Journal of Biological Chemistry

Published by Elsevier BV

Online ISSN: 1083-351X


Print ISSN: 0021-9258


Not Available
  • Article

May 1958


53 Reads




Structural and functional studies on the G(o) protein
  • Article
  • Full-text available

April 1993


127 Reads

Monoclonal antibodies were raised against purified bovine and human brain G-proteins. The epitopes recognized by three monoclonal antibodies (MONO, 3C2, and 3E7) were mapped by expressing defined parts of the bovine G(o) alpha-cDNA in bacteria, followed by immunoblotting. All three antibodies recognize the recombinant bovine alpha o-protein, but at distinct sites. The epitopes of MONO and 3C2 were mapped between alpha o amino acids 80 and 145, and both antibodies recognize alpha o exclusively. Heterotrimeric G(o)-proteins as well as guanosine 5'-3-O-(thio)triphosphate-liganded free alpha o-subunits are readily immunoprecipitated by these monoclonal antibodies. Binding of MONO or 3C2 does not affect ADP-ribosylation of the alpha o-subunit by pertussis toxin. Apparently, the antibodies do not bind to or induce large conformational changes in regions of the alpha o-subunit that are involved in association with beta gamma-subunits or ADP-ribosylation. 3E7 behaves as an anti common alpha-subunit antibody when used in immunoblots. However, under nondenaturing conditions, 3E7 recognizes alpha o exclusively. After binding of 3E7, the pertussis toxin-dependent ADP-ribosylation of alpha o is effectively blocked, while the ADP-ribosylation of the various alpha i-subunits is not affected. The epitope of 3E7 was mapped between alpha o amino acids 13-88, a region which has been implicated in the interaction between alpha- and beta gamma-subunits. Possibly, the inhibitory effect of 3E7 on ADP-ribosylation of G(o) is secondary to the loss of beta gamma-subunits that is observed upon binding of 3E7.

FIGURE 1. MES at a pulse width of 0.1 ms induces phosphorylation of p53. A, pulse shape with the indicated pulse width in milliseconds of the electrical stimulation used for treatment. DC, direct current. B, HCT116 cells were treated for 10 min with MES (1 V/cm, 55 pps) at various pulse widths (0.01, 0.1, 1, and 10 ms) or in the absence of pulse ( ms). Cell lysates were extracted 1 h after MES treatment. Phospho-p53 (Ser-15; p-p53) and p53 were detected by Western blot analysis. Actin served as a loading control. con, control. C, blots of phospho-p53 were quantified by densitometry using Image Gauge software (version 4.23, Fujifilm). Quantified blots were normalized to actin and are expressed as -fold increase relative to the control. Error bars indicate the mean S.D. (n 3). *, p 0.05 (assessed by analysis of variance with Dunnett's multiple comparison test). D, cells were treated for 30 min with MES (0.1 ms) at an amplitude of 6 V with a reference voltage (V 0 ) at 3 or 0 V, and lysates were recovered 1 h after treatment. Cell lysates were analyzed by immunoblotting with the indicated antibodies. Actin served as an internal control.  
FIGURE 3. MES enhances phosphorylation of p53 at Ser-15 and p53 recruitment to the nucleus. A, HCT116 cells were treated with MES (0.1 ms) for 30 min, and lysates were recovered 1 h after MES treatment; or cells were treated with 5-FU at the indicated concentrations for 24 h. Protein lysates were analyzed by Western blotting using the indicated antibodies. p-p53, phospho-p53; con, control. B, HCT116 cells were cross-linked 1 h after MES treatment. Protein lysates were immunoprecipitated (IP) with anti-p53 antibody (DO-1) or mouse IgG. Immunoprecipitated lysates and input samples were immunoblotted (IB) and analyzed using the indicated antibodies. C, cytosolic proteins and nuclear extract of HCT116 cells were recovered 1 h after MES treatment. Protein lysates were analyzed by Western blotting using the indicated antibodies. -Tubulin and HDAC2 serve as internal controls of the cytosol and nuclear fractions, respectively.  
FIGURE 4. MES induces p53 activation via the MKK3b-MKK6b-p38 pathway. A, HCT116 cells were cultured for 1 h in the presence of dimethyl sulfoxide (control (con)) or 10 M SB203580 and then treated with MES (0.1 ms, 1 V/cm, 55 pps) for 30 min. Protein lysates were extracted 1 h after MES treatment and analyzed by Western blotting using the indicated antibodies. The experiments were performed in triplicate. p-p53, phospho-p53. B, densitometric analysis of phosphorylated p53 expression was performed using Image Gauge software. The quantified blots were normalized to actin. Error bars indicate the mean S.D. (n 3). *, p 0.05 versus the control (analyzed by Student's t test); n.s., not significant. DMSO, dimethyl sulfoxide. C, HCT116 cells were transfected with the pcDNA3.1 (control vector), p38-DN, MKK3b-DN, or MKK6b-DN plasmid. 36 h after transfection, cells were treated with MES (0.1 ms, 1 V/cm, 55 pps) for 30 min. Protein lysates were extracted 1 h after MES treatment, and Western blotting was performed.  
FIGURE 5. p53 target genes are up-regulated by MES in HCT116 wild-type (p53 / ) cells. A–E, HCT116 p53 / and HCT116 p53 / cells were treated with MES (0.1 ms, 1 V/cm, 55 pps) for 30 min, and total RNA was extracted 4 h after treatment. The expression levels of p53 target genes (p21, BAX, NOXA, PUMA, and IRF9) were analyzed by quantitative RT-PCR. GAPDH was used as an internal control. The experiments were performed in triplicate. Error bars indicate the mean S.E. *, p 0.05 versus the control (assessed by Student's t test); **, p 0.01 versus the control; n.s., not significant. con, control. F, nuclear extracts were obtained from HCT116 p53 / cells 3 h after treatment with MES (0.1 ms, 1 V/cm, 55 pps, 30 min) or 12 h after treatment with 5-FU (50 M). p53 binding to the p21 promoter was assessed by conventional PCR using primers that recognize the p53 consensus site in the p21 promoter. The GAPDH promoter was used as negative control. IP, immunoprecipitate.  
FIGURE 2. MES phosphorylates p53 transiently and cumulatively in several epithelial cell lines. A, HCT116 cells were treated with MES (0.1 ms) for 10 min, and protein lysates were isolated at 0, 1, 3, or 6 h after treatment. p-p53, phospho-p53. B, cells were treated with MES (0.1 ms) for 0, 10, 20, or 30 min, and protein lysates were recovered 1 h after treatment. C, HCT116, A549, HEK293, and HepG2 cells were treated with MES (0.1 ms) for 10 min, and lysates were extracted 1 h after treatment. con, control. For A–C, cell lysates were analyzed by immunoblotting with the indicated antibodies. Actin served as an internal control.  
Mild Electrical Stimulation at 0.1-ms Pulse Width Induces p53 Protein Phosphorylation and G2 Arrest in Human Epithelial Cells

