.

Functional and possible physical association of scavenger receptor with cytoplasmic tyrosine kinase Lyn in monocytic THP-1-derived macrophages.

Acetyl LDL (modified low-density lipoprotein), which is thought to be taken up through scavenger receptor A (SR-A), rapidly induced the appearance of phosphotyrosine proteins in monocytic THP-1-derived macrophages in vitro. The two alternative forms of Lyn (p53 and p56) were found to be tyrosine-phosphorylated within 30 s after the stimulation with acetyl LDL. The catalytic activity of Lyn measured by an in vitro kinase assay had also increased in acetyl LDL-stimulated THP-1-derived macrophages. Furthermore, Lyn could be co-immunoprecipitated with SR-A from the cell lysate. These observations suggest a functional and possible physical association of SR-A with Lyn in THP-1-derived macrophages, and also imply a possible involvement of Lyn in SR-A signal transduction.

Amino acid sequence of a heat-stable enterotoxin isolated from enterotoxigenic Escherichia coli strain 18D.

A heat-stable enterotoxin produced by a strain of enterotoxigenic Escherichia coli 18D was purified by ion-exchange and reversed-phase high-pressure liquid chromatography. The amino acid sequence of the purified toxin was determined by Edman-degradation and a combination of fast atom bombardment mass spectrometry and carboxypeptidase digestion to be Asn-Thr-Phe-Tyr-Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys-Tyr.

Essential structure for full enterotoxigenic activity of heat-stable enterotoxin produced by enterotoxigenic Escherichia coli.

Several analogues of heat-stable enterotoxins (STh and STp) produced by enterotoxigenic Escherichia coli were synthesized. Peptides (STh[6-18] and STp[5-17]) consisting of 13 amino acid residues from the Cys residue near the N-terminus to the Cys residue near the C-terminus and linked by three disulfide bonds had the same biological and immunological properties as native STh and STp, respectively. The results indicated that the sequence with the 13 amino acid residues and three disulfide linkages is essential for full biological activity of ST.

1H-NMR study of Ca(2+)-and Mg(2+)-dependent interaction between troponin C and troponin I inhibitory peptide (96-116).

The Ca(2+)-and Mg(2+)-dependence of the interaction between rabbit skeletal muscle troponin C (TnC) and a 21 residue peptide corresponding to 96-116 of troponin I (denoted as CN4) was examined by means of 1H-NMR spectroscopy. The spectral changes of TnC with 4 mol of Ca2+ (Ca4TnC) and TnC with 4 mol of Mg2+ (Mg4TnC) were observed as a function of CN4 concentration. As CN4 was added to Ca4TnC, resonances of the following residues changed in chemical shift: Tyr10, Phe23, Phe72, Ala106, Gly108, Tyr109, Ile110, His125, Gly144, Ile146, Phe102 or Phe151, and Phe148 located in the N- and C-domains of Ca4TnC. Such CN4-induced change was also observed for resonances of Phe19, 26, and 75 in the N-domain of Ca4TnC by means of NOESY and HOHAHA experiments. The presence of CN4 increased the native-to-unfolded transition temperature of the N-domain of Ca4TnC. On the basis of these results, we conclude that CN4 binds to both the C- and N-domains of Ca4TnC ([CN4]:[TnC] = 1:1) and stabilizes the structure of the N-domain. The CN4-binding constant was estimated to be 1.1 x 10(5) M-1. As CN4 was added to Mg4TnC, chemical shift change was observed for resonances of Phe99, Tyr109, and Ile110 in the C-domain, while no change was observed for resonances arising from the N-domain. The presence of CN4 did not change the thermal stability of the N- and C-domains of Mg4TnC. The CN4-binding constant of Mg4TnC was obtained as 0.9 x 10(4) M-1, which is one-tenth of that of Ca4TnC.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP-induced conformational transition of denatured proteins.

