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Role of acidic residues in helices TH8-TH9 in membrane interactions of the diphtheria toxin T domain.

Ghatak C, Rodnin MV, Vargas-Uribe M, McCluskey AJ, Flores-Canales JC, Kurnikova M, Ladokhin AS - Toxins (Basel) (2015)

Bottom Line: Thermal unfolding and fluorescence measurements, complemented with molecular dynamics simulations, suggest that the mutant E362Q is more susceptible to acid destabilization because of disruption of native intramolecular contacts.Both mutants adopt a final functional state upon further acidification.We conclude that these acidic residues are involved in the pH-dependent action of the T domain, and their replacements can be used for fine tuning the pH range of membrane interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA. c.ghatak79@gmail.com.

ABSTRACT
The pH-triggered membrane insertion of the diphtheria toxin translocation domain (T domain) results in transferring the catalytic domain into the cytosol, which is relevant to potential biomedical applications as a cargo-delivery system. Protonation of residues is suggested to play a key role in the process, and residues E349, D352 and E362 are of particular interest because of their location within the membrane insertion unit TH8-TH9. We have used various spectroscopic, computational and functional assays to characterize the properties of the T domain carrying the double mutation E349Q/D352N or the single mutation E362Q. Vesicle leakage measurements indicate that both mutants interact with the membrane under less acidic conditions than the wild-type. Thermal unfolding and fluorescence measurements, complemented with molecular dynamics simulations, suggest that the mutant E362Q is more susceptible to acid destabilization because of disruption of native intramolecular contacts. Fluorescence experiments show that removal of the charge in E362Q, and not in E349Q/D352N, is important for insertion of TH8-TH9. Both mutants adopt a final functional state upon further acidification. We conclude that these acidic residues are involved in the pH-dependent action of the T domain, and their replacements can be used for fine tuning the pH range of membrane interactions.

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Related in: MedlinePlus

Size exclusion chromatography shows that the WT T domain (black) and the mutants E362Q (red) and E349Q/D352N (blue) elute as a single peak in buffer at pH 8.0 (A), which corresponds to the retention volume of the monomeric form of the protein. At pH 6.5 (B), both mutants showed an additional peak of earlier elution (~13 mL) that suggests the presence of dimers.
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toxins-07-01303-f005: Size exclusion chromatography shows that the WT T domain (black) and the mutants E362Q (red) and E349Q/D352N (blue) elute as a single peak in buffer at pH 8.0 (A), which corresponds to the retention volume of the monomeric form of the protein. At pH 6.5 (B), both mutants showed an additional peak of earlier elution (~13 mL) that suggests the presence of dimers.

Mentions: The conformational changes induced by lowering the pH lead to partial exposure of the hydrophobic core of the T domain, which in the absence of membranes, leads to oligomerization [32]. In the WT protein, the dimers start forming at pH < 6 [34], but in mutants with an altered distribution of charges, the dimerization may occur at higher pH. To test this hypothesis, we carried out size exclusion chromatography for the WT and the two mutants. Our results show that either of the proteins eluted as a single peak at pH 8.0 (Figure 5A), which corresponds to the monomeric T domain. The mutants, however, eluted as two peaks at pH 6.5 (Figure 5B), suggesting the formation of dimers.


Role of acidic residues in helices TH8-TH9 in membrane interactions of the diphtheria toxin T domain.

Ghatak C, Rodnin MV, Vargas-Uribe M, McCluskey AJ, Flores-Canales JC, Kurnikova M, Ladokhin AS - Toxins (Basel) (2015)

Size exclusion chromatography shows that the WT T domain (black) and the mutants E362Q (red) and E349Q/D352N (blue) elute as a single peak in buffer at pH 8.0 (A), which corresponds to the retention volume of the monomeric form of the protein. At pH 6.5 (B), both mutants showed an additional peak of earlier elution (~13 mL) that suggests the presence of dimers.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4417968&req=5

toxins-07-01303-f005: Size exclusion chromatography shows that the WT T domain (black) and the mutants E362Q (red) and E349Q/D352N (blue) elute as a single peak in buffer at pH 8.0 (A), which corresponds to the retention volume of the monomeric form of the protein. At pH 6.5 (B), both mutants showed an additional peak of earlier elution (~13 mL) that suggests the presence of dimers.
Mentions: The conformational changes induced by lowering the pH lead to partial exposure of the hydrophobic core of the T domain, which in the absence of membranes, leads to oligomerization [32]. In the WT protein, the dimers start forming at pH < 6 [34], but in mutants with an altered distribution of charges, the dimerization may occur at higher pH. To test this hypothesis, we carried out size exclusion chromatography for the WT and the two mutants. Our results show that either of the proteins eluted as a single peak at pH 8.0 (Figure 5A), which corresponds to the monomeric T domain. The mutants, however, eluted as two peaks at pH 6.5 (Figure 5B), suggesting the formation of dimers.

Bottom Line: Thermal unfolding and fluorescence measurements, complemented with molecular dynamics simulations, suggest that the mutant E362Q is more susceptible to acid destabilization because of disruption of native intramolecular contacts.Both mutants adopt a final functional state upon further acidification.We conclude that these acidic residues are involved in the pH-dependent action of the T domain, and their replacements can be used for fine tuning the pH range of membrane interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA. c.ghatak79@gmail.com.

ABSTRACT
The pH-triggered membrane insertion of the diphtheria toxin translocation domain (T domain) results in transferring the catalytic domain into the cytosol, which is relevant to potential biomedical applications as a cargo-delivery system. Protonation of residues is suggested to play a key role in the process, and residues E349, D352 and E362 are of particular interest because of their location within the membrane insertion unit TH8-TH9. We have used various spectroscopic, computational and functional assays to characterize the properties of the T domain carrying the double mutation E349Q/D352N or the single mutation E362Q. Vesicle leakage measurements indicate that both mutants interact with the membrane under less acidic conditions than the wild-type. Thermal unfolding and fluorescence measurements, complemented with molecular dynamics simulations, suggest that the mutant E362Q is more susceptible to acid destabilization because of disruption of native intramolecular contacts. Fluorescence experiments show that removal of the charge in E362Q, and not in E349Q/D352N, is important for insertion of TH8-TH9. Both mutants adopt a final functional state upon further acidification. We conclude that these acidic residues are involved in the pH-dependent action of the T domain, and their replacements can be used for fine tuning the pH range of membrane interactions.

Show MeSH
Related in: MedlinePlus