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Site-directed mutagenesis of the heterotrimeric killer toxin zymocin identifies residues required for early steps in toxin action.

ABSTRACT: Zymocin is a Kluyveromyces lactis protein toxin composed of ??? subunits encoded by the cytoplasmic virus-like element k1 and functions by ??-assisted delivery of the anticodon nuclease (ACNase) ? into target cells. The toxin binds to cells' chitin and exhibits chitinase activity in vitro that might be important during ? import. Saccharomyces cerevisiae strains carrying k1-derived hybrid elements deficient in either ?? (k1ORF2) or ? (k1ORF4) were generated. Loss of either gene abrogates toxicity, and unexpectedly, Orf2 secretion depends on Orf4 cosecretion. Functional zymocin assembly can be restored by nuclear expression of k1ORF2 or k1ORF4, providing an opportunity to conduct site-directed mutagenesis of holozymocin. Complementation required active site residues of ?'s chitinase domain and the sole cysteine residue of ? (Cys250). Since ?? are reportedly disulfide linked, the requirement for the conserved ? C231 was probed. Toxicity of intracellularly expressed ? C231A indicated no major defect in ACNase activity, while complementation of k1?ORF4 by ? C231A was lost, consistent with a role of ? C250 and ? C231 in zymocin assembly. To test the capability of ?? to carry alternative cargos, the heterologous ACNase from Pichia acaciae (P. acaciae Orf2 [PaOrf2]) was expressed, along with its immunity gene, in k1?ORF4. While efficient secretion of PaOrf2 was detected, suppression of the k1?ORF4-derived k1Orf2 secretion defect was not observed. Thus, the dependency of k1Orf2 on k1Orf4 cosecretion needs to be overcome prior to studying ??'s capability to deliver other cargo proteins into target cells.

SUBMITTER: Wemhoff S

PROVIDER: S-EPMC4178643 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Site-directed mutagenesis of the heterotrimeric killer toxin zymocin identifies residues required for early steps in toxin action.

Wemhoff Sabrina S Klassen Roland R Meinhardt Friedhelm F

Applied and environmental microbiology 20140815 20

Zymocin is a Kluyveromyces lactis protein toxin composed of αβγ subunits encoded by the cytoplasmic virus-like element k1 and functions by αβ-assisted delivery of the anticodon nuclease (ACNase) γ into target cells. The toxin binds to cells' chitin and exhibits chitinase activity in vitro that might be important during γ import. Saccharomyces cerevisiae strains carrying k1-derived hybrid elements deficient in either αβ (k1ORF2) or γ (k1ORF4) were generated. Loss of either gene abrogates toxicity ...[more]

PMID: 25128337

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Project description:The A2a adenosine receptor is a member of the G-protein coupled receptor family, and its activation stimulates cyclic AMP production. To determine the residues which are involved in ligand binding, several residues in transmembrane domains 5-7 were individually replaced with alanine and other amino acids. The binding properties of the resultant mutant receptors were determined in transfected COS-7 cells. To study the expression levels in COS-7 cells, mutant receptors were tagged at their amino terminus with a hemagglutinin epitope, which allowed their immunological detection in the plasma membrane by the monoclonal antibody 12CA5. The functional properties of mutant receptors were determined by measuring stimulation of adenylate cyclase. Specific binding of [3H]CGS 21680 (15 nM) and [3H]XAC (4 nM), an A2a agonist and antagonist, respectively, was absent in the following Ala mutants: F182A, H250A, N253A, I274A, H278A, and S281A, although they were well expressed in the plasma membrane. The hydroxy group of Ser-277 is required for high affinity binding of agonists, but not antagonists. An N181S mutant lost affinity for adenosine agonists substituted at N6 or C-2, but not at C-5'. The mutant receptors I274A, S277A, and H278A showed full stimulation of adenylate cyclase at high concentrations of CGS 21680. The functional agonist potencies at mutant receptors that lacked radioligand binding were > 30-fold less than those at the wild type receptor. His-250 appears to be a required component of a hydrophobic pocket, and H-bonding to this residue is not essential. On the other hand, replacement of His-278 with other aromatic residues was not tolerated in ligand binding. Thus, some of the residues targeted in this study may be involved in the direct interaction with ligands in the human A2a adenosine receptor. A molecular model based on the structure of rhodopsin, in which the 5'-NH in NECA is hydrogen bonded to Ser-277 and His-278, was developed in order to visualize the environment of the ligand binding site.

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Site-directed mutagenesis of the heterotrimeric killer toxin zymocin identifies residues required for early steps in toxin action.

Publications

Site-directed mutagenesis of the heterotrimeric killer toxin zymocin identifies residues required for early steps in toxin action.

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