April 2013


166 Reads






Exogenous low-intensity electrical stimulation has been used for treatment of various intractable diseases despite the dearth of information on the molecular underpinnings of its effects. Our work and that of others have demonstrated that applied electrical stimulation at physiological strength or mild electrical stimulation (MES) activates the PI3K-Akt pathway, but whether MES activates other molecules remains unknown. Considering that MES is a form of physiological stress, we hypothesized that it can activate the tumor suppressor p53, which is a key modulator of the cell cycle and apoptosis in response to cell stresses. The potential response of p53 to an applied electrical current of low intensity has not been investigated. Here, we show that p53 was transiently phosphorylated at Ser-15 in epithelial cells treated with an imperceptible voltage (1 V/cm) and a 0.1-ms pulse width. MES-induced p53 phosphorylation was inhibited by pretreatment with a p38 MAPK inhibitor and transfection of dominant-negative mutants of p38, MKK3b, and MKK6b, implying the involvement of the p38 MAPK signaling pathway. Furthermore, MES treatment enhanced p53 transcriptional function and increased the expression of p53 target genes p21, BAX, PUMA, NOXA, and IRF9. Importantly, MES treatment triggered G2 cell cycle arrest, but not cell apoptosis. MES treatment had no effect on the cell cycle in HCT116 p53−/− cells, suggesting a dependence on p53. These findings identify some molecular targets of electrical stimulation and incorporate the p38-p53 signaling pathway among the transduction pathways that MES affects.

Quantitative evaluation for the role of β146 His and β143 His residues in the Bohr effect of human hemoglobin in the presence of 0.1 M chloride ion

October 1984


74 Reads

Two different methods were used to determine the number of Bohr protons released upon oxygenation of human hemoglobin (Hb A) and Hb A lacking beta 146 His (des-His Hb A) at the pH ranging from pH 5.0 to 9.0 in the presence of 0.1 M Cl- at 25 degrees C. One is the direct differential titration method, the other is based on the measurement of oxygen affinity as a function of pH. The results obtained for Hb A or des-His Hb A with two methods were completely mutually consistent. The number of Bohr protons released from des-His Hb A upon oxygenation at pH 7.5 was about 44% less than that from Hb A, while at pH 5.5 the number of Bohr protons taken up by des-His Hb A was 20% greater than that by Hb A. The differences in the number of Bohr protons between Hb A and des-His Hb A could not be simply ascribed to the lack of beta 146 His from Hb A. The pK alpha values, which were determined by the deuterium exchange method using 1H NMR, were 8.0 for beta 146 His of deoxy-Hb A and 6.5 for that of CO Hb A, while those of beta 143 His were 5.2 for deoxy-Hb A and 6.0 for CO Hb A. From these pK alpha values, in addition to those of alpha 1 Val proposed for the modified CO and deoxy-Hb A with carbamylated beta chains by Van Beek and De Bruin (Van Beek, G. M., and De Bruin, S. H. (1980) Eur. J. Biochem. 105, 353-360), it became evident that almost all (about 92%) of the alkaline Bohr protons released upon oxygenation of Hb A in the presence of 0.1 M Cl- could be accounted for by the protons from these 2 residues, although the involvement of other histidine residues could not be denied. About half the acid Bohr protons from Hb A, which corresponds to the higher pH part (above pH 5.0) of the acid Bohr effect, could be explained by the involvement of beta 143 His residue. The residual acid Bohr effect in the more acidic pH region was presumably contributed by an amino acid residue with pK alpha values of 4.05 and 5.95 for the deoxy- and CO Hb A, respectively, although the amino acid residue was unspecified.(ABSTRACT TRUNCATED AT 400 WORDS)

