title: Accessory cholera enterotoxin, Ace, from Vibrio cholerae: structure, unfolding, and virstatin binding.
abstract: Vibrio cholerae accessory cholera enterotoxin (Ace) is the third toxin, along with cholera toxin (CT) and zonula occludens toxin (Zot), that causes the endemic disease cholera. Structural characterization of Ace has been restricted because of the limited production of this toxic protein by V. cholerae. We have cloned, overexpressed, and purified Ace from V. cholerae strain O395 in Escherichia coli to homogeneity and determined its biological activity. The unfolding of the purified protein was investigated using circular dichroism and intrinsic tryptophan fluorescence. Because Ace is predominantly a hydrophobic protein, the degree of exposure of hydrophobic regions was identified from the spectral changes of the environment-sensitive fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) that quenches the fluorescence of tryptophan residues of Ace in a concentration-dependent manner. Results showed that bis-ANS binds one monomeric unit of Ace with a 1:1 stoichiometry and a K' of 0.72 μM. Ace exists as a dimer, with higher oligomeric forms appearing upon glutaraldehyde cross-linking. This study also reports the binding of virstatin, a small molecule that inhibits virulence regulation in V. cholerae, to Ace. The binding constant (K=9×10(4) M(-1)) and the standard free energy change (ΔG°=-12 kcal mol(-1)) of Ace-virstatin interaction have been evaluated by the fluorescence quenching method. The binding does not affect the oligomeric status of Ace. A cell viability assay of the antibacterial activity of Ace has been performed using various microbial strains. A homology model of Ace, consistent with the experimental results, has been constructed.
authors.affiliation: Department of Biochemistry, Bioinformatics Centre, Bose Institute, P-1/12 CIT Scheme VIIM, Kolkata 700054, India. tanaya_chatterjee@yahoo.com
authors.forename: Tanaya
authors.initials: T
authors.lastname: Chatterjee
authors.forename: Debadrita
authors.initials: D
authors.lastname: Mukherjee
authors.forename: Sucharita
authors.initials: S
authors.lastname: Dey
authors.forename: Aritrika
authors.initials: A
authors.lastname: Pal
authors.forename: Kazi Mirajul
authors.initials: KM
authors.lastname: Hoque
authors.forename: Pinak
authors.initials: P
authors.lastname: Chakrabarti
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D001419:Bacteria / Q000187:drug effects / Q000254:growth & development; D001483:Base Sequence; D002087:Butyrates / Q000737:chemistry / Q000378:metabolism; D002772:Cholera Toxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D005976:Glutaral / Q000737:chemistry; D007700:Kinetics; D050296:Microbial Viability / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D053644:Naphthalimides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013050:Spectrometry, Fluorescence; D014734:Vibrio cholerae / Q000235:genetics / Q000378:metabolism*
chemical_list: C081530:Ace toxin; D002087:Butyrates; D053644:Naphthalimides; D011994:Recombinant Proteins; C505381:virstatin; D002772:Cholera Toxin; D005976:Glutaral