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S Choe

Publications and source records attributed to S Choe.

66 records · Page 4Linked to original sources
PubMed1994

Domain swapping: entangling alliances between proteins.

The comparison of monomeric and dimeric diphtheria toxin (DT) reveals a mode for protein association which we call domain swapping. The structure of dimeric DT has been extensively refined against data to 2.0-A resolution and a three-residue loop has been corrected as compared with our published 2.5-A-resolution structure. The monomeric DT structure has also been determined, at 2.3-A resolution. Monomeric DT is a Y-shaped molecule with three domains: catalytic (C), transmembrane (T), and receptor binding (R). Upon freezing in phosphate buffer, DT forms a long-lived, metastable dimer. The protein chain tracing discloses that upon dimerization an unprecedented conformational rearrangement occurs: the entire R domain from each molecule of the dimer is exchanged for the R domain from the other. This involves breaking the noncovalent interactions between the R domain and the C and T domains, rotating the R domain by 180 degrees with atomic movements up to 65 A, and re-forming the same noncovalent interactions between the R domain and the C and T domains of the other chain of the dimer. This conformational transition explains the long life and metastability of the DT dimer. Several other intertwined, dimeric protein structures satisfy our definition of domain swapping and suggest that domain swapping may be the molecular mechanism for evolution of these oligomers and possibly of oligomeric proteins in general.

Bacterial Proteins↗
PubMed1994

Inhibition of membrane translocation of diphtheria toxin A-fragment by internal disulfide bridges.

Fragment A of diphtheria toxin is translocated to the cytosol when the toxin is presented to receptor-positive cells. The toxin binds to cell surface receptors through its B-fragment, and after endocytotic uptake, the low endosomal pH triggers translocation of the A-fragment across the membrane. Translocation can also be induced at the level of the plasma membrane by exposure to low pH medium. Based on the diphtheria toxin crystal structure, we made five double cysteine mutants of the A-fragment, each expected to form an intramolecular disulfide bond. Four of the double cysteine mutants efficiently formed an intramolecular disulfide bridge, and these same mutants showed a strong reduction in their translocating ability. The inhibition of translocation was observed both when the toxin was endocytosed and when translocation was induced by exposing surface-bound toxin to low pH. The data indicate that extensive unfolding of the A-fragment is required for translocation.

Adenosine Diphosphate Ribose↗
PubMed1994

Refined structure of dimeric diphtheria toxin at 2.0 A resolution.

The refined structure of dimeric diphtheria toxin (DT) at 2.0 A resolution, based on 37,727 unique reflections (F > 1 sigma (F)), yields a final R factor of 19.5% with a model obeying standard geometry. The refined model consists of 523 amino acid residues, 1 molecule of the bound dinucleotide inhibitor adenylyl 3'-5' uridine 3' monophosphate (ApUp), and 405 well-ordered water molecules. The 2.0-A refined model reveals that the binding motif for ApUp includes residues in the catalytic and receptor-binding domains and is different from the Rossmann dinucleotide-binding fold. ApUp is bound in part by a long loop (residues 34-52) that crosses the active site. Several residues in the active site were previously identified as NAD-binding residues. Glu 148, previously identified as playing a catalytic role in ADP-ribosylation of elongation factor 2 by DT, is about 5 A from uracil in ApUp. The trigger for insertion of the transmembrane domain of DT into the endosomal membrane at low pH may involve 3 intradomain and 4 interdomain salt bridges that will be weakened at low pH by protonation of their acidic residues. The refined model also reveals that each molecule in dimeric DT has an "open" structure unlike most globular proteins, which we call an open monomer. Two open monomers interact by "domain swapping" to form a compact, globular dimeric DT structure. The possibility that the open monomer resembles a membrane insertion intermediate is discussed.

Amino Acid Sequence↗
PubMed1993

Crystal structure of a synthetic triple-stranded alpha-helical bundle.

The x-ray crystal structure of a peptide designed to form a double-stranded parallel coiled coil shows that it is actually a triple-stranded coiled coil formed by three alpha-helices. Unlike the designed parallel coiled coil, the helices run up-up-down. The structure is stabilized by a distinctive hydrophobic interface consisting of eight layers. As in the design, each alpha-helix in the coiled coil contributes one leucine side chain to each layer. The structure suggests that hydrophobic interactions are a dominant factor in the stabilization of coiled coils. The stoichiometry and geometry of coiled coils are primarily determined by side chain packing in the solvent-inaccessible interior, but electrostatic interactions also contribute.