Although the conformational change occurring in proteins upon ATP binding is important in many biological reactions, the mechanism by which ATP binding induces the conformational change is unknown. We found that ATP induces acid-unfolded (pH 2) ferricytochrome c or apomyoglobin to adopt a compact structure with a significant amount of alpha-helix and increased hydrophobicity. A very similar conformational transition was observed at neutral pH for an amphiphilic model polypeptide. The effectiveness of various adenine nucleotides in inducing the conformational transition was found to be proportional to their phosphate group contents, i.e., adenosine tetraphosphate greater than ATP greater than ADP greater than AMP. These results should be important when considering the mechanism of the ATP-induced conformational change in proteins during various biological reactions.

Molecular conformation of porcine amelogenin in solution: three folding units at the N-terminal, central, and C-terminal regions.

Circular dichroism (CD) studies were conducted to gain a better insight into the conformation of amelogenins, which were isolated from developing enamel of piglets. The intact porcine amelogenin and its degraded products were purified chromatographically. The 25-residue peptide corresponding to the segment at the C-terminus was synthesized. CD spectra of these samples were measured at pH 5.0-5.3 in the temperature range between 4 and 90 degrees C. The most remarkable finding was that the CD spectrum of the intact amelogenin was accounted for by the sum of the spectra of the three fragments at the N-terminal, central, and C-terminal regions, supporting the hypothesis that the structure of the whole protein consists of discrete folding units. Furthermore, low-angle laser light scattering analysis provided evidence that the 20 kDa amelogenin, the most abundant extracellular matrix protein in forming enamel tissue, exists in a monomeric form at pH 5.3 and 25 degrees C. It was tentatively concluded that the N-terminal region contains beta-sheet structures, while the spectral characteristics of the C-terminal region are similar to those of a random coil conformation. The conformation of the central region was characterized by a strong negative ellipticity at 203 nm, although its nature remains to be defined.

13C-NMR relaxation study on mobility of the DNA-binding arm of HU.

The mobility of the DNA-binding arm of HU protein was studied by 13C-NMR spectroscopy. The correlation times tau c of Phe47C alpha in the body and Gly60C alpha in the arm of HU were determined for HU and HU-DNA complex. The value of tau c of Phe47C alpha is 2-4 times larger than that of Gly60C alpha irrespective of the presence or absence of DNA. These results show that Gly60C alpha undergoes more rapid motion than Phe47C alpha. The increase in correlation time on addition of DNA is greater for Gly60C alpha than for Phe47C alpha. This suggests that the addition of DNA influences more significantly the motion of Gly60C alpha than that of Phe47C alpha. These results are in accord with the X-ray result, in which the top part of the arm is not visible. Gly60C alpha in the arm is thus more mobile than Phe47C alpha in the body, and the mobility of Gly60C alpha is reduced by the DNA binding.

Xenopus eggs express an identical DNA methyltransferase, Dnmt1, to somatic cells.

In mouse, an oocyte-specific short isoform of DNA methyltransferase-1 (Dnmt1) lacking amino terminal 118 amino acid residues exists and plays a crucial role in maintaining the methylation state of imprinted genes during early embryogenesis [Howell et al. (2001) Cell 104, 829-838]. To address the question of whether or not Xenopus oocyte expresses such a short isoform, we raised monoclonal antibodies against the amino-terminal portion of Xenopus Dnmt1. Two of the isolated monoclonal antibodies, 3C6 and 4A8, were determined to recognize (1-32) and (115-126) of Xenopus Dnmt1, respectively. The amounts of Dnmt1 in Xenopus eggs were determined to be similar, 10.0 2.5, 8.0 0.8, and 8.2 0.2 ng per egg with monoclonal antibodies 3C6 and 4A8, and polyclonal antibodies, respectively. This indicated that Dnmt1 in Xenopus mature eggs had an identical amino-terminal sequence to the amino acid sequence deduced from the cDNA. Together with the fact that Dnmt1 in A6 cells immuno-reacted with all the monoclonal antibodies isolated and with the polyclonal antibodies, we concluded that Dnmt1 expressed in Xenopus mature eggs possesses an identical amino-terminal sequence to that in somatic cells. Immuno-purified Xenopus Dnmt1 in mature eggs showed similar specific activity to that in proliferating A6 cells and that of mouse recombinant Dnmt1.