Mistranslation of the mRNA encoding bacteriophage T7 0.3 protein

June 1984


54 Reads

We have devised an experimental system using the T7 phage 0.3 protein to accurately quantitate in vivo errors in protein synthesis. The 0.3 protein is well suited for mistranslation studies because it is easy to purify, its precise amino acid and RNA sequences are known, and it contains no cysteine. Utilizing [35S]cysteine as a precursor we found an average of 1 cysteine residue misincorporated for every 43.5 molecules of 0.3 protein synthesized. Since there are 116 amino acids in 0.3 protein, 1 cysteine residue was misincorporated /5000 codons translated. If all 20 amino acids were misincorporated at the same frequency, the overall frequency of misincorporation of amino acids into 0.3 protein would be 4 X 10(-3)/codon translated. Parallel experiments measuring [35S]methionine incorporation into 0.3 protein supported the accuracy of our findings for cysteine misincorporation. We found an average of 5.7 methionine residues incorporated/molecule of 0.3 protein synthesized; the actual number from sequence data is known to be 6. Antibiotics which stimulate mistranslation (gentamicin and streptomycin) caused a modest increase in the number of cysteine residues misincorporated into 0.3 protein. The use of Escherichia coli strains, identical except for mutations in ribosomal protein genes known to affect the fidelity of translation, supported the contention that the errors being quantitated were mainly due to mistranslation rather than mistranscription .

Purification of the gene 0.3 protein of bacteriophage T7, an inhibitor of the DNA restriction system of Escherichia coli

April 1981


38 Reads

The gene 0.3 protein of bacteriophage T7 prevents the DNA restriction system of EScherichia coli from interfering with T7 infection. A mutant strain of T7 that greatly overproduces the 0.3 protein has been constructed and used for purification of this protein. The 0.3 protein ws found to be extremely acidic and can be separated from virtually all other proteins of the infected cell by chromatography on DEAE-cellulose. Residual contaminating proteins and nucleic acids can be removed by gel filtration, but an even simpler final purification is possible, because under appropriate conditions the 0.3 protein is soluble in high concentrations of ethanol. Thus, a simple, essentially two-step purification can produce about 50 mg of pure 0.3 protein from 30 liters of culture. The purified protein appears to be a dimer of identical subunits. AS expected from its known function during infection, the purified 0.3 protein inhibits the nuclease and ATPase activities of partially purified Eco B, the DNA restriction enzyme of E. coli B, but it does not interfere with several different type II endonucleases tested. The inhibition of Eco B appears to require stoichiometric rather than catalytic amounts of 0.3 protein.

Amino acid sequence of the gene 0.3 protein of bacteriophage T7 and nucleotide sequence of its mRNA

April 1981


31 Reads

The amino acid sequence of purified gene 0.3 protein of T7, the protein responsible for overcoming host restriction, has been determined. The nucleotide sequence of the 0.3 RNA, the messenger RNA that codes for both the 0.3 protein and the gene 0.4 protein, a T7 protein of unknown function, has also been determined. The 0.3 RNA is 578 nucleotides long, 509 of which are used to code for the 2 proteins. The coding sequences do not overlap, but the termination codon for the 0.3 protein and the presumed initiation codon for the 0.4 protein do overlap in the sequence UAAUG. The 0.3 protein is very acidic: 34 of its 116 amino acids are aspartic or glutamic acid and only 6 are arginine or lysine. The 0.3 protein contains no cysteine. The nucleotide sequence predicts that the 0.4 protein consists of 50 amino acids and contains no histidine or proline. The effects of different mutations indicate that a protein which contains only the first 87 amino acids of the 0.3 protein is unable to prevent host restriction in vivo; one that contains te first 93 amino acids has weak function; and one that has the first 94 amino acids (plus 2 that are not in the wild type sequence) is fully able to prevent host restriction. The apparently critical 94th amino acid is tryptophan. The mutant 0.3 proteins that contain 87 or more amino acids appear to be reasonably stable in vivo, but those that contain 78 or fewer are apparently too unstable to have been observed by gel electrophoresis.

Determination of the P/2e- stoichiometries at the individual coupling sites in mitochondrial oxidative phosphorylation. Evidence for maximum values of 1.0, 0.5, and 1.0 at sites 1, 2, and 3

September 1987


42 Reads

P/2e- stoichiometries in six assay systems spanning different portions of the respiratory chain were estimated by direct determinations of Pi uptake in suspensions of bovine heart mitochondria containing a hexokinase trap. The electron donors were malate + pyruvate, succinate, and ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine, and the electron acceptors were ferricyanide (Site 1, Site 2, and Sites 1 + 2) and O2 (Sites 1 + 2 + 3, Sites 2 + 3, and Site 3). A major objective was to find conditions in which the six systems yield results in sufficiently good agreement to allow confidence as to their reliability. This objective was achieved, and maximum values of 1.1, 0.5, and 1.0 were observed in the Sites 1, 2, and 3 systems, respectively. This required that the energy-conserving reactions be relatively nonlimiting and that the P/2e- ratios be estimated from the slopes of plots of respiration rate versus phosphorylation rate obtained by inhibiting oxidative phosphorylation with respiratory chain inhibitors. The latter requirement allows avoidance of the effect of an apparent endogenous uncoupler and is based on the observation (Tsou, C. S., and Van Dam, K. (1969) Biochim. Biophys. Acta 172, 174-176) that uncoupling agents at low concentrations decrease the rate of phosphorylation nearly as much in absolute amount at low rates of respiration as at high rates. The maximum P/2e- stoichiometry at Site 1 is considered to be 1.0, and the value observed in the Site 1 system is suggested to be higher as a result of H+ ejection at the transhydrogenase level. Respiratory control due to carboxyatractyloside inhibition was examined and found to differ greatly among the systems. It is pointed out that this observation is not consistent with the lack of complete control being due primarily to ion cycling and that, in view of this, the relatively meager control at Site 3 is not consistent with O2 being reduced on the matrix side of the coupling membrane.

