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Erkang Fan

Publications and source records attributed to Erkang Fan.

7 recordsLinked to original sources
PubMed2003

Analysis of the sequence motifs responsible for the interactions of peroxins 14 and 5, which are involved in glycosome biogenesis in Trypanosoma brucei.

Glycosome biogenesis in trypanosomatids occurs via a process that is homologous to peroxisome biogenesis in other eukaryotes. Glycosomal matrix proteins are synthesized in the cytosol and imported posttranslationally. The import process involves a series of protein-protein interactions starting by recognition of glycosomal matrix proteins by a receptor in the cytosol. Most proteins to be imported contain so-called PTS-1 or PTS-2 targeting sequences recognized by, respectively, the receptor proteins PEX5 and PEX7. PEX14, a protein associated with the peroxisomal membrane, has been identified as a component of the docking complex and a point of convergence of the PEX5- and PEX7-dependent import pathways. In this paper, the strength of the interactions between Trypanosoma brucei PEX14 and PEX5 was studied by a fluorescence assay, using (i) a panel of N-terminal regions of TbPEX14 protein variants and (ii) a series of different peptides derived from TbPEX5, each containing one of the three WXXXF/Y motifs present in this receptor protein. On the PEX14 side, the N-terminal region of TbPEX14 including residues 1-84 appeared to be responsible for TbPEX5 binding. The results from PEX14 mutants identified specific residues in the N-terminal region of TbPEX14 involved in PEX5 binding and showed that in particular hydrophobic residues F35 and F52 are critical. On the PEX5 side, 13-mer peptides incorporating the first or the third WXXXF/Y motif bind to PEX14 with an affinity in the nanomolar range. However, the second WXXXF/Y motif peptide did not show any detectable affinity. Studies using variants of second and third motif peptides suggest that the alpha-helical content of the peptides as well as the charge of a residue at position 9 in the motif may be important for PEX14 binding. Assays with 7-, 10-, 13-, and 16-mer third motif peptides showed that 16-mers and 13-mers have comparable binding affinity for PEX14, whereas 10-mers and 7-mers have about 10- and 100-fold lower affinity than the 16-mers, respectively. The low sequence identities of PEX14 and PEX5 between parasite and its human host, and the vital importance of proper glycosome biogenesis to the parasite, render these peroxins highly promising drug targets.

Amino Acid Motifs↗
PubMed2003

TFA-sensitive arylsulfonylthiourea-assisted synthesis of N,N'-substituted guanidines.

An efficient synthesis of N,N'-substituted guanidine derivatives was developed via an aromatic sulfonyl-activated thiourea intermediate. The use of certain aromatic sulfonamides, such as PbfNH(2), as the key reagent to incorporate a TFA-labile guanidine protection group greatly facilitates solid-phase synthesis of N,N'-substituted guanidine compounds.

Catalysis↗
PubMed2002

Solution and crystallographic studies of branched multivalent ligands that inhibit the receptor-binding of cholera toxin.

The structure-based design of multivalent ligands offers an attractive strategy toward high affinity protein inhibitors. The spatial arrangement of the receptor-binding sites of cholera toxin, the causative agent of the severe diarrheal disease cholera and a member of the AB(5) bacterial toxin family, provides the opportunity of designing branched multivalent ligands with 5-fold symmetry. Our modular synthesis enabled the construction of a family of complex ligands with five flexible arms each ending with a bivalent ligand. The largest of these ligands has a molecular weight of 10.6 kDa. These ligands are capable of simultaneously binding to two toxin B pentamer molecules with high affinity, thus blocking the receptor-binding process of cholera toxin. A more than million-fold improvement over the monovalent ligand in inhibitory power was achieved with the best branched decavalent ligand. This is better than the improvement observed earlier for the corresponding nonbranched pentavalent ligand. Dynamic light scattering studies demonstrate the formation of concentration-dependent unique 1:1 and 1:2 ligand/toxin complexes in solution with no sign of nonspecific aggregation. This is in complete agreement with a crystal structure of the branched multivalent ligand/toxin B pentamer complex solved at 1.45 A resolution that shows the specific 1:2 ligand/toxin complex formation in the solid state. These results reiterate the power of the structure-based design of multivalent protein ligands as a general strategy for achieving high affinity and potent inhibition.

Binding Sites↗
PubMed2002

Characterization and crystal structure of a high-affinity pentavalent receptor-binding inhibitor for cholera toxin and E. coli heat-labile enterotoxin.

Multivalent ligand design constitutes an attractive avenue to the inhibition of receptor recognition and other biological events mediated by oligomeric proteins with multiple binding sites. One example is the design of multivalent receptor blockers targeting members of the AB(5) bacterial toxin family. We report here the synthesis and characterization of a pentavalent inhibitor for cholera toxin and Escherichia coli heat-labile enterotoxin. This inhibitor is an advance over the symmetric pentacyclen-derived inhibitor described in our earlier work in that it presents five copies of m-nitrophenyl-alpha-D-galactoside (MNPG) rather than five copies of beta-D-galactose. The approximately 100-fold higher single-site affinity of MNPG for the toxin receptor binding site relative to galactose is found to yield a proportionate increase in the affinity and IC50 measured for the respective pentavalent constructs. We show by dynamic light scattering that inhibition of receptor binding by the pentavalent inhibitor is due to 1:1 inhibitor:toxin association rather than to inhibitor-mediated aggregation. This 1:1 association is in complete agreement with a 1.46 A resolution crystal structure of the pentavalent inhibitor:toxin complex, which shows that the favorable single-site binding interactions of MNPG are retained by the five arms of the 5256 Da pentavalent MNPG-based inhibitor and that the initial segment of the linking groups interacts with the surface of the toxin B pentamer.

Bacterial Toxins↗
PubMed2002

Anchor-based design of improved cholera toxin and E. coli heat-labile enterotoxin receptor binding antagonists that display multiple binding modes.

The action of cholera toxin and E. coli heat-labile enterotoxin can be inhibited by blocking their binding to the cell-surface receptor GM1. We have used anchor-based design to create 15 receptor binding inhibitors that contain the previously characterized inhibitor MNPG as a substructure. In ELISA assays, all 15 compounds exhibited increased potency relative to MNPG. Binding affinities for two compounds, each containing a morpholine ring linked to MNPG via a hydrophobic tail, were characterized by pulsed ultrafiltration (PUF) and isothermal titration calorimetry (ITC). Crystal structures for these compounds bound to toxin B pentamer revealed a conserved binding mode for the MNPG moiety, with multiple binding modes adopted by the attached morpholine derivatives. The observed binding interactions can be exploited in the design of improved toxin binding inhibitors.

Bacterial Toxins↗
PubMed2002

Combating infectious diseases through multivalent design.

Many biological interactions are multivalent, linking two particles via many copies of the same ligand-receptor binding pair. Examples of multivalent binding range from cell-cell adhesion to the assembly of large protein complexes from constituent multimers to the binding of AB5 bacterial toxins at the cell surface. Multivalent interactions can be effectively mimicked, inhibited, or disrupted through the design of suitable multivalent ligands. We review here recent work on multivalent ligand design based on a number of different chemical scaffolds, with a specific emphasis on the use of structure-based ligand design to target multimeric bacterial toxins.

Anti-Bacterial Agents↗