Amino Acid Sequence↗
PubMed1993

Overexpression and purification of avian ovomucoid third domains in Escherichia coli.

Genetic engineering studies of ovomucoid domains have been hindered by the lack of an efficient procedure for overproducing this protein. The novel scheme presented here has led to the isolation of chicken ovomucoid third domain (OMCHI3) at a level of 22 mg pure protein/l Escherichia coli culture medium. The gene coding for OMCHI3 was fused to the 3' end of the gene encoding staphylococcal nuclease (SNase). Expression of the chimeric gene was placed under control of the strong transcription and translation signals of the phage T7 promoter. Upon isopropyl-beta-D-galactopyranoside induction, the cells harboring the target plasmid efficiently overproduced the protein (30% of the total soluble protein). The 56-residue fragment corresponding to OMCHI3 was then liberated by cyanogen bromide (CNBr) cleavage at a genetically engineered methionine residue located at the nuclease--OMCHI3 junction (OMCHI3 lacks an internal methionine). SDS--PAGE, enzyme inhibition studies and NMR spectroscopy all indicated that the recombinant OMCHI3 has properties identical to those of OMCHI3 isolated from its natural source. The expression system was easily adapted for the production of [98% U 15N] OMCHI3. The expression vector was mutated for overexpression of turkey ovomucoid third domain (OMTKY3), which differs from OMCHI3 by three amino acid substitutions. Since many other avian ovomucoid domains also lack methionine residues, this approach should be suitable for large-scale production and isotope labeling of homologous proteinase inhibitors with a variety of inhibitory specificities.

Amino Acid Sequence↗
PubMed1992

pH-dependent insertion of proteins into membranes: B-chain mutation of diphtheria toxin that inhibits membrane translocation, Glu-349----Lys.

To investigate how diphtheria toxin (DT) undergoes pH-dependent membrane translocation in mammalian cells, we have isolated and characterized mutants of the toxin that are defective in acidic-pH-dependent killing of Escherichia coli. Cloned DT secreted to the periplasm of E. coli kills the bacteria under acidic conditions (near pH 5.0) by inserting into and permeabilizing the inner membrane (a mechanism independent of the toxin's ADP-ribosylation activity). Mutant forms of DT with reduced lethality for E. coli were selected by plating the bacteria under acidic conditions. CRM503, one of the full-length mutants selected by this protocol, also showed diminished cytotoxicity for mammalian cells. We traced the altered cytotoxicity of CRM503 to a Glu-349----Lys mutation (E349K), one of three point mutations, within the B fragment. The E349K mutation alone inhibited cytotoxicity and membrane translocation in mammalian cells and lethality for E. coli but did not affect enzymic activity or receptor binding. The recently determined crystallographic model of DT shows that Glu-349 resides within a short loop connecting two long hydrophobic alpha-helices of the translocation domain. Protonation of Glu-349 and two other nearby acidic residues, Asp-352 and Glu-362, may enable these helices to undergo membrane insertion and the intervening loop to be transferred to the opposite face of the bilayer. The E349K mutation introduces a positive charge at this site, which would be expected to inhibit membrane insertion and the insertion-dependent activities of DT. These results suggest that protonation of Glu-349 and nearby acidic residues may be important in triggering the translocation step of toxin action.

Animals↗
PubMed1992

The crystal structure of diphtheria toxin.

The crystal structure of the diphtheria toxin dimer at 2.5 A resolution reveals a Y-shaped molecule of three domains. The catalytic domain, called fragment A, is of the alpha + beta type. Fragment B actually consists of two domains. The transmembrane domain consists of nine alpha-helices, two pairs of which are unusually apolar and may participate in pH-triggered membrane insertion and translocation. The receptor-binding domain is a flattened beta-barrel with a jelly-roll-like topology. Three distinct functions of the toxin, each carried out by a separate structural domain, can be useful in designing chimaeric proteins, such as immunotoxins, in which the receptor-binding domain is substituted with antibodies to target other cell types.

Bacterial Toxins↗
PubMed1992

Three-dimensional profiles for analysing protein sequence-structure relationships.