FIG. 2. van't Hoff plots for the binding of wild-type and mutant RP135 peptides to the 0.5 Fv fragment. Binding experiments were carried out at different temperatures in 50 mM sodium phosphate buffer (pH 7.5) containing different concentrations of Tween-20 in the case of wild-type peptide (panel A) and 0.05% Tween-20 in the case of the mutants (panels B-D). For additional details, see " Experimental Procedures. " The data were fitted to Equation 1.  
FIG. 3. Plot of the change in heat capacity upon binding of wild-type RP135 peptide to the 0.5 Fv fragment as a function of the concentration of Tween-20. Binding experiments were carried out in the presence of different fixed concentrations of Tween-20 as described under " Experimental Procedures. " A linear relationship is observed with a correlation coefficient of 0.89. FIG. 4. Differential scanning calorimetry curves showing the change with temperature of the apparent heat capacity of the 0.5 Fv fragment in 50 mM sodium phosphate buffer (pH 7.5) without (—) or with (---) 0.05% Tween-20. The DSC curves shown were base-line corrected by subtracting the curve of the change with the temperature of the apparent heat capacity of buffer alone or buffer with 0.05% Tween-20 as appropriate.  
FIG. 1. Fluorescence-monitored stability of the 0.5 Fv fragment . The fluorescence of the 0.5 Fv fragment was measured as a function of its concentration in the presence of 50 mM sodium phosphate buffer (pH 7.5) containing 0.05% Tween-20 at temperatures of 10, 25, and 35 °C. The excitation wavelength was 280 nm with a bandpass of 10 nm, and the emission wavelength was 330 nm with a bandpass of 30 nm. A linear dependence (r 0.999) is observed for all three temperatures , indicating that there is negligible dissociation.  
Thermodynamic Analysis of the Interaction between the 0.5 Fv Fragment and the RP135 Peptide Antigen Derived from the V3 Loop of HIV-1 gp120

January 1998


81 Reads

The Fv fragment of the 0.5beta monoclonal antibody has recently been constructed, expressed, and purified. It binds with nanomolar affinity to the immunogenic RP135 peptide that is derived from the principal neutralizing determinant of HIV-1 in the third hypervariable region of gp120. Here, we analyzed the temperature-dependence of binding of the 0.5beta Fv fragment to the RP135 peptide and a series of mutants thereof. Our results show that there is almost complete enthalpy-entropy compensation in the effects of mutations in the peptide on binding to the Fv, indicating that the mutations do not change the binding mechanism. There is good correlation, for residues within the antigenic epitope, between mutational effects on DeltaCp and calculated values of DeltaDeltaCp based on the extent of burial of polar and non-polar surface areas of amino acids. The value of DeltaCp for the binding of the 0.5beta Fv fragment to the wild-type RP135 peptide is found to be -5.0 (+/- 0.9) kcal K-1 mol-1 in the presence of 0.1% Tween-20 but only -0.1 (+/- 0.9) kcal K-1 mol-1 in its absence. This result has important implications for the successful application of the structural parameterization approach to predicting changes in heat capacity that accompany binding reactions carried out in the presence of detergent or protein-stabilizing agents.

FIG. 1. Analysis by SDS-polyacrylamide gel electrophoresis of steps in the purification of the 0.5 Fv antibody fragment. In lane 1 is shown total protein of IPTG-induced E. coli TG2 cells harboring the pT11 plasmid. Samples from fractions A, B, and C of the purification procedure and of the purified Fv are shown in lanes 2–5, respectively. M indicates molecular mass markers (kDa). 15% SDS-polyacrylamide gel electrophoresis was carried as described by Laemmli (31).  
FIG. 2. Fluorescence emission spectra of the 0.5 Fv fragment and its complex with the RP135 peptide antigen. Fluorescence emission spectra of 300 nM 0.5 Fv fragment without (continuous line) and with (broken line) 1 M RP135 peptide antigen were measured at 25.0 ( 0.1) °C in 50 mM phosphate buffer (pH 7.5) using an excitation wavelength of 280 nm with a band pass of 10 nm. Also shown is the spectrum of the buffer with and without peptide under the same conditions (dashed line).  
FIG. 4. Thermodynamic cycles showing the coupling between effects of high salt and mutations in the peptide antigen on its binding to the 0.5 Fv fragment. The Fv fragment and the wild-type peptide antigen are designated by Ab and Ag(wt), respectively. Single-letter notation for amino acids is used. Free energy values are given in kcal mol 1 (Table I). The coupling free energies , G int , were calculated using Equation 4.  
FIG. 3. Titration of the 0.5 Fv fragment with wild-type peptide monitored by fluorescence enhancement. The concentration of the 0.5 Fv fragment was 0.5 M. The data were fitted to Equation 1. For further details, see " Experimental Procedures. "  
Contribution of Arginine Residues in the RP135 Peptide Derived from the V3 Loop of gp120 to Its Interaction with the Fv Fragment of the 0.5beta HIV-1 Neutralizing Antibody

July 1996


69 Reads

The construction, expression, and purification of an active Fv fragment of the 0.5beta monoclonal human immunodeficiency virus type 1 (HIV-1) neutralizing antibody is reported. The interaction between the Fv fragment and the RP135 peptide derived from the V3 loop of gp120 from HIV-1IIIB was studied by varying the salt concentration and by mutating arginine residues in the peptide. The mutations R4A, R8A and R11A (which correspond to residues 311, 315, and 318 in gp120 of HIV-1IIIB) reduce the binding free energy by 0.22 (+/- 0. 20), 4.32 (+/- 0.16), and 1.58 (+/- 0.17) kcal mol-1, respectively. The salt-dependent components of their contributions to binding are 0.02 (+/- 0.22), -0.55 (+/- 0.18), and -0.97 (+/- 0.19) kcal mol-1, respectively. The magnitudes of the mutational effects and the extent of shielding by 1 M NaCl suggest that Arg-8 is involved in a buried salt bridge in the peptide-Fv fragment complex, whereas Arg-11 is involved in a more solvent-exposed electrostatic interaction.

Comparison and Evolution of Human Immunoglobulin VH Segments Located in the 3′ 0.8-Megabase Region: Evidence for Unidirectional Transfer of Segmental Gene Sequences

February 1994


88 Reads

Nucleotide sequences of 64 VH segments within the 3' 0.8-megabase region of the human immunoglobulin germ line VH locus were compared with trace evolution of human VH segments. Based on alignment of the deduced amino acid sequences of 37 functional germ line VH segments, a phylogenetic tree was generated using the neighbor-joining method. The phylogenetic tree clearly supports the previous classification of human VH segments into six families, which correlate roughly with mouse VH families with varying conservation. The human VH-III family is most homologous to mouse VH segments, suggesting that members of the VH-III family may be conserved by some functional constraint. The 5'-flanking region of each family has a family-specific structure. The sequenced 64 VH segments include 31 pseudogenes, of which 24 were highly conserved. Unidirectional transfer of segmental sequences was identified within the VH-III and VH-IV families, providing clear examples of germ line gene conversion. Such gene conversion may contribute to conserve structures of pseudo-VH segments. Comparison of the VH-IV family members indicates that recent repeated duplications and frequent gene conversions are responsible for strong conservation of this family, although functional selection is not completely excluded.

Crystal Structure of Triosephosphate Isomerase Complexed with 2-Phosphoglycolate at 0.83-A Resolution

April 2003


41 Reads

The atomic resolution structure ofLeishmania mexicana triosephosphate isomerase complexed with 2-phosphoglycolate shows that this transition state analogue is bound in two conformations. Also for the side chain of the catalytic glutamate, Glu167, two conformations are observed. In both conformations, a very short hydrogen bond exists between the carboxylate group of the ligand and the catalytic glutamate. The distance between O11 of PGA and Oε2 of Glu167 is 2.61 and 2.55 Å for the major and minor conformations, respectively. In either conformation, Oε1 of Glu167 is hydrogen-bonded to a water network connecting the side chain with bulk solvent. This network also occurs in two mutually exclusive arrangements. Despite the structural disorder in the active site, the C termini of the β strands that construct the active site display the least anisotropy compared with the rest of the protein. The loops following these β strands display various degrees of anisotropy, with the tip of the dimer interface loop 3 having very low anisotropy and the C-terminal region of the active site loop 6 having the highest anisotropy. The pyrrolidine ring of Pro168 at the N-terminal region of loop 6 is in a strained planar conformation to facilitate loop opening and product release.

Agonist binding site of Torpedo electric tissue nicotinic acetylcholine receptor. A negatively charged region of the δ subunit within 0.9 nm of the α subunit binding site disulfide

December 1991


56 Reads

The positively charged quaternary ammonium group of agonists of the nicotinic acetylcholine (ACh) receptor binds to a negative subsite at most about 1 nm from a readily reducible disulfide. This disulfide is formed by alpha Cys192 and Cys193 (Kao and Karlin, 1986). In order to identify Asp or Glu residues that may contribute to the negative subsite, we synthesized S-(2-[3H]glycylamidoethyl)dithio-2-pyridine. Purified ACh receptor from Torpedo californica was mildly reduced and reacted with S-(2-[3H]glycylamidoethyl)dithio-2-pyridine. The predominant product was a mixed disulfide between the 3H-N-glycylcysteamine moiety and alpha Cys192 or Cys193. In the extended conformation of [3H] N-glycylcysteamine, the distance from the glycyl amino group to the cysteamine thio group is 0.9 nm. Thus, the amino group of disulfide-linked [3H]N-glycylcysteamine could react with carboxyls within 0.9 nm of Cys192/Cys193. To promote amide bond formation between the tethered amino group and receptor carboxyls, we added 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide. The predominant sites of amide coupling were on the delta subunit, in CNBr fragment 4 (delta 164-257). This reaction was inhibited by ACh. Only the first 61 residues of delta CNBr 4 are predicted to be extracellular, and there are 11 Asp or Gly residues in this region. One or more of these residues is likely to contribute to the binding of ACh.