In the method of 3D (three-dimensional) profiles, each residue position in a protein is characterized by its environment and is represented by a row of 20 numbers in a table, the profile. These numbers are the statistical preferences (called 3D-1D scores) of each of the 20 amino acids for this environment. A profile is computed from the coordinates of a protein model, and it gives a score S for any amino acid sequence folded as the model. To date 3D profiles have found three applications. The first is to identify other protein sequences which are folded in the same general pattern as the structure from which the profile was prepared. These are sequences which have high scores for the profile computed from the model. The second is to assess the validity of protein models, however determined. Correct models are found to give profiles that have high scores for their own amino acid sequences, and incorrect models are found to have lower scores. The example of the X-ray structure determination of diphtheria toxin is discussed. The third application is to assess which is the stable oligomeric state of a folded protein. Several examples suggest that the highest profile score for a sequence is achieved when the protein is aggregated into its most stable oligomeric state.

Amino Acid Sequence↗
PubMed1991

Chronic murine experimental myasthenia gravis: strength testing and serology.

CB6 (Balb/c x C57Bl/6 F1) and C57Bl/6 (B6) mice were hyperimmunized with Torpedo acetylcholine receptor (AChR) for 7 months. Control groups were hyperimmunized with bovine serum albumin. Antibody titers against Torpedo AChR rose quickly, reaching plateau levels by 3-4 months, while antibody to mouse AChR lagged by a few months, reaching plateau levels in 5 months. After the last immunization the mice maintained a state of stable autoimmunity for 9 months with high levels of antibodies against Torpedo and mouse AChR. Fatigability was measured on a programmable treadmill and remained present through the 9 months after the last immunization. CB6 mice had less weakness than the B6 mice, but the latter strain when immunized with BSA had more "false-positive" weakness. Titers of antibodies did not correlate with the degree of weakness measured on the treadmill. Despite the weakness and the high titers of anti-AChR antibodies, sera from myasthenic mice, in contrast to sera from myasthenic humans, were not able to block bungarotoxin binding to native AChR on the surface of BC3H1 cells.

Animals↗
PubMed1988

New surgical method to prevent pelvic radiation enteropathy.

Pelvic radiation doses exceeding 4,000-4,500 cGy are known to be associated with acute and chronic radiation enteropathy. This same radiation dose is, at the same time, only moderately effective in the elimination of microscopic malignancy, let alone gross clinical disease. Numerous medical and surgical attempts to minimize this complication have been uniformly unsuccessful. With the availability of a new synthetic, absorbable, polyglycolic acid mesh, an intestinal sling surgical procedure has been devised to exclude the small bowel from the pelvis and subsequent radiation fields. Twenty-five patients have been treated by this new technique with only one complication presenting as a fungal infection. Small-bowel barium contrast studies in 16 patients referred for postoperative radiation demonstrated 13 satisfactory exclusions of the small bowel from the translateral pelvic irradiation field. In 16 evaluable patients, three had unsatisfactory exclusion two of which were due to technical error. This has permitted high-dose (5,500-6,500 cGy) radiotherapy to the critical treatment volume without posttreatment complication. Mean follow-up time is 14.8 months. Several patients have been reexplored demonstrating complete absorption of the mesh without fibrinous adhesions or other foreign body reaction. It is concluded that this new technique of small bowel exclusion will permit the routine delivery of much higher doses of radiation in patients requiring improved local-regional control of their pelvic cancers and without morbidity from radiation-associated small bowel injury.

Aged↗
PubMedDate not supplied

Overview of regulatory cytochrome P450 enzymes of the vitamin D pathway.

Cytochromes P450c1 and P450c24 are regulated hydroxylase enzymes that direct the bioactivation and metabolic degradation of vitamin D. The bioactivation pathway is regulated by cytochrome P450c1 through its synthesis of 1alpha,25(OH)(2)D(3), the hormonally active form of the vitamin. Expression of the P450c1 gene is regulated at the transcription level. Promoter regions within the P450c1 gene have been identified that respond to cAMP and 1alpha,25(OH)(2)D(3) during the respective up- and down-regulation of P450c1 gene expression. The diametric action of 1alpha,25(OH)(2)D(3) to up-regulate P450c24 gene expression is discussed in the context of two vitamin D response elements (VDREs) that are linked functionally to an adjoining Ets-binding site. It is apparent from sequence-derived data that the P450c1 and P450c24 enzymes share only 10-25% sequence identity, yet they display functionally similar domains that are conserved across the family of cytochrome P450 enzymes. Expression of E. coli recombinant P450c1 and P450c24 enzymes, and the substrate-binding parameters for P450c24 are discussed. Finally, the natural point mutations in human P540c1 from patients with pseudovitamin D-deficiency rickets (PDDR) are discussed in the context of the enzyme's structure and function.

Amino Acid Sequence↗