Table 1 Data collection, phasing and refinement statistics
Fig. 2  
Crystal Structure of Human Parathyroid Hormone 1–34 at 0.9-Å Resolution

October 2000


240 Reads

The N-terminal fragment 1-34 of parathyroid hormone (PTH), administered intermittently, results in increased bone formation in patients with osteoporosis. PTH and a related molecule, parathyroid hormone-related peptide (PTHrP), act on cells via a common PTH/PTHrP receptor. To define more precisely the ligand-receptor interactions, we have crystallized human PTH (hPTH)-(1-34) and determined the structure to 0.9-A resolution. hPTH-(1-34) crystallizes as a slightly bent, long helical dimer. Analysis reveals that the extended helical conformation of hPTH-(1-34) is the likely bioactive conformation. We have developed molecular models for the interaction of hPTH-(1-34) and hPTHrP-(1-34) with the PTH/PTHrP receptor. A receptor binding pocket for the N terminus of hPTH-(1-34) and a hydrophobic interface with the receptor for the C terminus of hPTH-(1-34) are proposed.

Structure of the nicotinic receptor acetylcholine-binding site. Identification of acidic residues in the ?? subunit within 0.9 nm of the ?? subunit-binding site disulfide

March 1995


37 Reads

In the nicotinic receptor, the quaternary ammonium group of acetylcholine (ACh) binds to a negative subsite at most 1 nm from a readily reducible disulfide formed between α-subunit residues Cys and Cys. The cross-linker S-(2-[3H]glycylamidoethyl)dithio-2-pyridine formed a disulfide bond with reduced αCys/Cys and an amide bond with an acidic residue in the subunit (Czajkowski, C., and Karlin, A. (1991) J. Biol. Chem. 266, 22603-22612). The fully extended cross-linking moiety -NHCH2CONHCH2CH2S- is 0.9 nm long. After the disulfide bond linking -NHCH2CONHCH2CH2S- to the α subunit was reduced, -NHCH2CONHCH2CH2SH remained linked to the subunit by an amide bond. The subunit was cleaved at Met residues, and the radioactive fragments were isolated and sequenced by automated Edman degradation. Additionally, the isolated radioactive fragments were further cleaved at Trp residues and sequenced. Peaks of release of radioactivity were obtained in the sequencing cycles corresponding to Asp, Asp, and Glu. The mutation of Asp to Asn decreased the affinity of the receptor for ACh 100-fold, whereas the mutation of either Asp, Glu, or 8 other acidic residues in the same region of decreased the affinity by 3-fold or less (Czajkowski, C., Kaufmann, C., and Karlin, A.(1993) Proc. Natl. Acad. Sci. U. S. A 90, 6285-6289). Because Asp both contributes to ACh binding and is suitably close to the binding site disulfide, it is likely to be part of the ACh-binding site formed in the interface between the α and the subunits.

Fig. 1 A shows the 2 F obs Ϫ F calc SIGMAA weighted electron 
FIG. 5. Structural rearrangements of the catalytic residues that occur during enzyme catalysis. A, error scaled distance difference matrix (ESCET plot) calculated between the 0.95-Å resolution acyl-enzyme intermediate structure and the 1.1-Å resolution native elastase structure. B, error scaled distance difference matrix calculated between the 0.95-Å resolution acyl-enzyme intermediate structure and the 1.4-Å tetrahedral intermediate structure. C, superposition of the active site of the tetrahedral intermediate (orange) and the acyl-enzyme (magenta) structures based on the ESCET structural alignment.
X-ray Structure of a Serine Protease Acyl-Enzyme Complex at 0.95-A Resolution

June 2002


145 Reads

Kinetic analyses led to the discovery that N-acetylated tripeptides with polar residues at P3 are inhibitors of porcine pancreatic elastase (PPE) that form unusually stable acyl-enzyme complexes. Peptides terminating in a C-terminal carboxylate were more potent than those terminating in a C-terminal amide, suggesting recognition by the oxy-anion hole is important in binding. X-ray diffraction data were recorded to 0.95-A resolution for an acyl-enzyme complex formed between PPE and N-acetyl-Asn-Pro-Ile-CO2H at approximately pH 5. The accuracy of the crystallographic coordinates allows structural issues concerning the mechanism of serine proteases to be addressed. Significantly, the ester bond of the acyl-enzyme showed a high level of planarity, suggesting geometric strain of the ester link is not important during catalysis. Several hydrogen atoms could be clearly identified and were included within the model. In keeping with a recent x-ray structure of subtilisin at 0.78 A (1), limited electron density is visible consistent with the putative location of a hydrogen atom approximately equidistant between the histidine and aspartate residues of the catalytic triad. Comparison of this high resolution crystal structure of the acyl-enzyme complex with that of native elastase at 1.1 A (2) showed that binding of the N-terminal part of the substrate can be accommodated with negligible structural rearrangements. In contrast, comparison with structures obtained as part of "time-resolved" studies on the reacting acyl-enzyme complex at >pH 7 (3) indicate small but significant structural differences, consistent with the proposed synchronization of ester hydrolysis and substrate release.

Cohen S, Ushiro H, Stoscheck C, Chinkers MA native 170 000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles. J Biol Chem 257: 1523-1531

March 1982


170 Reads

A method is presented for the preparation of a "native" epidermal growth factor (EGF) receptor-kinase complex of molecular weight 170,000 from A-431 cells. Although this receptor complex is capable of binding EGF, noncovalently, in quantities similar to the previously isolated 150,000 complex (Cohen, S., Carpenter, G., and King, L., Jr. (1980) J. Biol. Chem. 255, 4834-4842), the 170,000 preparation has 5 to 10 times the intrinsic kinase activity (autophosphorylation). However, the 170,000 kinase activity toward other proteins is lower than that of the 150,000 preparation. Both the 170,000 and 150,000 kinase activities are enhanced by EGF. The 170,000 and 150,000 proteins are also capable of forming covalent linkages to 125I-EGF, and each is precipitated by antisera directed against the 170,000 protein. We suggest the 150,000 protein is a proteolytic degradation product of the 170,000 protein. The EGF-enhanced kinase activity of the 170,000 preparation remains associated with the 125I-EGF-binding activity following EGF affinity chromatography, electrophoresis in nondenaturing gels, or immunoprecipitation with antisera directed against the sodium dodecyl sulfate (SDS) gel-purified 170,000 protein. These results indicate that the receptor, kinase, and substrate domains are linked, possibly covalently.

Fig. 1. Protein sequence comparison 
Fig. 2. Electron density representation of the dimer interface of the DNA-binding domain 
Fig. 3. IclR topology and its dimer and tetramer arrangement 
Fig. 4. Putative signal-binding pocket 
Fig. 5. DNA binding model 
Crystal Structure of Thermotoga maritima 0065, a Member of the IclR Transcriptional Factor Family

June 2002


174 Reads

Members of the IclR family of transcription regulators modulate signal-dependent expression of genes involved in carbon metabolism in bacteria and archaea. The Thermotoga maritima TM0065 gene codes for a protein (TM-IclR) that is homologous to the IclR family. We have determined the crystal structure of TM-IclR at 2.2 A resolution using MAD phasing and synchrotron radiation. The protein is composed of two domains: the N-terminal DNA-binding domain contains the winged helix-turn-helix motif, and the C-terminal presumed regulatory domain is involved in binding signal molecule. In a proposed signal-binding site, a bound Zn(2+) ion was found. In the crystal, TM-IclR forms a dimer through interactions between DNA-binding domains. In the dimer, the DNA-binding domains are 2-fold related, but the dimer is asymmetric with respect to the orientation of signal-binding domains. Crystal packing analysis showed that TM-IclR dimers form a tetramer through interactions exclusively by signal-binding domains. A model is proposed for binding of IclR-like factors to DNA, and it suggests that signal-dependent transcription regulation is accomplished by affecting an oligomerization state of IclR and therefore its affinity for DNA target.

FIGURE 1. Schematic diagrams of mesothelin and MORAb-009 Fab. A, the upper panel shows the precursor protein encoded by the human MSLN gene. The 622-residue precursor is subsequently processed by the endoprotease furin into the mature form of mesothelin containing residues from 296 to 598. For convenience, the mature mesothelin is renumbered from residue 1 to 303. The mature form of mesothelin together with two additional residues and a hexahistidine tag (the lower panel) was expressed for analysis. The three N-glycosylation sites in mesothelin (Asn-93, Asn-193, and Asn-220) are indicated. B, shown is sequence alignment of the N-terminal 64 residues of mesothelins from human, mouse, and rat. The conserved Cys-7 and Cys-31 that form a disulfide bridge are highlighted in boldface. Residues that form -helices in the structure are shown as rectangles below the sequence alignment. C, schematics of Fab light and heavy chains are shown. Residue numbering is consistent with prior literature, and CDRs in shaded boxes are assigned according to the improved Chothia method. CDRs are labeled as L1, L2, and L3 for the light chain and H1, H2, and H3 for the heavy chain. The variable and constant domains are indicated as V and C, respectively. MPF, megakaryocyte potentiating factor.  
FIGURE 3. Molecular interactions between mesothelin and Fab. A, shown is a ribbon diagram of the structure of the Msln-(7– 64)-Fab complex. The mesothelin fragment is colored in magenta. The N-terminal disulfide bond is illustrated as sticks in yellow. The Fab light chain and heavy chain are colored in cyan and blue, respectively. Variable and constant domains of the light chain (V L and C L ) and the heavy chain (V H and C H ) are indicated. CDRs of the light chain are labeled as L1, L2, and L3, respectively, and those of heavy chain are labeled H1, H2, and H3, respectively. Inset, shown is an enlarged view of the hydrophobic and aromatic-aromatic interactions between Phe-22 of mesothelin and Fab. Residues involving in interacting with Phe-22 of mesothelin are labeled. B, a close up view of the structure of Msln-(7– 64) is shown. Right panel, the five -helices are labeled as 1, 2, 3, 4, and 5, sequentially , from the N to C terminus. Residues important for interacting with Fab are labeled in magenta, and those for interacting with CA-125 are shown in cyan. Left panel, a semitransparent electrostatic potential surface is plotted covering the structure of the fragment with positive and negative potentials in blue and red, respectively. CDRs for the light chain are colored in cyan, and those for the heavy chain are in blue. C, a stereoscopic pair shows a region of mesothelin in stick model around the disulfide bond between Cys-7 and Cys- 31. Overlapping this model is a 2F o F c map contoured at 1.0 level showing the quality of electron density. D, the electrostatic potential surfaces show the chemical and shape complementarity between the interacting mesothelin and Fab. The mesothelin molecule is intentionally flipped by 180° to expose the interacting surfaces, revealing a concave surface on the side of Fab and a convex one on the mesothelin side. The two surfaces are in perfect shape and  
FIGURE 4. Conformational adaptation in Fab upon binding to mesothelin. A, shown is superposition of the antigen-free Fab to the one bound with mesothelin. The light and heavy chains of the antigen bound Fab are shown as ribbon diagrams in cyan and blue, respectively; the free Fab is shown in gray. The bound mesothelin is shown as a ribbon model in magenta. CDRs of both the light chain and the heavy chain are labeled. B, shown are conformational changes in side chains of Fab upon binding to mesothelin. Mesothelin is displayed in magenta. The residues involving in significant conformational transitions are shown in stick model, and direction of the transition from the  
FIGURE 2. Binding of MORAb-009 to full-length mesothelin and identification of the minimal epitope-containing fragment recognized by MORAb-009. A, SDS-PAGE analysis of the purified wild-type mesothelin and triple mutant expressed in insect cells is shown. The wild-type mesothelin is glycosylated, whereas the N93Q/N193Q/N220Q triple mutant shows a significant downward shift in molecular weight. B, BN-PAGE analysis of MORAb-009 and its complexes with wild-type and triple mutant mesothelin is shown. When mixed with MORAb-009 Fab, both wild type and triple mutant can form a complex as shown by the single band on the BN-PAGE shifted to a much higher molecular weight compared with the mesothelin alone. The Fab fragment shows a broad smeared band on the BN-PAGE gel. C, identification of the minimal fragment of mesothelin for MORAb-009 binding is shown. Cell extracts from E. coli expressing TrxA-fused mesothelin fragments were analyzed by Western blot using MORAb-009 as the primary antibody. The expression levels of these fragments were confirmed using antibody against the hexahistidine tag. The number of residues included in each fragment is indicated . The C7S in the lower panel is the C7S mutant of TrxA-Msln-(1– 64), whereas the 64 indicates the fragment for TrxA-Msln-(1– 64).  
Correlation between structure and activity of mesothelin-interacting antibodies
Recognition of mesothelin by the therapeutic antibody MORAb-009: Structural and mechanistic insights

July 2012


218 Reads

Mesothelin is a tumor differentiation antigen that is highly expressed in many epithelial cancers, with limited expression in normal human tissues. Binding of mesothelin on normal mesothelial cells lining the pleura or peritoneum to the tumor-associated cancer antigen 125 (CA-125) can lead to heterotypic cell adhesion and tumor metastasis within the pleural and peritoneal cavities. This binding can be prevented by MORAb-009, a humanized monoclonal antibody against mesothelin currently under clinical trials. We show here that MORAb-009 recognizes a non-linear epitope that is contained in the first 64-residue fragment of the mesothelin. We further demonstrate that the recognition is independent of glycosylation state of the protein but sensitive to the loss of a disulfide bond linking residues Cys-7 and Cys-31. The crystal structure of the complex between the mesothelin N-terminal fragment and Fab of MORAb-009 at 2.6 Å resolution reveals an epitope encompassing multiple secondary structural elements of the mesothelin, including residues from helix α1, the loops linking helices α1 and α2, and between helices α4 and α5. The mesothelin fragment has a compact, right-handed superhelix structure consisting of five short helices and connecting loops. A residue essential for complex formation has been identified as Phe-22, which projects its side chain into a hydrophobic niche formed on the antibody recognition surface upon antigen-antibody contact. The overlapping binding footprints of both the monoclonal antibody and the cancer antigen CA-125 explains the therapeutic effect and provides a basis for further antibody improvement.

Characterization of volume-sensitive, calcium-permeating pathways in the osteosarcoma cell line UMR-106-01

April 1989


37 Reads

Measurements of cell volume changes, free cytosolic Ca2+ concentration [( Ca2+]i) with Fura 2 and cell membrane potential with 3,3'-dipropylthiodicarbocyanine iodide were used to study the effect of cell volume change on Ca2+ influx and the membrane potential of the osteoblastic osteosarcoma cell line, UMR-106-01. Swelling the cells by hypo-osmotic stress was followed by reduction in cell volume which was markedly impaired by removal of medium Ca2+. Accordingly, cell swelling resulted in [Ca2+]i increase only in the presence of medium Ca2+. The cell swelling-activated Ca2+ entry pathway was active at resting membrane potentials, and Ca2+ influx through this pathway markedly increased upon cell hyperpolarization. A linear relationship between Ca2+ entry and the potential across the plasma membrane was observed. Thus, the volume-activated Ca2+ permeating pathway in UMR-106-01 cells has conductive properties. These pathways do not spontaneously inactivate with time when the cells are not allowed to volume regulate. The pathway can be blocked by micromolar concentrations of nicardipine and La3+ but display very low sensitivity to diltiazem and verapamil. Activation of the volume-sensitive, Ca2+ permeating pathway was not dependent on an increase in [Ca2+]i. Likewise, activation of the pathway was independent of a change in membrane potential between -85 and -3 mV. The increase in [Ca2+]i resulted in hyperpolarization of the cells, probably due to activation of Ca2+-activated K+ channels. The volume-sensitive pathways were partially active under isotonic conditions. Their activity was inhibited by cell shrinkage and increased by cell swelling. The pathways were sensitive to small changes in cell volume, particularly around a medium osmolarity of 310 mosM.

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